RecJ exonuclease plays crucial roles in several DNA repair and recombina-tion pathways, and its ubiquity in bacterial species points to its ancientorigin and vital cellular function. RecJ exonuclease from Haemophilusinfluenzae is a 575-amino-acid protein that harbors the characteristic motifsconserved among RecJ homologs. The purified protein exhibits a processive5′–3′ single-stranded-DNA-specific exonuclease activity. The exonucleaseactivity of H. influenzae RecJ (HiRecJ) was supported by Mg2+ or Mn2+ andinhibited by Cd2+, suggesting a different mode of metal binding in HiRecJas compared to Escherichia coli RecJ (EcoRecJ). Site-directed mutagenesis ofhighly conserved residues in HiRecJ abolished enzymatic activity. Interest-ingly, substitution of alanine for aspartate 77 resulted in a catalyticallyinactive enzyme that bound to DNA with a significantly higher affinity ascompared to the wild-type enzyme. Noticeably, steady-state kinetic studiesshowed that H. influenzae single-stranded DNA-binding protein (HiSSB)increased the affinity of HiRecJ for single-stranded DNA and stimulated itsexonuclease activity. HiSSB, whose C-terminal tail had been deleted, failedto enhance RecJ exonuclease activity. More importantly, HiRecJ was foundto directly associate with its cognate single-stranded DNA-binding protein(SSB), as demonstrated by various in vitro assays. Interaction studies carriedout with the truncated variants of HiRecJ and HiSSB revealed that the twoproteins interact via the C-terminus of SSB protein and the core-catalyticdomain of RecJ. Taken together, these results emphasize direct interactionbetween RecJ and SSB, which confers functional cooperativity to these twoproteins. In addition, these results implicate SSB as being involved in therecruitment of RecJ to DNA and provide insights into the interplay betweenthese proteins in repair and recombination pathways.
Exonucleases are key players in themaintenance ofgenome integrity, pertaining to their critical roles inall DNAmetabolismpathways spanning replication,repair, and recombination. Of the several exonu-cleases identified in Escherichia coli, four exo-nucleases are involved in the DNA mismatchrepair (MMR) pathway: exonuclease I (ExoI), exo-nuclease VII (ExoVII), exonuclease X (ExoX), andRecJ. These exonucleasesmediate the excision step ofMMR subsequent to unwinding of nascent DNAstrand byUvrD helicase. Three of these exonucleases(RecJ, ExoI, and ExoVII) are processive single-stranded-DNA-specific enzymes, while ExoX can
d.
1376 Interaction of RecJ and SSB
hydrolyze both single-stranded and double-stranded DNA in a distributive manner.1 WhileExoI and ExoX degrade DNA strictly in the 3′–5′direction, RecJ functions unidirectionally at 5′-termini, and ExoVII can degrade a DNA strand ofeither polarity. The presence of these exonucleaseswith different polarities allows the MMR to occurindependent of the orientation of a d(GATC) site innascent DNA strands.2 While extracts of all possiblesingle-, double-, and triple-exonuclease mutantsdisplay significant residual MMR, extracts deficientin RecJ, ExoI, ExoVII, and ExoX exonucleases aredevoid of normal repair activity.3 Cells that are defi-cient in all the four exonucleases are associated withlow rates of mutation, albeit consequent to reducedviability and/or chromosome loss associated withactivation of the MMR system in the absence ofassociated exonucleases.4
RecJ, broadly classified as a DNA repair protein,apart from its role in MMR, is further associatedwith recombinational repair and base-excisionrepair. E. coli recJwas first identified as a gene essen-tial for RecBCD-independent pathways, such asRecF and RecE pathways, for conjugational recombi-nation and repair of UV damage.5 RecJ, in combina-tion with RecQ helicase, produces 3′-single-strandedDNA tails required to initiate recombination from adouble-stranded break, as well as functions indis-pensably to rescue replication from a stalled replica-tion fork with DNA damage.6 A role for RecJ hasbeen postulated in aberrant recombination betweenshort direct oligonucleotide repeats located withinthe gonococcal chromosome.7 RecJ−ExoVII− doublemutant is extremely sensitive to UV irradiation and,against the recD background, exhibits significantlyreduced viability and compromised λ-phage recom-bination.1,8,9 All these studies emphasize that 5′–3′exonucleases are more important than 3′–5′ acti-vities in DNA repair reactions in both wild-type andrecD backgrounds, and that RecJ exonuclease facil-itates recombination. In addition, RecJ can remove a5′-terminal 2′-deoxyribose-5-phosphate residue(dRPase activity) that is produced in base-excisionrepair pathway.10 These functions of the RecJ exonu-clease are consequential for genetic stability inpathogenic organisms such asHaemophilus influenzae.H. influenzae is a naturally transformable Gram-nega-tive bacterium that is widespread in its distributionamong the human population. This organism hasthe potential for rapid generation of geneticvariability through the phenomenon of phasevariation, which can confer drug resistance andfacilitate its pathogenesis and persistence in hosts.Sequencing of H. influenzae genome revealed thepresence of homologs of MMR-associated proteins,except for ExoX.11 More recently, RecJ has beenimplicated to be important for genome maintenancein H. influenzae.12
RecJ belongs to the DHH superfamily, which in-cludes several phosphoesterases from archaea, bac-teria, yeast, and orthologs in higher eukaryotes andhumans.13 RecJ protein and its homologs possessfour highly conservedmotifs of charged residues that
are essential for metal ion binding and catalysis ofphosphoesterase reactions. The family derives itsname from the characteristic signature sequence(DHH: aspartate–histidine–histidine) in the thirdmotif. Mutational analysis of E. coli RecJ (EcoRecJ)showed that all the motifs are essential for exonu-clease activity.14 While EcoRecJ requires Mg2+ for itsactivity, RecJ homologs from Thermus thermophilusand Saccharomyces cerevisiae are active in the pre-sence of Mg2+, Mn2+, or Co2+.15–17 RecJ has a strongspecificity for single-stranded DNA, while double-stranded DNA is neither a substrate nor a competi-tive inhibitor of single-stranded DNA exonucleaseactivity. The crystal structure of T. thermophilus RecJ(TtRecJ) bound to Mn2+ shows a novel fold in whichtwo domains are interconnected by a long helix,forming a characteristic narrow central groove that iswide enough to accommodate single-strandedDNA.18 Mn2+ is located on the wall of the grooveand is coordinated by conserved aspartate andhistidine residues. RecJ releases mononucleotideproducts during degradation of single-strandedDNA.19 However, transient secondary structureelements within single-stranded DNA can impedethe activity of RecJ exonuclease and, in bacterial cells,these secondary structures of DNA are removed bysingle-stranded DNA-binding protein (SSB). In con-trast to the roles of SSB proteins in covering bacterialsingle-stranded DNA and protecting it from degra-dation by exonucleases,E. coli SSB (EcoSSB) is knownto stimulate the activity of RecJ and ExoI.19,20Furthermore, EcoSSB was found to copurify withRecJ exonuclease.21
Despite a plethora of studies, regulation of theexonucleolytic activity of RecJ and its interactionwith other proteins of DNA metabolism pathwaysremains unclear. Given that RecJ is essential forgenomemaintenance inH. influenzae and is involvedin numerous vital DNA metabolism pathways, itwas of interest to carry out a biochemical analysis ofRecJ protein from H. influenzae, which could lead tobetter insights on the physiological roles of thisprotein. Despite several lines of evidence suggestinga functional interaction between SSB and RecJ, thereis no discrete evidence of a direct interaction betweenthe two proteins. Furthermore, the regions of SSBand RecJ proteins that are essential for their interac-tions have not yet been identified. Importantly, inthis study, a direct protein–protein interactionbetween RecJ and SSB has been demonstrated,suggesting that the two proteins function in closecoordination within pathways of DNA repair andrecombination.
Results and Discussion
Cloning, overexpression, and purification ofH. influenzae RecJ
H. influenzae RecJ (HiRecJ) was heterologouslyoverexpressed in E. coli strain BL21(DE3) pLysS as
1377Interaction of RecJ and SSB
an N-terminal maltose-binding protein (MBP)-tagged protein. After induction with isopropyl-1-thio-β-D-galactopyranoside (IPTG), a protein withmobility corresponding to ∼107 kDa, which is thesame as the theoretical molecular mass calculatedfrom the amino acid sequence of the H. influenzaeMBP–RecJ fusion protein, was observed (Fig. 1a,lane 3). The H. influenzae MBP–RecJ protein waspurified to homogeneity, as judged by SDS-PAGE(Fig. 1a, lane 4). In addition, HiRecJ was alsoexpressed and purified as a His-tagged protein,which was found to be insoluble (data not shown).However, it was used to generate polyclonal anti-bodies against HiRecJ, and Western blot analysisdetected a single band in crude cell lysates, as wellas that of the purified protein corresponding toMBP–RecJ (Fig. 1b, lanes 2–4). A faint band was seenin the uninduced cell lysate (Fig. 1b, lane 2), possiblydue to leaky expression of protein in the absence ofIPTG.TheMBP tagwas removed from the fusion protein
by treatment of the MBP–RecJ protein bound to anamylose resin column with Factor Xa protease. Ascan be seen from Fig. 1c, treatment of MBP–RecJ(lane 2) with Factor Xa resulted in cleavage of thefusion protein to MBP (∼45 kDa) and RecJ(∼64 kDa; lane 3). Since the fusion protein wasbound to the affinity matrix, uncleaved MBP–RecJand MBP generated after cleavage remained bound
Fig. 1. Expression, purification, and cleavage of HiRecJ fuanalysis of MBP–RecJ purification: lane 1, molecular weight m2, total cell extract of uninduced culture; lane 3, total cell extraPAGE and (d) Western blot analysis of the cleavage of Rec[prestained molecular weight markers in (d)]; lane 2, MBP–Rlane 4, HiRecJ.
to the matrix, while RecJ was released in the super-natant with small amounts of Factor Xa protease(Fig. 1c, lane 4). In Western blot analysis with RecJantiserum, a 64-kDa single band corresponding tocleaved RecJ protein was observed (Fig. 1d, lane 4).However, subsequent to its cleavage from MBP, thecleaved RecJ protein began to precipitate on storage,suggesting that MBP enhanced the solubility of theprotein.
