doi:10.1016/j.jmb.2010.05.030 J. Mol. Biol. (2010) 400, 295– 308
Available online at www.sciencedirect.com
Identification of a New Motif in Family B DNAPolymerases by Mutational Analyses of theBacteriophage T4 DNA Polymerase
Vincent Li1, Matthew Hogg2 and Linda J. Reha-Krantz1⁎
1Department of BiologicalSciences, University of Alberta,Edmonton, Alberta, CanadaT6G 2E92Department of Microbiologyand Molecular Genetics,University of Vermont,Burlington, VT 05405, USA
Received 4 March 2010;received in revised form28 April 2010;accepted 14 May 2010Available online21 May 2010
Structure-based protein sequence alignments of family B DNA polymerasesrevealed a conservedmotif that is formed from interacting residues betweenloops from the N-terminal and palm domains and between the N-terminalloop and a conserved proline residue. The importance of the motif forfunction of the bacteriophage T4 DNA polymerase was revealed bysuppressor analysis. T4 DNA polymerases that form weak replicatingcomplexes cannot replicate DNA when the dGTP pool is reduced. Theconditional lethality provides the means to identify amino acid substitu-tions that restore replication activity under low-dGTP conditions either bycorrecting the defect produced by the first amino acid substitution or bygenerally increasing the stability of polymerase complexes; the second typeare global suppressors that can effectively counter the reduced stabilitycaused by a variety of amino acid substitutions. Some amino acidsubstitutions that increase the stability of polymerase complexes producea new phenotype—sensitivity to the antiviral drug phosphonoacetic acid.Amino acid substitutions that confer decreased ability to replicate DNAunder low-dGTP conditions or drug sensitivity were identified in the newmotif, which suggests that the motif functions in regulating the stability ofpolymerase complexes. Additional suppressor analyses revealed anapparent network of interactions that link the new motif to the fingersdomain and to two patches of conserved residues that bind DNA. Thecollection of mutant T4 DNA polymerases provides a foundation for futurebiochemical studies to determine how DNA polymerases remain stablyassociated with DNA while waiting for the next available dNTP, how DNApolymerases translocate, and the biochemical basis for sensitivity toantiviral drugs.
Keywords: NPL motif in family B DNA polymerases; stability of DNApolymerase complexes; DNA replication fidelity; sensitivity to phosphono-acetic acid; DNA polymerase translocation
Edited by J. Karn
Introduction
DNA polymerases interact with DNA in thepolymerase active site and at several locationsalong the DNA binding groove (Fig. 1a). While theDNA polymerase holds the DNA primer-terminalregion in the correct alignment for nucleotideincorporation, these interactions must relax toallow the DNA polymerase to reposition on the
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ress:
onoacetic acid; PPi,ank.
DNA template in order to bind the next incomingnucleotide. DNA polymerases must also be able todissociate when DNA damage is encountered and toproofread, which requires strand separation andrepositioning of the primer terminus in the exonu-clease active site to form exonuclease complexes.2
The processivity of many DNA polymerases isenhanced by association with “clamp” proteinssuch as proliferating cell nuclear antigen and relateddoughnut-shaped structures that tether the DNApolymerase to DNA.3 DNA polymerases, however,control the stability of replicating complexes, sincesingle amino acid substitutions in the DNA poly-merase can significantly affect stability. Asexplained below, the I417V and A737V substitutions
when replication pauses because of reduced nucle-otide pools,8 which is in sharp contrast to the almostimmediate release (half-life of ∼1 s) when theholoenzyme encounters a blocking DNA hairpinstructure in the template strand.9 Biochemicalexperiments reveal that the I417V- and A737V-DNA polymerases form less stable polymerasecomplexes with or without the clamp, and instabil-ity is exacerbated when dNTP pools are reduced.4–7
In vivo, the instability of polymerase complexesformed with the I417V- and A737V-DNA poly-merases results in lethality when the mutant phageare propagated under low-dGTP conditions on theoptA1 bacterial host,10 which expresses an elevatedlevel of a dGTPase that degrades dGTP to thedeoxynucleoside and tri-polyphosphate.11,12 Wild-type phage T4 DNA replication is only slightlyaffected by reduced dGTP, but a reduced dGTP poolseverely inhibits replication by the I417V- andA737V-DNA polymerases.4,10 The reduced abilityof the I417V- and A737V-DNA polymerases to fromstable polymerase complexes produces anothercharacteristic called the antimutator phenotype. Ifpolymerase complexes cannot be formed, thisincreases the opportunity to form exonucleasecomplexes with DNAs with mismatched and evenmatched primer termini.2,4–6,13–18