Purification and characterization of H. influenzaesingle-stranded DNA-binding protein
Pairwise amino acid sequence alignment of H.influenzae single-stranded DNA-binding protein(HiSSB) and EcoSSB shows that the two proteinsshare a 59% identity and a 70% similarity over theirentire length (data not shown). The distal C-terminalend of HiSSB contains a stretch of residues(−FDDDIPF) that is identical with its E. coli counter-part and highly conserved in other bacterial homo-logs. The C-terminal domain of bacterial SSBproteins comprising this well-conserved stretch ofacidic and hydrophobic residues is essential for SSB-mediated modulation of enzymatic activities andinteraction with different proteins of replication andrepair machinery. To determine whether HiSSB hasany effect on the exonuclease activity of its cognateRecJ and whether the two proteins interact, HiSSB
sion protein. (a) SDS-PAGE analysis and (b) Western blotarkers [prestained molecular weight markers in (b)]; lanect of induced culture; lane 4, purified MBP–RecJ. (c) SDS-J from MBP protein: lane 1, molecular weight markersecJ; lane 3, MBP–RecJ treated with Factor Xa (1 μg/ml);
1378 Interaction of RecJ and SSB
was purified. In addition, a variant of HiSSB in whichtheC-terminal 41 residuesweredeletedwas generatedto ascertain whether the C-terminal domain of HiSSBis essential for its interaction with other proteins.HiSSB and HiSSBΔC proteins were overex-
pressed and purified to homogeneity using recom-binant DNA constructs harboring the wild-typeand mutant H. influenzae ssb genes, respectively(Fig. S1a). Polyclonal antiserum generated usingpurified HiSSB identified protein bands correspon-ding to HiSSB and HiSSBΔC on Western blotanalysis (Fig. S1b), while no cross-reactivity wasobserved with MBP–RecJ (data not shown). TheDNA binding properties of the two proteins wereinvestigated using electrophoretic mobility shiftassay (EMSA) and surface plasmon resonance(SPR) analysis. Radiolabeled 45-mer single-stranded DNA was incubated with increasing con-centrations of either wild-type HiSSB or HiSSBΔC,and electrophoresed through native PAGE. BothSSB and SSBΔC formed a single protein–DNAcomplex, as is evident from Fig. S1c and d, respec-tively. The faster mobility of the HiSSBΔC–single-stranded DNA complex (Fig. S1d) is indicative ofits relatively smaller size as compared to wild-typeSSB. At higher concentrations of HiSSBΔC, bandsof radiolabeled single-stranded DNA stuck in thewells were observed, the exact identity of which isunclear at present (Fig. S1d). SPR analysis using a65-mer single-stranded DNA immobilized on thesurface of a streptavidin sensor chip was furtherused to determine the binding constants for thesetwo proteins (data not shown). The Kd value ofwild-type SSB was determined to be 2.08×10−9 M,while that for the SSBΔC–single-stranded DNAinteraction was found to be 1.18×10−7 M. Thissuggests that binding of SSBΔC to single-strandedDNA is relatively weaker as compared to nativeSSB. This observation is similar to that reported forSSB protein and its C-terminal deletion mutantfrom Mycobacterium smegmatis.22
Characterization of HiRecJ activity
Exonuclease activity of purified H. influenzaeMBP–RecJ protein
EcoRecJ is a single-stranded-DNA-specific exonu-clease that hydrolyzes the DNA in a 5′–3′direction.15 The exonuclease activity of the purifiedH. influenzae MBP–RecJ protein was checked usingdifferent DNA substrates. MBP–RecJ protein couldonly hydrolyze linear single-stranded DNA (Fig.S2a). In addition, the protein was able to hydrolyze7.2 kb of linear M13 single-stranded DNA efficiently(Fig. S2a), suggesting that HiRecJ protein is not asequence-specific exonuclease. No nuclease activitywas detected with supercoiled, circular single-stranded, or linear double-stranded M13 virionDNA or mouse ribosomal RNA (Fig. S2), suggestingthat purified MBP–RecJ did not have any endonu-clease activity on double-stranded or single-stranded DNA as well as RNase activity. These
results indicate that RecJ protein fromH. influenzae isa single-stranded-DNA-specific exonuclease.The exonuclease activity of MBP–RecJ fusion
protein and cleaved RecJ protein obtained aftercleavage from MBP was further assayed on radio-actively labeled single-stranded oligonucleotide assubstrate (Fig. S3a and b, respectively). The sub-strate was hydrolyzed by both MBP–RecJ and RecJwith similar efficiencies (Fig. S3c), as no major diffe-rence was observed in the extent to which the sub-strate was degraded by the two proteins at differenttime points. MBP did not exhibit any nucleaseactivity (data not shown). This suggests that exo-nuclease activity is specific to the RecJ protein andthat MBP has no effect on the exonuclease activity ofRecJ. Since the MBP–RecJ fusion protein exhibitedhigher solubility than cleaved RecJ, with exonu-clease efficiency almost equal to that of the latter, thefusion protein was used for all subsequent assays.
Processivity
EcoRecJ is a processive exonuclease (i.e., it remainsbound to single-stranded DNA substrate until themajority of the phosphodiester bonds have beenhydrolyzed). RecJ homolog from S. cerevisiae, thecytosolic PPX1 exopolyphosphatase, processivelycleaves the terminal phosphate group from thepolyphosphate chain.23 Similarly, λ exonuclease isa 5′–3′ exonuclease with a very high processivity(N3000 bp).As an initial step in characterizing HiRecJ exonu-
clease, its processive nature was investigated. Aheat-denatured 234-nucleotide DNA (1 μM) labeledinternally with 3H and at the 3′-end with 32P wasincubated with 50 nM RecJ such that the rate ofhydrolysis was limited by the amount of enzymepresent. Under these conditions, the two radio-isotopes were released at the same rates (Fig. 2,inset) such that the 3H/32P ratio was always ∼1,suggesting a processive mode of catalysis (Fig. 2).This method, however, may provide an underesti-mation of the extent of processivity of the enzymegiven the short length of the substrate. Therefore, theprocessivity of HiRecJ was further quantified usinga 2686-nucleotide denatured single-stranded DNA.The DNA was labeled internally with 3H and at its5′-end with 32P. Preincubation of RecJ and thesingle-stranded DNA substrate was performed toallow RecJ to bind to DNA. The reaction was initia-ted by the addition of Mg2+, with concomitantaddition of a 50-fold molar excess of cold single-stranded DNA. The excess of cold substrate wasused to compete out any unbound RecJ or moleculesof RecJ that fall off from the DNA after a first roundof catalysis so that they cannot initiate the secondround of catalysis. RecJ molecules associated withthe DNA would degrade the substrate in a 5′–3′direction, thereby releasing the terminal 32P and theinterior 3H, and the ratio of the internal 3H to the5′-32P can be used to determine the extent of proces-sivity. The ratio of internal 3H to the 5′-32P presentwithin the acid-soluble fraction was determined to
Fig. 2. Processivity of MBP–RecJ. MBP–RecJ (50 nM)was incubated with 1 μM double-labeled denatured 234-nucleotide single-stranded DNA in a 500-μl reaction, andthe progress of the reaction was monitored at differenttime intervals by withdrawing 50-μl aliquots (inset). Allthe samples were analyzed for soluble radioactivity asdescribed under Experimental Procedures. The extent of32P (3′-terminal label) release was plotted against that of3H (5′-terminal) digestion.
1379Interaction of RecJ and SSB
be 0.26 at initial time points, suggesting that 26% ofthe 2686-nucleotide substrate was hydrolyzed forevery 5′-terminus, thereby revealing the processi-vity of HiRecJ to be ∼700 nucleotides. The proces-sivity of HiRecJ is comparable to the processivity ofEcoRecJ, which is ∼1000 nucleotides.19 The proces-sive nature of the RecJ exonuclease is advantageousin the context of its diverse in vivo functions. Itallows a limited number of RecJ molecules to func-tion simultaneously in ongoing multiple cellularDNA metabolism pathways, with only a few mole-cules being sufficient to carry out the entire reactionat each focus of repair or recombination.
Activity of HiRecJ protein on different DNA structures
Various branched DNA intermediates generatedduringmost of theDNAmetabolic processes, such asreplication, repair, and recombination, act as sub-strates for cellular nucleases. For instance, EXO-I, aRAD2 class III family nuclease found in S. cerevisiae,Drosophila, and humans, harbors 5′–3′ exonucleaseand 5′-flap endonuclease activities in vitro andfunctions at the excision step of eukaryotic MMR.24
The 5′-exonuclease domains of the DNA polymeraseI of eubacteria and the FEN1 of archaea and eukar-yotes are other representatives of structure-specific5′-exonucleases.25 These proteins contain motifssimilar to motifs I and II of RecJ, which are consi-dered to be involved in metal binding.14
EcoRecJ is able to degrade single-stranded DNAtails of 5′-overhangs.15,19 However, the activity ofa RecJ protein on branched DNA substrates suchas flaps, Y-junctions, replicative forks, and othershas not been studied in detail. Therefore, to further
investigate the substrate specificity of HiRecJ,different radioactively-labeled branched DNA sub-strates were used. These include overhangs, Y-junctions, flap structures, and replicative forks with5′ and 3′ single-stranded tails. Each of the DNAsubstrates (10 nM) was incubated with 50 nMMBP–RecJ, and the progress of the reaction was monitoredat different time intervals. As can be seen from Fig. 3,H. influenzae MBP–RecJ was able to rapidly hydro-lyze substrates with free 5′-ends with equal effi-ciency (i.e., 5′-overhang, 5′-flap, and pseudo-Y) (Fig.3b, d, and f, respectively). However, RecJ did notexhibit any exonuclease activity on DNA substratesin which the 3′-end was free, while the 5′-end wasblocked by base pairing with complementary DNA(Fig. 3a, c, e, and g). This clearly indicates thatHiRecJ has a strong unidirectional specificity forDNA substrates with free 5′-ends, but does notexhibit specificity for any DNA structure. The acti-vity of HiRecJ exonuclease was further assayed onsingle-stranded DNA oligonucleotides labeled with32P at the 3′-end and phosphorylated or unpho-sphorylated at the 5′-end. Unlike the λ exonuclease,which specifically degrades 5′-phosphorylated sub-strates, RecJ exonuclease could efficiently degradeboth the phosphorylated and the unphosphorylatedsubstrates (data not shown).
Catalytic properties of HiRecJ
Metal ions are essential cofactors for almost allenzymes that catalyze the hydrolysis of phospho-diester bonds and may have structural and catalyticroles. To investigate the requirements of metal ionsfor the activity of H. influenzae MBP–RecJ, exonu-clease assay was carried out using 5′-end-labeledsingle-stranded DNA oligonucleotide as substrate inthe presence of increasing concentrations (0–50 mM)of either Mg2+, Mn2+, Ca2+, or Cd2+. H. influenzaeMBP–RecJ exhibited activity in the presence of Mg2+
and Mn2+, while Ca2+ and Cd2+ did not signifi-cantly support its activity (Fig. 4a). Exonucleaseactivity was not observed in the absence of Mg2+ orMn2+, suggesting that metal ions are essential forH. influenzae MBP–RecJ activity (Fig. 4a). Theoptimal concentration of Mg2+ required for MBP–RecJ activity was determined to be 2.5 mM. Mono-valent metal ions such as Na+ and K+ did notsupport the nuclease activity of HiRecJ (data notshown). It is noteworthy that the metal ionrequirements of HiRecJ are significantly differentfrom those of EcoRecJ, which is active only in thepresence of Mg2+, while Mn2+ inhibited its acti-vity.15 In contrast, H. influenzae MBP–RecJ exhibitssimilar metal ion requirements as have been repor-ted for TtRecJ and the yeast exopolyphosphatasescPPX1 (a RecJ homolog), both of which are activein the presence of either Mg2+, Mn2+, or Co2+, butnot in the presence of Ca2+ or Zn2+.16 In the crystalstructure of TtRecJ, Mn2+ was found to be coor-dinated to four invariant aspartate and histidineresidues.18 However, different metal ion require-ments of EcoRecJ and HiRecJ may suggest a diffe-
Fig. 3. Exonuclease activity of H. influenzae MBP–RecJ on different DNA structures. Reactions were performed with10 nM each of the indicated 32P-labeled substrates and 50 nMMBP–RecJ, and the progress of the reaction was monitoredby withdrawing aliquots at different time intervals (0, 1, 2, 4, 6, 8, 10, 15, 20, 30, 35, 45, and 60 min). These were processedas described under Experimental Procedures. (a) Double-stranded DNA; (b) 5′-overhang; (c) 3′-overhang; (d) 5′-flap; (e)3′-flap; (f) pseudo-Y; (g) replication fork.
1380 Interaction of RecJ and SSB
rent mode of metal binding or plasticity of themetal-binding pocket within HiRecJ.Trace amounts of divalent metal ions are essential
nutrients for bacterial cells. However, heavy metal
ions such as Cd2+ are toxic to bacteria. Cd2+ is apotent carcinogen that is known to inactivate theeukaryotic MMR pathway and several other DNArepair proteins.26 However, the effects of Cd2+ on
Fig. 4. Exonuclease activity of H. influenzae MBP–RecJin the presence of different metal ions. (a) Nuclease assayscontaining 10 nM (5′-32P)-labeled substrate (ODN1; TableS1) and 50 nM RecJ were performed with increasingconcentrations (0–50 mM) of Mg2+, Mn2+, Ca2+, or Cd2+.The reactions were performed for 10 min at 37 °C andsubjected to a denaturing PAGE, which was visualized byPhosphorImager. The percentage of digestion was plottedagainst metal ion concentration. (b) Nuclease assays wereperformed in the presence of 10 mM Mg2+ and increasingconcentrations of Cd2+ or Ca2+ (0–50 mM). The reactionwas analyzed as described under Experimental Proce-dures. The percentage of degradation was calculated fromthe amount of undegraded substrate and plotted againstthe concentrations of Cd2+ and Ca2+. Error bars indicatethe standard deviation of three different measurements.
1381Interaction of RecJ and SSB
prokaryotic MMR proteins have not been studied.Therefore, the effect of cadmium on the activity ofRecJ was further investigated. In the presence ofCd2+, a concentration-dependent inhibition of theexonuclease activity of RecJ was observed (Fig. 4b).Interestingly, in another set of reactions, while 1 mMCd2+ was able to abrogate the activity of RecJ even inthe presence of a 10-fold molar excess of Mg2+, equi-molar or higher concentrations of Ca2+ did notsignificantly inhibit the activity of RecJ under thesame reaction conditions (Fig. 4b). This indicates thatcadmium inhibits the activity of RecJ exonuclease,which cannot be overcome byMg2+. In addition, RecJwas found to be inactive in another experimentwherein it was first incubated with 1 mM Cd2+ andthen assayed with increasing concentrations of Mg2+,
thus suggesting that Cd2+-bound RecJ is inactive(data not shown). Cadmium is reported to bind to thefree thiol and sulfhydryl groups in proteins and tocofactors such as glutathione, resulting in inhibition oftheir enzymatic activity, and the presence of freecysteine and histidine amino acids in enzymaticreactions in vitro can overcome this inhibition.27
Therefore, cadmium may inhibit the activity of RecJby forming an adduct with the cysteine and histidineresidues in the protein. Furthermore, Cd2+ could alsocompete withMg2+ for binding the active site of RecJ.
Mutational analysis of HiRecJ
Based on a sequence analysis of RecJ protein andits homologs from various species, four highly con-served motifs have been identified.13 These motifsharbor highly invariant aspartate residues that formcarboxylate cluster (Fig. 5a), which may be essentialfor metal ion coordination and catalysis. Mutationalanalysis of various conserved residues within thesemotifs of EcoRecJ showed that all motifs were essen-tial for RecJ function.14 Three different mutants ofHiRecJ—D77A, D156A, and H157A—were gene-rated. The mutant proteins were purified to homo-geneity as MBP-fusion proteins and had the samemolecular weight as the wild-type protein, as can bejudged from their similar mobilities on SDS-PAGEand Western blot analysis (Fig. 5b and c, respec-tively). No gross conformational changes wereobserved in the mutant proteins as compared to thewild-type RecJ protein, as determined from circulardichroism (CD) spectra (data not shown). As isevident from Fig. 5d, all three mutants failed toexhibit any significant exonuclease activity. Even ashigh as a 200-fold molar excess of these mutant pro-teins exhibited only∼10% of activity as compared tothe wild-type MBP–RecJ.
DNA binding studies
TheDNAbinding properties ofH. influenzaeMBP–RecJ were investigated by EMSA using a 32P-labeled42-mer single-stranded DNA (5 nM), which wasincubated with increasing concentrations of MBP–RecJ. In the absence of Mg2+, RecJ binding could bedetected by mobility shift without degradation ofsubstrate. HiRecJ was able to bind the single-stranded DNA as indicated by the reduced mobilityof the single-stranded DNAwith increasing concen-trations of the protein (Fig. 6a). Only a single pro-tein–DNA complex was observed. As expected, nobinding was detected for double-stranded DNAsubstrate or single-stranded DNA in which the 3′-end was free and the 5′-end was blocked (data notshown). The affinity and kinetic parameters ofinteraction between H. influenzae MBP–RecJ and 65-mer single-stranded DNA were further examinedusing SPR. Binding was carried out in the absence ofmetal ions. The binding sensorgram showed thatMBP–RecJ was able to quantitatively bind single-stranded DNA (Fig. 6b). Global fitting of the datausing 1:1 Langmuir binding equation yielded a Kd
Fig. 5. (a) Multiple sequence alignment of RecJ proteins from different bacterial species. The residues that have beenmutated in this study are marked by ‘⁎’. (b) SDS-PAGE and (c) Western blot analysis of purified HiRecJ point mutants:lane 1, molecular weight markers [prestained molecular weight markers in (c)]; lane 2, MBP–RecJ; lane 3, MBP–RecJD77A;lane 4, MBP–RecJD156A; lane 5, MBP–RecJH157A. (d) Exonuclease activity of the mutant MBP–RecJ proteins comparedwith wild-type MBP–RecJ. Fifty nanomolar of wild-type (wt) MBP–RecJ (■) and either 1 μM RecJD77A (◊) or 1 μMRecJD156A (○) or 1 μM RecJH157A (▽) were incubated with 10 nM 3′-end radiolabeled single-stranded DNA (ODN1;Table S1). The progress of the reaction was monitored at different time points (0, 2, 5, 10, 15, 20, and 30 min) as describedearlier.
1382 Interaction of RecJ and SSB
value of 6.5×10−8 M. The modest Kd value of theinteraction of H. influenzae MBP–RecJ with single-stranded DNA could result from a greater potentialfor secondary structure formation in the long single-stranded DNA that can reduce RecJ binding. This issupported by an earlier study with EcoRecJ, whichsuggested that native and MBP-tagged EcoRecJproteins have similar DNA binding properties, butexhibit a weak interaction with a 65-mer oligonu-
cleotide or a single-stranded region of 5′-overhangthat is more than 10 nucleotides long.19
DNA binding studies were carried out to investi-gate whether the D77A, D156A, and H157A muta-tions introduced within HiRecJ affected the DNAbinding properties of these mutant proteins. ForEMSA, 5′-32P-labeled 42-mer single-stranded DNA(5 nM)was incubatedwith increasing concentrationsof the mutant proteins in separate reactions. All the
Fig. 6. Interaction of H. influenzae wild-type MBP–RecJ and MBP–RecJ D77A with single-stranded DNA. (a) EMSAwas performed by incubating different concentrations (0, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5, and 3 μM) ofMBP–RecJ with 32P-labeled 42-mer single-stranded DNA (5 nM). (b) Interaction of MBP–RecJ with DNA was furtheranalyzed using SPR. Different concentrations of wild-type MBP–RecJ were injected on the single-stranded DNAsurface in standard buffer containing 150 mM NaCl. Both sample injection (association phase) and buffer injection(dissociation phase) were carried out as described in Experimental Procedures. Representative sensorgrams depictingchanges in the SPR signal (the y-axis) as a function of time (the x-axis) are shown. (c and d) EMSA and SPR studies,respectively, performed with MBP–RecJ D77A.
1383Interaction of RecJ and SSB
mutants were able to bind to the single-strandedDNA, thus indicating that the loss of catalytic acti-vity was not due to their inability to bind DNA (datanot shown). The kinetics of interaction of the mutantproteins with single-stranded DNA were furtherexamined by SPR. Interestingly, D77A mutantprotein interacted with single-stranded DNA witha 10-fold higher affinity (Kd=7.3×10
−9 M) as com-pared to the wild-type protein (Fig. 6c and d). D156Aand H157A mutant proteins interacted with DNAwith comparable efficiencies as the wild-type RecJ(data not shown). From the structure of TtRecJ, it isevident that Asp82 (corresponding to Asp77 inHiRecJ) is present in the vicinity (at a distance ofabout 4Å) of themetal binding site, but is not directlycoordinated to the metal ion.18 Furthermore, the sidechain of this aspartate residue protrudes in the
single-stranded DNA-binding cleft of RecJ. There-fore, this aspartate residue may function to coordi-nate the binding of metal ion or DNA to thehydrolysis of DNA. Elimination of this negativelycharged acidic residue, which is in proximity to theDNA binding site, may result in reduced steric/electrostatic repulsion of negatively charged DNAbackbone, resulting in tight binding of DNA to themutant protein. Similar results were reported forEcoRII and HhaI DNA methyltransferases, whereinreplacement of a catalytic cysteine residue by aglycine resulted in an approximately 10-fold increasein the affinity of these enzymes for their DNAsubstrate.28,29 Although these mutations were cyto-toxic to E. coli cells expressing the mutant methyl-transferases, the D77A mutation of HiRecJ was notdetrimental to the host E. coli cells.
1384 Interaction of RecJ and SSB
Effect of HU on the activity of HiRecJ
E. coli HU (EcoHU) is a small, basic, heat-stableDNA-binding protein and is one of the most abun-dant proteins associated with the E. coli nucleoid.EcoHU shares a 90% sequence similarity with H.influenzae HU. In an earlier study, it was reportedthat EcoHU specifically binds to DNA repairintermediates such as single-stranded and double-stranded breaks, and protects them from exonu-clease degradation.30 In the absence of HU, E. colicells are more sensitive to γ and UV irradiation;therefore, it is conceivable that HU plays a directrole in repair mechanisms.31 In the context of theMMR pathway, EcoHU was found to inhibit thenicking activity of H. influenzae MutH protein.32
Recently, HU has been reported to specifically bindDNA containing mismatched regions longer than3 bp, as well as DNA bulges.33 In order to investi-gate the effect of EcoHU on the activity of HiRecJ,exonuclease assay—wherein 5 nM 3′-end-labeledsingle-stranded DNAwas preincubated with a satu-rating concentration of EcoHU prior to addition ofMBP-RecJ—was carried out. EcoHU was found toinhibit the activity of H. influenzae MBP–RecJ (Fig.S4a). While more than 60% of the substrate washydrolyzed by MBP–RecJ in the absence of EcoHUwithin 5 min of incubation, only about 30% of thesubstrate was hydrolyzed in the presence of EcoHU(Fig. S4b). Complete inhibition of exonuclease acti-vity was not observed perhaps because EcoHUcould get transiently dislodged from DNA in vitroduring which RecJ might get access to the substrate.This is supported by the fact that EcoHU bindsnoncooperatively to linear single-stranded DNAfragments with low affinity and only under low-salt conditions.33
Inhibition of HiRecJ by EcoHU could be due toaggregation of HU on DNA, thereby making thesubstrate DNA inaccessible to RecJ, or due to seques-tration of RecJ by HU through protein–protein inter-action. In a coimmunoprecipitation assay using anaffinity-purified HiRecJ antibody that was coincu-bated with MBP–RecJ and EcoHU, no interactionbetween RecJ and HU was observed (data notshown). Studies on HU reveal that it introducesstructural changes in the DNA. Recently, it has beenshown that binding of HU to single-stranded DNAresults in the formation of a hairpin-like U-loop inDNA,33 and introduction of this secondary structureinto the DNA substrate could extenuate hydrolysisby RecJ. Therefore, HU can cover the DNA andprotect it from degradation by RecJ.
Effect of SSB on the activity of HiRecJ
Within bacterial cells, single-strandedDNA is asso-ciatedwith SSBprotein. In the absence of SSBprotein,MMR activity is substantially reduced in vitro,possibly because of its role in repairDNAsynthesis.34
Moreover, although SSB has been implicated asbeing essential for mismatch-provoked DNA exci-sion, the function of SSB at the resection step ofMMR
pathway has not been studied in detail. Previously,EcoSSB has been shown to enhance the exonucleaseactivities of EcoRecJ and E. coli ExoI.19,20 Todetermine the effect of HiSSB on the activity ofHiRecJ, exonuclease assay was carried out in thepresence of a saturating concentration of HiSSB(25 nM monomer), which was preincubated with 3′-end-labeled single-stranded DNA (5 nM) to allowthe formation of a DNA–protein complex. Thereaction was initiated by the addition of 50 nM H.influenzae MBP–RecJ with Mg2+, and the progress ofthe reaction was monitored at different time inter-vals. In the presence of HiSSB, there was clearstimulation in the hydrolysis of the substrate by RecJas compared to reactions without SSB, as determinedby the quantitation of undegraded substrate in bothcases at different time points (Fig. 7a). The stimula-tory effect of HiSSB on H. influenzae MBP–RecJactivity was best observed at saturating concentra-tions of SSB. Moreover, HiSSB and EcoSSB were ableto stimulate the activities of EcoRecJ and HiRecJ,respectively (data not shown). More importantly,SSB was able to enhance the activity of RecJ on aDNA substrate that formed a strong secondarystructure (Fig. S5a), which may interfere with RecJactivity, as well as on a homopolymeric substratewithout any secondary structure (Fig. S5b). Thisindicates that SSB, in addition to stabilizing single-stranded DNA by melting any secondary structure,may perhaps stimulate RecJ activity by interactingwith it. Furthermore, to address the specificity of thefunctional interaction between HiSSB and HiRecJ,the ability of T4 gene 32 protein (a SSB homolog frombacteriophage T4) to stimulate the activity of H.influenzae MBP–RecJ was tested. No enhancement inthe exonuclease activity of H. influenzae MBP–RecJwas observed in the presence of the T4 gene 32protein (Fig. S5c), suggesting that stimulatory effectis specific to the HiSSB protein. Apart from stabiliz-ing single-stranded DNA, SSB regulates the enzy-matic activities of DNA-transacting proteins. SSBenhances the activity of ExoI, another exonucleaseinvolved in the MMR pathway, by direct protein–protein interactionmediated by the C-terminal tail ofEcoSSB.35 EcoSSB stimulates DNA binding andunwinding by the E. coli RecQ helicase,36 as well asthe activity of both E. coli and mycobacterial DNAtopoisomerase I.37 In contrast, Sulfobolus tokodaii SSBinhibits the exonuclease and endonuclease activitiesof S. tokodaii NurA 5′–3′ nuclease.38 Since the C-terminal tail of SSB protein is essential for its cellularfunctions by mediating its interactions with proteinsof DNA replication and repair machinery,39 it was ofinterest to further study the activity of RecJ in thepresence of the C-terminal truncated form of HiSSB(HiSSBΔC) protein. In contrast to the wild-typeprotein, the SSBΔC protein strongly inhibited theexonuclease activity of both HiRecJ (Fig. 7b) andEcoRecJ (data not shown).These observations are important in the cellular
context. For instance, during MMR, the exonucleo-lytic cleavage of nascent strand involves the degra-dation of as long as a kilobase or more of single-
Fig. 7. Effect of HiSSB andHiSSBΔC on the activity of HiRecJ.Exonuclease assays containing(5 nM) 45-mer single-strandedDNA radiolabeled at the 3′-endand 50 nM MBP–RecJ were per-formed (a) in the absence or in thepresence of 6 nM HiSSB tetramer or(b) in the presence of 10 nMHiSSBΔC tetramer. The progress ofthe reaction was monitored at dif-ferent time points and assayed asdescribed in Experimental Proce-dures. (c) Steady-state kinetics ofDNA hydrolysis by MBP–RecJ inthe presence (□) and in the absence(■) of HiSSB. DNA cleavage assayswere carried out in a reactionmixture containing 50 nM MBP–RecJ and increasing concentrations(2–14 μM) of 3′-32P-labeled single-stranded DNA (63-mer) with orwithout SSB (18 μM). The reactionswere incubated at 37 °C for 10 minand assayed as described underExperimental Procedures. Theamount of undegraded substrateremaining for each concentrationwas quantitated and used to calcu-late the velocity (v) of the reaction,the reciprocal of which was plottedagainst the reciprocal of substrate
concentration (1/[v] versus 1/[S]). Lineweaver–Burk equationwas used for the calculation of kinetic parameters. Error barsindicate standard deviation.
1385Interaction of RecJ and SSB
stranded DNA by one of the four exonucleases. SSBhas been implicated as being critical to this process,wherein it may function to stabilize the single-stranded DNA tracts generated by the action of theUvrD helicase and the exonucleases by preventingthe reannealing of single-stranded DNAs or the for-mation of secondary structures in them. In addition,by stimulating the activities of RecJ and ExoI, SSBmay allow enhanced rates of repair to occur in vivo.Similarly, SSB and RecJ may function in a coopera-tive manner during other repair and recombinationpathways.
Kinetic studies
To study the kinetics of single-stranded DNAdegradation by H. influenzae MBP–RecJ, steady-state conditions were used ([single-strandedDNA]⋙ [MBP–RecJ]). Under these conditions,hydrolysis of the total substrate would require multi-ple enzyme turnovers involving binding of RecJ to the5′-end of single-stranded DNA, hydrolysis of phos-phodiester bonds, and, subsequently, attack on a new5′-terminus. Increasing concentrations of 3′-end 32P-labeled 63-mer oligonucleotide (2–14 μM) wereincubated with 50 nM RecJ, in the presence or in theabsence of a saturating concentration of HiSSB(18 μM). The reaction rate [v] was estimated fromdensitometric quantitation of the amount of unde-
graded substrate remaining for each substrate con-centration. The plot of 1/[v] versus 1/[S] showed thebest fit to the Lineweaver–Burk equation and wasused to estimate Km and Vmax values (Fig. 7c). In thepresence of HiSSB, the reciprocal plot pivots clock-wise at about the point of intersection with thecontrol plot. The Km for MBP–RecJ was estimated tobe 10.9 μM in the absence of SSB and 3.3 μM in thepresence of SSB. Therefore, the apparent change inthe affinity (Ks) of HiRecJ in the presence of HiSSB isby a factor α (see the reaction scheme below), whichis equal to 0.3. Vmax (3.7×10−3 μM/s) remainedunaltered in both cases. This clearly suggested thatthe affinity of RecJ for single-stranded DNA wasenhanced in the presence of SSB, indicating that SSBcould function to recruit RecJ to DNA termini. TheKm and kcat values of the HiRecJ in the absence ofSSB are comparable to those of the EcoRecJ andTtRecJ.15,16
From the kinetic data, it is clear that SSB functionsas an activator of RecJ exonuclease. There are twomajor types of activation: (a) nonessential activa-tion, in which the reaction can occur in the absenceof the activator, and (b) essential activation, inwhich the true substrate is the one associated withthe activator.40 An activator enhances the enzyma-tic reaction by altering the rate of dissociation of theES complex into E and S by a factor α. For non-essential activators such as SSB, α is less than unity.
Fig. 8. In vitro interaction ofHiRecJ and HiSSB by coprecipi-tation. Five micromolar each of theMBP–RecJ monomers and HiSSBtetramers were incubated togetherfollowed by addition of 150 g/lammonium sulfate in reactions asindicated by ‘+’ symbols. Pellet (P)and supernatant (S) fractions areindicated.
1386 Interaction of RecJ and SSB
The equation given below is a representation ofnonessential activation:
Interaction of HiRecJ and cognate SSB
As HiSSBΔC failed to stimulate the activity ofHiRecJ, it is possible that a direct physical interac-tion between the two proteins may be essential forSSB-mediated stimulation of RecJ. To determinewhether HiRecJ and SSB physically interact, copre-cipitation by ammonium sulfate was initially used.Coprecipitation is a fast and simple qualitativemethod used to study the interaction of a proteinwith SSB, taking advantage of the fact that SSBprotein can be readily precipitated at very lowconcentrations of ammonium sulfate (Fig. 8, lane10), while most other proteins, including RecJ, aresoluble under these conditions (Fig. 8, lane 6). Theinteraction between H. influenzae MBP–RecJ andHiSSB was studied by coincubating the proteinstogether, followed by the addition of ammoniumsulfate to a final concentration of 150 g/l. In theabsence of ammonium sulfate, both RecJ and SSBremained completely in solution (Fig. 8, lanes 5, 9,and 13). When the two proteins were subjected toprecipitation with ammonium sulfate, a significantamount of MBP–RecJ was found to precipitate withSSB, suggesting an interaction between the twoproteins (Fig. 8, lane 14). Some amount of MBP–RecJwas present in the supernatant obtained afterammonium sulfate fractionation (Fig. 8, lane 15),suggesting that these two proteins interact with anaffinity in the range of the concentrations of theproteins used in this assay. The interaction between
SSB and RecJ was not disrupted by DNase Itreatment, thereby excluding the possibility of indi-rect interaction between the two proteins mediatedby DNA (Fig. S6a). This interaction was specific forRecJ and SSB proteins, as MBP and EcoP15I methyl-transferase (a DNA-binding protein) failed tocoprecipitate with SSB (Fig. S6b). This assay there-fore qualitatively suggested a direct physical inter-action between the two proteins. Significantly,HiRecJ failed to coprecipitate with HiSSBΔC uponaddition of ammonium sulfate (data not shown),indicating that the C-terminus of SSB mediates itsinteraction with RecJ.A direct interaction between HiSSB and HiRecJ
was also observed in the far Western analysis. SSBinteracts with RNA polymerase,41 and this wasused as positive control in this experiment. Similarto RNA polymerase, positive interaction wasobserved for RecJ, suggesting that it interacts directlywith SSB, while MBP failed to show any interaction(Fig. 9a). Far Western analysis performed withHiSSBΔC failed to show any positive interactionwith RecJ, as well as with RNA polymerase (data notshown), suggesting that the interaction between RecJand SSB was mediated through the C-terminus ofSSB. The C-terminus of SSB proteins is known tomediate the direct interaction of SSB with a numberof DNA-transacting proteins, including E. coliExoI,35 RecA,22 RNApolymerase,41 primase,42 uracilDNA glycosylase,43 DNA pol III χ subunit,44 PriAhelicase,45 and RecO.46
To better address the direct interaction betweenRecJ and SSB, in vitro coimmunoprecipitationstudies using antibodies against the two proteinswere carried out. Using an affinity-purified anti-body against HiRecJ that was coincubated withpurifiedH. influenzaeMBP–RecJ and HiSSB proteins,both RecJ and SSB were precipitated (Fig. 9b, lane 9).The HiRecJ–HiSSB complex was resistant to dis-sociation after extensive washing of the Protein ASepharose–immunoglobulin complex bound to theproteins with a buffer containing 150 mM NaCl,indicating a specific and stable interaction betweenthese two proteins. In addition, the interactionbetween the two proteins was resistant to DNase I
Fig. 9. (a) Far Western analysis of interaction between HiSSB and HiRecJ. Different concentrations (0.2–1.6 μM) ofMBP-RecJ, E. coli RNA polymerase, and MBP were applied to a nitrocellulose membrane. Interaction was studied byincubation of the membrane with 2 μM HiSSB tetramer and immunoblotting with HiSSB antiserum to detect any boundSSB on the membrane. (a) Interaction between H. influenzae MBP–RecJ and HiSSB by coimmunoprecipitation. Fivemicromolar each of the MBP–RecJ monomers and SSB tetramers were incubated either with rabbit pre-immune serum(lanes 3 and 4, respectively), anti-RecJ serum (lane 9) or anti-SSB serum (lane 10). To rule out cross-reactivity, MBP–RecJwas incubated with SSB antiserum (lane 5) in the absence of SSB, and SSBwas incubated with anti-RecJ antibodies (lane 6)in the absence of RecJ. The protein–antibody complexes were then separated using Protein A Sepharose beads. Theimmunoprecipitates were subjected to SDS-PAGE, followed by immunoblotting (IB) with either RecJ antiserum (a) or SSBantiserum (b). Purified MBP–RecJ (Lane 1) and SSB (lane 2) were loaded as markers and readily immunoprecipitated withtheir respective antibodies (lanes 7 and 8, respectively).
1387Interaction of RecJ and SSB
treatment, thus excluding the possibility of anindirect interaction between the two proteinsmediated by contaminating DNA in the proteinpreparations (data not shown). Moreover, the anti-body was able to pull down SSB only in the presenceof RecJ (Fig. 9b, lane 9), while normal rabbit serumwas unable to precipitate either RecJ or SSB (Fig. 9b,lanes 3 and 4, respectively). Similarly, in a reciprocalexperiment with purified antibodies against HiSSB,RecJ could be immunoprecipitated from solutiononly in the presence of SSB (Fig. 9b, lane 10).Furthermore, MBP did not coimmunoprecipitatewith SSB, suggesting a direct interaction betweenRecJ and SSB (data not shown). The antibodiesgenerated against the two proteins did not showany cross-reactivity (Fig. 9b, lanes 5 and 6).In order to validate and quantitate the interactions,
SPR analysis was employed. The interaction betweenH. influenzae MBP–RecJ and HiSSB was first investi-gated in the presence of DNA by saturation of single-stranded DNAwith HiSSB. Interaction between RecJand the SSB–single-stranded DNA complex wasinvestigated by injection of different concentrationsof RecJ over the SSB–DNAcomplex. Figure 10a shows
the sensorgram for the interaction of RecJ with SSB inthe presence of DNA showing a significant increase inresponse proportional to the increasing concentra-tions of RecJ. No interaction was observed betweenSSB and MBP (data not shown). The data wereanalyzed by global fit according to a simple 1:1Langmuir equation yielding a binding constant of1.5×10−6 M and a stoichiometry of approximately3.67 RecJ monomers per HiSSB tetramer. It isinteresting to note that EcoSSB interacts with theDNA polymerase III χ subunit in the presence ofDNA, with a binding constant of 4.2×10−5 M and astoichiometry of approximately 4.4 χ subunits perSSB tetramer, as determined using SPR.44 In a recentstudy on the interaction between E. coli RecQ andEcoSSB, oneC-terminal peptide of EcoSSBwas shownto bind one monomer of RecQ with a Kd of ∼6 μMusing isothermal titration calorimetry.36 In a similarexperiment using SPR, a saturating concentration ofHiSSBΔC was bound to DNA, followed by injectionof MBP–RecJ. However, we observed a negativechange in resonance, indicating the absence of anyinteraction between the two proteins (Fig. 10a). Thisobservation is consistent with coprecipitation and far
Fig. 10. SPR sensorgrams show-ing the interactions between HiSSBand H. influenzae MBP–RecJ. (a)Interaction between HiSSB andHiSSBΔC with MBP–RecJ in thepresence of single-stranded DNA.One hundred fifty response units of65-mer oligonucleotide was immo-bilized on the surface of streptavidinsensor chip. Varying concentrationsof MBP–RecJ in standard buffercontaining 150 mM NaCl werepassed over a saturated concentra-tion (400 RU) of bound HiSSB orHiSSBΔC. Data collected from theinjections of MBP–RecJ over anempty reference cell used as controlwere subtracted as blank. The globalfit of the data was used to calculatethe binding constant. (b) Directinteraction ofMBP–RecJwith immo-bilized HiSSB in the absence ofDNA. Varying concentrations ofMBP–RecJ in standard buffer con-taining 150 mM NaCl were passedover 220 RU of HiSSB immobilizedon a CM5 sensor chip. Data collectedfrom control injections of MBP–RecJover a reference cell reacted withethanolamine were subtracted asblank. Inset shows a plot of equili-brium values of the individual pro-files as a function of HiRecJ con-centration, which was used to deter-mine the affinity constant Kd.
1388 Interaction of RecJ and SSB
Western data, further substantiating that the inter-action of SSBwith RecJ is mediated by the C-terminusof SSB. Recently, the cocrystal structure of the C-terminal peptide of SSB bound to ExoI of E. coliprovided evidence that a stable interaction betweenthe two proteins via the C-terminus of SSB isimportant for the stimulation of ExoI activity bySSB, and that SSB stimulates enzyme activity byrecruiting ExoI to its substrate.47
SPR was further used to study the direct interac-tion of HiSSB with H. influenzae MBP–RecJ. HiSSBwas immobilized on a CM-5 sensor chip, andvarying concentrations of HiRecJ were injected onthis surface. SPR profiles showed an increase inresonance signal proportional to the concentrationsof MBP–RecJ. However, the sensorgram showed afast association, but a slow and negligible disso-ciation (Fig. 10b). Therefore, an overestimation of koffvalues was obtained probably due to the negativelycharged surface of the chip, which may interferewith the dissociation of RecJ. Thus, a plot of equili-brium values of the individual profiles as a functionof RecJ concentration was used to determine bindingaffinity (Fig. 10b, inset), which yielded an affinityconstant of 1.0×10−6 M.
To determine the regions of HiRecJ that mayinteract with SSB, various truncated forms of RecJ(i.e., RecJΔN, RecJΔC, and the core-catalytic domainof HiRecJ) were generated. The amino acid residueswere deleted from the regions of RecJ, which areexterior to the conserved catalytic motifs of theprotein (Fig. 11a). All the three truncated variants ofRecJ were purified as MBP-fused proteins similar tothe wild-type protein and were analyzed using SDS-PAGE and Western blot analysis (Fig. 11b). Thedeletion mutants showed a faster mobility on SDS-PAGE as compared to the wild-type, correspondingto a decrease in their molecular masses. Similar tothe wild-type protein, the CD spectra of each of thedeletion mutants of RecJ showed negative doublemaxima at ∼222 and 210 nm, which are character-istic of α-helical structures. This suggests that all thetruncated proteins remained folded (data notshown). However, similar to the TtRecJ core-cata-lytic domain,16 the CD spectra of the HiRecJ deletionmutants showed relatively less intensity as com-pared to the wild-type, perhaps due to deletions ofregions external to the core-catalytic domain of RecJ.The interaction of HiSSB with the truncated variantsof HiRecJ was studied by far Western analysis. All
Fig. 11. Interaction of HiSSB with HiRecJ deletion mutants. (a) MBP–RecJ and its deletion variants used in this study.The different catalytic motifs are marked. (b) SDS-PAGE (a) and Western blot analysis (b) of purified MBP–RecJ deletionmutant proteins: lane 1, molecular weight markers; lane 2, wild-type MBP–RecJ; lane 3, MBP–RecJ ΔN34; lane 4, MBP–RecJ ΔC76; lane 5, MBP–RecJ–core-catalytic domain. (c) Far Western analysis of interaction between HiSSB and MBP–RecJ deletion mutants. Varying concentrations (0.2–1.6 μM) of MBP–RecJ, MBP–RecJ ΔN34, MBP–RecJ ΔC76, MBP–RecJ–core-catalytic domain, E. coli RNA polymerase, and MBP were applied on a nitrocellulose membrane. Interactionwas studied by incubation of the membrane with 2 μM HiSSB tetramer and immunoblotting with SSB antiserum todetect any bound SSB on the membrane. (d) SPR sensorgrams showing the interactions between HiSSB and MBP–RecJ–core-catalytic domain. Varying concentrations of MBP–RecJ–core-catalytic domain in standard buffer containing150 mM NaCl were passed over HiSSB immobilized on a CM5 sensor chip. Interaction between the two proteins wasanalyzed as described in the legend to Fig. 10b.
1389Interaction of RecJ and SSB
three truncated forms of HiRecJ showed a positiveinteraction with SSB similar to the full-length RecJand E. coli RNA polymerase (Fig. 11c), suggesting
that the core-catalytic domain of RecJ, whichharbors all the conserved motifs essential for RecJfunction, possesses a region essential for the
1390 Interaction of RecJ and SSB
interaction of RecJ with SSB. Interaction betweenHiSSB and the core-catalytic domain of HiRecJ wasfurther investigated by SPR. Figure 11d clearlyshows that SSB interacts with the core-catalyticdomain of HiRecJ with an affinity (Kd=1.1×10
−6 M)similar to that of the wild-type protein (Fig. 11d,inset). These data further reinforce the resultobtained from far Western analysis that SSB inter-acts with RecJ within its core-catalytic region.In previous studies, binding of EcoRecJ to single-
stranded DNAwas enhanced by EcoSSB, suggestingthe formation of an SSB and RecJ co-complex in thepresence of DNA.19 Although RecJ copurifies withaffinity-tagged SSB,21 no direct interaction has beendemonstrated for these proteins. The results pre-sented in this study elucidate that a direct physicalinteraction between SSB and RecJ is essential for SSB-mediated enhancement in the activity of RecJexonuclease. These results further establish that theC-terminal of SSB is important for this interaction,and that SSB recognizes a region within the core-catalytic domain of RecJ. SSB has been implicated asbeing essential for the resection step ofMMR, and theresults presented here and in other studies suggestthat SSB may function to recruit the exonucleaseswith different polarities at their respective DNAtermini generated after nicking activity of MutH andunwinding by UvrD. The ability of SSB to interactsimultaneously with both single-stranded DNA andRecJ possibly allows SSB to recruit RecJ to the 5′-termini of single-stranded DNA and subsequentlykeep it associated with single-stranded DNA, thusenhancing the processivity of the exonuclease. How-ever, the possibility that SSB may modulate theexonuclease activity by bringing about conformationchanges in the RecJ protein cannot be ruled out. SSBmay further mediate interactions of RecJ with otherprotein partners such as the RecQ protein by simul-taneous binding of both proteins to an SSB homo-tetramer. Assembly of SSB at single-stranded DNAregions generated by these helicase activities and itsinteraction with the exonucleases would furtherallow close coordination of the displacement ofsingle-stranded DNA brought about by a helicaseand its rapid exonucleolytic removal by the exonu-cleases. RecJ may function in conjunction with SSB invivo, and SSBmay target RecJ to specific DNA lesionsand stalled replication forks through SSB-mediatedcomplexes. In addition, considering that both HiSSBand EcoSSB proteins can stimulate their cognate andheterologous RecJ exonucleases, it is likely thatfunctional cooperation and physical interactionbetween RecJ and SSB may be evolutionarily con-served phenomena with an essential role in bacterialspecies. SSB associates with numerous genomemaintenance proteins, further corroborating a newemerging role of SSB as a scaffold for DNA repairand recombination complexes. Therefore, SSB mayplay an important role in integrating and modulat-ing important enzyme activities at specific sites onDNA.In summary, this study focuses on the biochemical
analysis of RecJ exonuclease from H. influenzae and
provides insights into the molecular mechanisms ofthis enzyme activity. HiRecJ was found to function asa processive exonuclease with a strong preference forthe 5′-termini of single-stranded DNA. In contrast toEcoRecJ, HiRecJ was active in the presence of Mg2+
and Mn2+, suggesting a different mode of metalbinding in HiRecJ. In addition, Cd2+ inhibited theMg2+-dependent exonuclease activity of HiRecJ.Interestingly, mutational analysis of conserved resi-dues in the active site of HiRecJ showed that aspartate77 may have a regulatory role in coordinating DNA/metal ion bindingwithDNAhydrolysis. Furthermore,HiRecJ was found to be stimulated by HiSSB, and thetwo proteins were found to interact directly, suggest-ing an interplay between HiRecJ and its cognate SSB.Given that the C-terminus of SSB is essential forfunctional stimulation and interaction with RecJ asdescribed in this work, it is likely that SSB interactionfacilitates recruitment of RecJ to foci of DNA repair.Finally, this study identified the core-catalytic regionof HiRecJ to harbor the site for association with SSB.Our results therefore suggest a close functional andphysical interaction between SSB and RecJ, whichreflects on a complex protein interaction networkwithin the DNA metabolism pathways.
Experimental Procedures
Bacterial strains and plasmids
H. influenzae Rd KW20 was used to isolate genomicDNA, as described earlier.32 E. coli strain DH5α was usedas host for the preparation of plasmid DNA. PlasmidDNAs pMAL-c2x or pRSET B, M13mp18 RFI, and RFIIDNA were prepared as previously described.48 DNAconstructs derived from pMAL-c2x (New England Bio-labs, USA) were used for the overexpression andpurification of wild-type and mutant HiRecJ proteins.Wild-type and truncated SSB proteins were overexpressedand purified from DNA constructs derived from pRSET B(Novagen, Inc.). These proteins were expressed in E. coliBL21(DE3) pLysS cells by transformation with appropriateplasmid constructs, using standard protocol.48
Chemicals
Restriction endonucleases, T4 DNA ligase, T4 polynu-cleotide kinase, amylose resin, EcoRecJf, and Factor Xaprotease were obtained from New England Biolabs.Phusion DNA polymerase was obtained from Finnzymes.Ampicillin, Coomassie brilliant blue R-250, proteinase K,Hepes, polyethyleneimine, protease inhibitor cocktail,phenylmethylsulfonyl fluoride (PMSF), and IPTG wereprocured from Sigma Chemical Company (USA). Protein ASepharose was obtained from GE Healthcare Lifescience(Uppsala, Sweden). [γ-32P]ATP (5000 Ci/mmol) and [α-32P]dATP (3500 Ci/mmol) were obtained fromBRIT (India). Allother reagents used were of analytical or ultrapure grade.
Oligonucleotides and radiolabeling
Oligonucleotides used in this study were synthesizedby Sigma Genosys, and their sequences are given in TableS1. Concentrations of oligonucleotides were determined
1391Interaction of RecJ and SSB
by UV absorbance at 260 nm using the sum of theextinction coefficients of the individual bases. Oligonu-cleotides (ODN) (Table S1) were labeled at the 5′-end with[γ-32P]ATP (30 μCi) using T4 polynucleotide kinase or atthe 3′-end with [α-32P]dATP using terminal deoxynucleo-tide transferase. The labeled oligonucleotides were puri-fied by Sephadex G-25 spun-column chromatography.For exonuclease assays of RecJ involving radioactive
single-stranded DNA substrates, ODN1, ODN2, andODN3 (Table S1) were radiolabeled at the 3′-end. ODN1was used as substrate for reactions in the presence of SSBproteins and EcoHU. Radiolabeled ODN3 was used forDNA binding studies of HiRecJ. For kinetic experiments,ODN7 (Table S1) radiolabeled at the 3′-end was used. Inorder to generate DNA substrates with different structuressuch as overhangs, flaps, Y-shaped substrates, and othersubstrates with 3′ and 5′ single-stranded tails, stoichio-metric concentrations of different oligonucleotides wereannealed by incubation in 100 μl of 0.3 M sodium citratebuffer (pH 7) containing 0.1 M NaCl for 5 min at 95 °C,followed by gradual cooling to room temperature. Toprepare 5′-overhangs, 5′-end radiolabeled ODN4 wasannealed with ODN5; 3′-overhang (ODN6 annealed toODN7 that was radiolabeled at the 3′-end); 5′-flap (ODN4and ODN5 annealed to ODN7 that was radiolabeled at the5′-end); 3′-flap (ODN4 was radiolabeled at the 3′-end andannealed to ODN6 and ODN7); pseudo-Y (ODN4annealed to ODN7 that was radiolabeled at the 5′-end);and replication fork (ODN4, ODN5, and ODN6 annealedto ODN7 that was radiolabeled at the 5′-end). The DNAmixture was separated on a 10% (wt/vol) native poly-acrylamide gel in 89 mM Tris–borate buffer (pH 8.3)containing 1 mM ethylenediaminetetraacetic acid (EDTA)at 10 V/cm for 4 h. The bands corresponding to annealedDNA structures were excised from the gel and eluted intoTE buffer [10 mM Tris–HCl (pH 8.0) and 1 mM EDTA]containing 0.1 M NaCl.For processivity experiments, pUC19 DNA labeled
internally with [3H]thymidine was prepared as describedearlier.49 A 234-bp substrate was generated by digestion ofthe tritiated pUC19 DNAwith NdeI and PstI, followed bylabeling of this fragment at the 3′-end with 32P. The linear2.6-kb double-labeled substrate was generated by restric-tion digestion of tritiated pUC19 DNA with NdeI,following which the free 5′-end was labeled with 32P.The substrates were denatured prior to the assay byboiling at 95 °C for 5 min and rapid snap chilling on ice togenerate linear single-stranded DNA.
Amplification and cloning of H. influenzae Rd recJ
The 1728-bp-long recJ was amplified by polymerasechain reaction (PCR) using H. influenzae Rd genomic DNAas template with Phusion DNA polymerase, using theprimer pair recJF and recJR (Table S1). The primers weredesigned with the help of the annotated complete genomesequence of H. influenzae Rd by identifying the putativegene sequence of recJ. The underlined sequence shows theBamHI site in the forward primer recJF, and PstI site in thereverse primer recJR.The amplified PCR fragmentwas cloned into the bacterial
expression vector pMAL-c2X using the restriction enzymesites mentioned. The PCR product was inserted into thevector in the same translational frame as the malE gene(encoding for MBP) such that the MBP tag was introducedat the N-terminus of the RecJ protein. The DNA constructcontaining the recJ gene was confirmed by restrictiondigestion and sequencing, and designated as pRS1.
Overexpression and purification of H. influenzae RdMBP–RecJ
E. coli BL21(DE3) pLysS cells harboring the pRS1construct were grown at 37 °C in LB broth containing100 μg/ml ampicillin and 35 μg/ml chloramphenicol toan A600 nm∼0.6. MBP–RecJ production was induced byaddition of 0.3 mM IPTG. After 10 h of incubation at18 °C, bacterial cells were harvested by centrifugation at3500g for 10 min. Bacterial cell pellets were resuspendedin sample loading buffer [125 mM Tris–HCl (pH 6.8),4% SDS, 10% glycerol, 0.06% bromophenol blue, and25 mM β-mercaptoethanol] and lysed by sonication.Inductions were checked by subjecting bacterial cellextracts to 0.1% SDS–10% PAGE, followed by stainingof protein bands with Coomassie brilliant blue R-250.As control, inductions were checked in E. coli BL21(DE3) pLysS cells and in the same cells containing thepMAL-c2X plasmid.All subsequent steps were performed at 4 °C or on ice.
Bacterial cells (8 g) were resuspended in buffer A [20 mMTris–HCl buffer (pH 7.4) containing 300 mM NaCl, 1 mMEDTA, 13% glycerol, 10 mM β-mercaptoethanol, and1 mM PMSF], using four times the buffer volume as themass of cell pellets. The cells were lysed by sonication withstandard probe. Cell lysate was centrifuged at 27,200g for60 min at 4 °C, and supernatant was loaded onto anamylose resin affinity column equilibrated with buffer A.The columnwas washedwith 100 bed volumes of buffer Acontaining 1 mM maltose, and protein was eluted with10 mM maltose present in the same buffer. Fractionscontaining pure MBP–RecJ protein were pooled anddialyzed against buffer B [20 mM Tris–HCl (pH 7.4)containing 150 mM NaCl, 0.1 mM EDTA, 50% glycerol,and 10 mM β-mercaptoethanol] and stored at −20 °C. ForFactor Xa digestion of MBP–RecJ protein, the amyloseresin with bound protein was washed extensively withbuffer A and incubated with 10 μg/ml Factor Xa proteasefor 16 h at 4 °C. The resin was centrifuged at 8500g for5 min, and the supernatant was collected. Cleavage of theRecJ protein from MBP was observed by 0.1% SDS–10%PAGE of the supernatant obtained. The cleaved RecJprotein was present in the supernatant, while MBPremained bound to the amylose resin. Protein concentra-tions were estimated using the Bradford reagent (SigmaChemical Company), with bovine serum albumin asstandard.50 Polyclonal antiserum against H. influenzaeHis-tagged RecJ was generated and used for Western blotanalysis as described previously.48 RecJ antibody waspurified from the rabbit serum using Protein A Sepharoseaffinity column.
Overexpression and purification of MBP
Purified H. influenzae Rd RecJ protein is a fusion pro-tein with a 42.5-kDa MBP tag present at its N-terminus.Therefore, MBP protein was used as control in assayswith H. influenzae MBP–RecJ protein to rule out thepossibility that MBP altered the exonuclease activity ofRecJ. E. coli BL21(DE3) pLysS cells harboring the pMAL-c2X plasmid were used for overexpression and purifica-tion of MBP, using the same protocol as described forMBP–RecJ.
Purification of EcoHU protein
EcoHUproteinwas purified as describedby Joseph et al.32
1392 Interaction of RecJ and SSB
Amplification and cloning of H. influenzae ssb
H. influenzae ssb (507 bp) was amplified by PCR usingH. influenzae Rd genomic DNA as template and PhusionDNA polymerase with gene-specific primers (ssbF andssbR; Table S1) containing appropriate 5′-overhangs withrestriction endonuclease recognition sites. The PCRproduct was digested with NdeI and BamHI, and clonedinto pRSET B vector. The resulting plasmid was referredto as pRS2. The authenticity of the clone was confirmedby restriction digestion and ascertained by sequencing ofthe complete DNA fragment.
Generation of C-terminal deletion of HiSSB
A pRS2 construct that carries the full-length ssb gene ofH. influenzaewas used as template for the generation of theC-terminal deletion mutant. C-terminal deletion wasgenerated by introducing a stop codon in the open readingframe of ssb gene such that the encoded protein would lackthe last 41 amino acid residues from the C-terminus(SSBΔC41). The stop codon was introduced by site-direc-ted mutagenesis using the two-stage megaprimer PCR-based technique.51 PCR was carried out using specificprimers (ssbΔC-sense and ssbΔC-antisense; Table S1), andan appropriate restriction site (PsiI) was introduced at thesite ofmutagenesis to score formutants. The constructwiththe truncated ssb gene was confirmed by sequencing.
Overexpression and purification of H. influenzae SSBand SSBΔC
HiSSB was overexpressed in E. coli BL21(DE3) plysSharboring the pRS2 DNA. Six hundred milliliters of LBmedium containing ampicillin (100 μg/ml) and chloram-phenicol (35 μg/ml) was inoculated with 3 ml of anovernight culture of BL21(DE3) pLysS cells harboring pRS2DNA and allowed to grow to anA600 nm of 0.6 at 37 °C. Theexpression of the proteinwas achieved by addition of IPTGto a final concentration of 1 mM, and cells were allowed togrow for another 4 h at 37 °C. The culture was cooled onice, and bacterial cells were harvested by centrifugation at3500g for 10 min.HiSSB and HiSSBΔC proteins were purified using the
same protocol as described previously for EcoSSB.52
Purified HiSSB was used to generate polyclonal antiserumin rabbits following standard protocol,48 and SSB anti-bodies were purified using Protein A Sepharose column.
Generation of RecJ deletion mutants
Three deletion mutants of RecJ were generated: ΔN34(in which 34 amino acid residues from the N-terminuswere deleted), ΔC76 (in which 76 amino acid residuesdistal to the C-terminus were deleted), and the core-catalytic domain (which contains amino acid residues35–498). The fragments corresponding to the deletionmutants were generated by PCR using pRS1 as templateunder the conditions described for the wild-type gene.For ΔN34, recJΔN sense and recJR antisense primerswere used; for ΔC77, recJF sense and recJΔC antisenseprimers were used; and for core-catalytic domain,recJΔN sense and recJΔC antisense primers were used.The forward and reverse primers in each case containedthe BamHI and PstI sites, respectively (Table S1). Thedesign of the primers for deletion mutants was based on
the results of multiple sequence alignment, such thatregions external to the conserved catalytic motifsrequired for RecJ activity were deleted. The PCR productwas cloned into pMAL-c2x as described earlier. MutantRecJ proteins were expressed and purified as describedfor the wild-type.
Site-directed mutagenesis of recJ
Site-directed mutagenesis was performed using pRS1DNA as template to replace required amino acids asdescribed earlier.51 For each substitution, recJF-sense pri-mer and mutagenic antisense primer were used (Table S1).The antisense mutagenic primers were designed in such away that the change in the respective amino acids resultedin the generation of a restriction enzyme site. Hence, theresultant plasmids could be screened easily. The constructscontaining the mutant recJ were used for the expressionand purification of mutant RecJ proteins. All mutationswere confirmed by DNA sequencing.
Exonuclease assays for RecJ activity
The exonuclease activity associated with RecJ wasassayed using 1 μg of the various DNA and RNAsubstrates. The different substrates used include thefollowing: M13mp18 viral supercoiled replicative form,single-stranded circular M13mp18 DNA, linear double-stranded M13mp18 viral DNA (obtained by EcoRI diges-tion of M13mp18 viral supercoiled DNA), linear single-stranded virion M13mp18 DNA (generated by heatdenaturation of linearized double-stranded M13mp18DNA), and mouse ribosomal RNA. Standard reactionmixtures contained 10 mM Tris–HCl (pH 7.5), 50 mMNaCl, 2.5 mMMgCl2, and 1 mMDTT. The substrates wereincubated at 37 °C for 20 min with increasing molarconcentrations of H. influenzae MBP–RecJ (0.5–3 μM) in a20-μl reaction. The reaction was terminated by addition of0.1% SDS, followed by proteinase K (0.2 mg/ml) treatmentat 37 °C for 15 min. All reaction products were analyzedwith a 1% agarose gel electrophoresis in 40 mM Tris–acetate/1 mM EDTA buffer (pH 8.3) at 5 V/cm for 60 min.The gels were stained with ethidium bromide (1 μg/ml)and visualized on a UV transilluminator.Exonuclease activity of MBP–RecJ was further checked
on various radiolabeled DNA substrates. Standard exo-nuclease assay reaction mixtures contained 10 nM radi-olabeled DNA substrates and 50 nM purified MBP–RecJ in10 mM Tris–HCl (pH 7.5), 50 mM NaCl, 2.5 mM MgCl2,and 1 mM DTT, in a total reaction volume of 20 μl. Inexonuclease reactions carried out in the presence of eitherSSB or HU, 25 nM HiSSB monomer and 8 μM EcoHUmonomer were used with 5 nM 3′-end-labeled single-stranded DNA. The reactions were incubated at 37 °C andstopped by addition of 0.1% SDS, followed by proteinaseK (0.2 mg/ml) treatment at 37 °C for 15 min. Fivemicroliters of formamide loading dye (95% deionizedformamide in TBE buffer) was added to each reactionmixture and loaded onto a 15% polyacrylamide gel con-taining 8 M urea and 89 mM Tris–borate/1 mM EDTAbuffer (pH 8.3). The gels were visualized by Phosphor-Imager (Fuji), and the amount of undegraded DNAsubstrates was quantified using ImageGauge V2.54 den-sitometry software.Processivity experiments were carried out as described
earlier19 using 2.6 kb of linear single-stranded DNAlabeled internally with 3H and at its 5′-end with 32P.
1393Interaction of RecJ and SSB
Purified MBP–RecJ (50 nM) was preincubated with 1 μMdouble-labeled DNA in the absence of Mg2+ in a buffercontaining 10 mM Tris–HCl (pH 7.5), 50 mM NaCl, and1 mMDTTon ice for 45 min. The reaction was initiated byaddition of 2.5 mM MgCl2 and a 50-fold excess ofunlabeled competitor single-stranded DNA derived byheat denaturation of linearized pUC19 DNA. The progressof the reaction was monitored from 0.5 to 5 min by releaseof acid-soluble radioactive nucleotides. Acid-solubleradioactivity was estimated and recorded as the percen-tage of total counts, as described earlier.53 The ratio ofinternal 3H to 5′-32P gave an estimate of the extent towhich MBP–RecJ degraded the substrate during a singlebinding event.To determine the kinetic parameters of MBP–RecJ
activity, 50 nM RecJ was incubated with increasingconcentrations of 3′-end-labeled single-stranded DNA(ODN7; 2–14 μM) in a 10-μl reaction volume containing10 mM Tris–HCl (pH 7.5), 50 mM NaCl, 2.5 mM MgCl2,and 1 mM DTT for 10 min at 37 °C. The reaction wasstopped by snap freezing in liquid nitrogen, and thereaction mixture was electrophoresed through a 15% poly-acrylamide gel containing 8 M urea and 89 mM Tris–borate/1 mM EDTA buffer (pH 8.3). The gels wereanalyzed using PhosphorImager (Fuji), and the amountof undegraded DNA substrates was quantified usingImageGauge V2.54 densitometry software. The velocityof the reaction was calculated from the concentration ofundegraded substrate, which was then plotted against thetotal substrate concentration to determine the Km and kcatvalues using the Lineweaver–Burk equation. Unlessotherwise indicated, the enzyme activity data were plottedin GraphPad Prism as the mean of at least triplicatedeterminations, with error bars representing standarddeviation.
Electrophoretic mobility shift assays
EMSAs for the interaction of MBP–RecJ with single-stranded DNA were performed in a 10-μl reactioncontaining 25 mM Hepes (pH 7.5), 50 mM NaCl, and0.1 mM EDTA. 5′-End-labeled single-stranded DNA(5 nM, ODN3; Table S1) was incubated with increasingconcentrations of RecJ fusion protein on ice for 1 h,followed by electrophoresis through a 6% nondenaturingPAGE using 25 mM Tris–HCl buffer (pH 8.0) containing200 mM glycine at 4 °C. The gels were visualized usingFuji PhosphorImager.EMSAwas performed in order to determine the satura-
ting concentrations of HiSSB, HiSSBΔC, and EcoHUproteins with respect to single-stranded DNA substrate.3′-End-labeled single-stranded DNA (5 nM, ODN1; TableS1) was incubated with increasing concentrations of eitherHiSSB or HiSSBΔC monomers (0–100 nM) or EcoHUmonomers (0–18 μM) in a buffer containing 20 mM Tris–HCl (pH 8.0), 50 mM NaCl, 2.5 mM MgCl2, 5% glycerol,and 50 μg/ml bovine serum albumin for 10 min at 37 °C.The samples were applied on an 8% nondenaturingpolyacrylamide gel prepared in 25 mM Tris–borate/1 mM EDTA buffer (pH 8.3) and analyzed as describedabove.
Ammonium sulfate coprecipitation
Coprecipitation experiments were performed essen-tially as described earlier.35,36 Briefly, H. influenzae MBP–RecJ (5 μM monomers) was incubated with HiSSB orHiSSBΔC (5 μM tetramers) in 100-μl reactions with
coprecipitation buffer [10 mM Hepes (pH 7.4), 150 mMNaCl, 10% vol/vol glycerol, 1 mM EDTA, 1 mM PMSF,and 1× protease inhibitor cocktail] on ice for 30 min. Incontrol experiments, 5 μM HiSSB tetramers were sepa-rately incubated with 5 μM MBP or 5 μM M.EcoP15Iproteins. Ammonium sulfate (150 g/l; final reactionvolume, 200 μl) was added to the protein mixture,incubated on ice for an additional 60 min, and then cen-trifuged for 10 min at 13,800g. Supernatant was removed,and pellets were washed thrice with 100 μl of coprecipita-tion buffer containing 150 g/l ammonium sulfate. Pelletfractions were resuspended in 50 μl of sample loadingbuffer [125 mM Tris–HCl (pH 6.8), 4% SDS, 10% glycerol,0.06% bromophenol blue, and 25mM β-mercaptoethanol].Fifteen microliters of each fraction was subjected to 0.1%SDS–12% PAGE. Proteins were visualized by silverstaining and Western blot analysis.
Coimmunoprecipitation
Coimmunoprecipitation was performed to determinethe specific interaction ofHiRecJwith SSB.HiSSB tetramers(5 μM) were preincubated with 5 μMMBP–RecJ in a buffercontaining 10 mM Hepes (pH 7.4), 150 mM NaCl, 1 mMEDTA, and 5% glycerol for 30 min at 4 °C. To each of thesamples, prechilled immunoprecipitation buffer [10 mMHepes (pH 7.4), 150 mM NaCl, and 1 mM EDTA] wasadded to a volume of 500 μl, followed by addition of 1:1000dilutions of either purified anti-RecJ antibodies or purifiedanti-SSB antibodies in separate reactions. In the controlreactions, the proteins were incubated with rabbit pre-immune serum. The immune complexwas allowed to formfor 4 h by continuous gentle mixing of the samples on arotator at 4 °C. The immune complex was separated byaddition of 100 μl of 50% Protein A Sepharose beads andgentle mixing on a rotator. The Protein A beads werewashed thricewith immunoprecipitation buffer containing0.1% Triton X-100, followed by three washes withimmunoprecipitation buffer. Immune complexes boundto the Protein A Sepharose beads were denatured byaddition of a sample loading buffer [125 mM Tris–HCl(pH 6.8), 4% SDS, 10% glycerol, 0.06% bromophenol blue],followed by boiling for 5 min. The sample supernatantswere loaded onto 0.1% SDS–12% PAGE. The gels wereprocessed for Western blot analysis with either HiRecJ orHiSSB antibodies.
Far Western analysis
Far Western studies were carried out by a similarmethod as described earlier.54 Briefly, 5 μl of increasingconcentrations (0.2, 0.4, 0.8, and 1.6 μM) of each of thefollowing proteins—H. influenzae MBP–RecJ, E. coli RNApolymerase (positive control), and MBP (negativecontrol)—was spotted on a nitrocellulose membrane. Toinvestigate the interaction of different deletion mutants ofRecJ with wild-type SSB, increasing concentrations ofRecJΔN, RecJΔC, and RecJ core-catalytic domain werealso spotted with appropriate controls on a nitrocellulosemembrane. Subsequent to its blocking with 5% (wt/vol)nonfat milk in buffer C [10 mM Hepes (pH 7.4), 150 mMNaCl, and 1 mM EDTA] for 2 h at 4 °C, the membranewas incubated with 2 μM HiSSB tetramers in buffer Ccontaining 5% (wt/vol) nonfat milk for 5 h at 4 °C. Themembrane was washed thrice with buffer C containing0.05% (vol/vol) Triton X-100 and then incubated withanti-SSB antiserum (1:5000 dilution) to determine theinteraction of the target proteins with SSB for 3 h at 4 °C.
1394 Interaction of RecJ and SSB
After three successive washes with buffer C containing0.05% (vol/vol) Triton X-100, the membrane was incu-bated with horseradish-peroxidase-conjugated goat anti-rabbit antibodies (1:2000 dilution) for 1 h at 4 °C andwashed thrice with phosphate-buffered saline buffercontaining 0.05% (vol/vol) Triton X-100. The blot wasfurther processed using ECL plus Western blot analysiskit from GE Healthcare (UK). The same experiment wasrepeated to determine the interaction of wild-type MBP–RecJ with HiSSBΔC.
CD spectral analysis
CD measurements were recorded on a Jasco J-810spectropolarimeter between 190 and 300 nm in a 2-mmpathlength quartz cuvette. All experiments were per-formed at 25 °C in 10 mM potassium phosphate buffer(pH 8.0). One micromolar of the wild-type RecJ protein orits mutants was incubated for 5 min in a final volume of400 μl prior to recording of the spectrum. Each experi-mental spectrum represents the best fit of at least 10determinations.
SPR studies
The binding kinetics of MBP–RecJ and its mutants withDNA was determined by SPR spectroscopy using theBIAcore3000 optical biosensor (GE Healthcare Life-science). A 3′-end-biotinylated 65-mer single-strandedDNA (ODN8; Table S1) was immobilized on the surfaceof a streptavidin-coated sensor chip (GE HealthcareLifescience) to a final concentration of 500 response units(RU), as per the manufacturer's recommendations. Thebinding reactions were carried out at 25 °C in a continuousflow of standard buffer containing 10 mM Hepes buffer(pH 7.4) 150 mMNaCl, 1 mM EDTA, and 0.05% surfactantP-20 at a flow rate of 20 μl/min. Increasing concentrationsof MBP–RecJ were injected onto the surface of thebiosensor chip for 120 s at a flow rate of 20 μl/min,followed by a dissociation period of 120 s. The requiredprotein concentrations were made by diluting withstandard buffer. The surface was regenerated by passing5 μl of 0.05% SDS, followed by 10 μl of the running bufferfor further binding reactions. One of the four surfaceswithout the biotinylated oligonucleotide was used asnegative control. Background nonspecific binding and thebulk concentration of H. influenzae MBP–RecJ wereexperimentally determined by simultaneous injectionsover a surface that lacked DNA. The association anddissociation of the protein from DNAwere monitored bychanges in resonance due to the change in mass on thesensor surface. Each experiment was repeated at leastthrice to ensure reproducibility of results. The affinity andkinetic parameters were determined by subjecting thesensorgrams of association and dissociation phases to aglobal analysis using BIAevaluation software version 3.0.Global fitting analyzes both association and dissociationdata for all concentrations simultaneously using a 1:1Langmuir binding model.The interaction between SSB and MBP–RecJ in the
presence of DNA was investigated by immobilization of150 RU of a 65-mer oligonucleotide (ODN8; Table S1) thatwas biotinylated at its 3′-end on a streptavidin sensorchip. The single-stranded DNAwas saturated with HiSSB,yielding 400 RU of bound SSB tetramer, followed bystabilization of SSB–DNA complex by including a 1000-swashing step. Interaction between RecJ and SSB–single-stranded DNA complex was investigated by injection of
different concentrations of MBP–RecJ (0.5–6 μM) over theresultant immobilized complex for 120 s, followed by adissociation step of 120 s. The surface was regeneratedusing 0.1% SDS, followed by reinjection of HiSSB to allowcomplete saturation of the single-stranded DNA surfacefor the next injection of MBP–RecJ. Similarly, the interac-tion between HiSSBΔC and MBP–RecJ was examined bysaturation of DNA with HiSSBΔC prior to injection ofMBP–RecJ. Stoichiometry (n) was derived using thefollowing equation:
n = RUmax=RU HiSSBð Þ� �
MHiSSB=MMBP�RecJ� �
where M denotes the molecular masses of the respectiveproteins, and RU denotes response units, assuming thatRU values are proportional to mass increases on the sensorsurface independent of the nature of bound protein.To study the direct interaction between SSB and MBP–
RecJ, HiSSB was immobilized on the surface of a CM5sensor chip (GE Healthcare Lifescience) using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuc-cinimide activation chemistry, as per the manufacturer'sinstructions. After coupling of the protein, the unboundcarbodiimide groups were blocked with ethanolamine.Coupling of SSB to the CM5 surface resulted in a responsesignal of 220 RU. Immobilization of MBP–RecJ resulted inthe loss of its ability to interact with SSB, possibly due tomasking of interaction sites. One of the flow cells wastreated with ethanolamine alone to serve as referencechannel for nonspecific binding. Binding experimentswere carried out in standard buffer at a flow rate of20 μl/min at 25 °C. Different concentrations of MBP–RecJ(0.25–4 μM) were injected over the SSB surface, followedby a dissociation period of 240 s. The surface wasregenerated with 0.1 M glycine (pH 9.5). All the datacollected for the interaction between SSB and RecJ werecorrected for nonspecific binding in the blank referenceflow cell. The binding data were analyzed using a 1:1Langmuir binding model in BIAevaluation software 3.0 todetermine the kinetic constants.
Acknowledgements
Madhu N. is acknowledged for assisting in theSPR experiments. Drs. V. Nagaraja, U. Varshney, andP. N. Rangarajan (Indian Institute of Science,Bangalore) are acknowledged for providing purifiedE. coli RNA polymerase, EcoSSB, and mouse riboso-mal RNA, respectively. Arathi S. is thanked forproviding technical assistance. Dr. V. Nagaraja andmembers of D.N.R.'s laboratory are acknowledgedfor critical reading of the manuscript and usefuldiscussions. This work was aided by a grant fromthe Department of Science and Technology, Gov-ernment of India, to D.N.R. R.S. was a recipient ofa Senior Research Fellowship from the UniversityGrants Commission, New Delhi.
Supplementary Data
Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2008.11.041
1395Interaction of RecJ and SSB
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