The conditional lethality of the optA1-sensitiveI417V- and A737V-DNA polymerases provides themeans to select for optA1-resistant phage that haveacquired a second mutation in the polymerase genethat restores polymerizing activity under low-dGTPconditions. The doubly mutant DNA polymerasesphenotypically resemble wild type because thesecond amino acid substitution provides a balancingchange that counters the reduced stability ofpolymerase complexes conferred by the I417V andA737V amino acid substitutions. Most of the optA1-resistant phage recovered retain the original muta-tions in the T4 DNA polymerase gene that encodethe I417V or A737V substitutions, but a secondmutation was acquired, which, in many cases,encoded the L412M substitution in the polymeraseactive site.19 T4 DNA polymerase residue L412corresponds to L415 in the phage RB69 DNApolymerase (Fig. 1a). The structure of the closelyrelated RB69 DNA polymerase is shown because astructure of the T4 DNA polymerase is not available,but the high amount of sequence and functionalsimilarities allows structural assumptions for the T4DNA polymerase based on RB69 DNA polymerasestructures.20
Fig. 1. Location of the NPL core in the structure of the ternais adapted from Franklin et al.1 (PDB ID: 1IG9). (a) The five domthe N-terminal domain is brown, the exonuclease domain is ligdomain is lime green and the P helix is yellow, and the thumb dthread. The DNA substrate is in orange and the deoxynuclexonuclease active sites are enclosed by black circles. Aminoand the corresponding amino acids in the phage T4 DNA poNPL core residues. Residue I52, which resides in a loop structuinteracts with P427, which resides in a loop structure in the pathe linker peptide. The corresponding residues in the phage T
The L412M substitution suppresses optA1 sensi-tivity but only partially rescues the antimutatorphenotype observed for the singly mutant I417V-and A737V-DNA polymerases.4 The biochemicalbasis for suppression of optA1 sensitivity is due tothe ability of the L412M substitution to increase thestability of polymerase complexes, even when dNTPpools are low.4,5,13 The increased stability ofpolymerase complexes has the potential to increasemismatch extension; hence, a mutator phenotype isobserved for the L412M-DNA polymerase.2,4 TheL412M-DNA polymerase is also sensitive to phos-phonoacetic acid (PAA),4,21 a pyrophosphate (PPi)analog that inhibits replication by the herpes viralDNA polymerase by inhibiting the PPi exchangeand pyrophosphorolysis reactions.22 PAA sensitiv-ity requires formation of stable polymerizing com-plexes, specifically complexes that have nottranslocated to be in position to bind the nextincoming nucleotide.23,24 PAA sensitivity can beused to identify mutations that suppress drugsensitivity, and many of these encode amino acidsubstitutions that decrease the stability of polymer-ase complexes and confer optA1 sensitivity.4,23 Thus,identification of suppressors of optA1 and PAAsensitivity can be used to gain insights into how theT4 DNA polymerase produces polymerase com-plexes that maintain stability under low dNTP poolsbut still discriminate against the extension ofmismatches that is required for high-fidelity DNAreplication.Here, we present structure-based protein se-
quence alignments and mutational studies of theT4 DNA polymerase that revealed a previouslyunidentified DNA polymerase motif that is con-served in many family B DNA polymerases. Thenew motif, which we call the NPL core, is formed atsites of interactions between residues in loops fromthe N-terminal and Palm domains, and interactionsbetween the N-terminal domain loop and a con-served proline residue in the Linker region, whichconnects the N-terminal and palm domains (Fig. 1).Amino acid substitutions in the NPL core conferoptA1 and PAA sensitivities, and thus, we proposethat the NPL core functions in regulating thestability of polymerase complexes. Although theNPL core does not contact DNA directly, furthergenetic studies revealed an apparent network ofinteractions that connect the NPL core to the fingersdomain and to two conserved patches of residuesthat contact DNA. Since the NPL core is conservedin family B DNA polymerases, we propose that the
ry complex of the phage RB69 DNA polymerase; the figureains of the RB69 DNA polymerase are colored as follows:
ht blue, the palm domain is green, the N helix of the fingersomain is magenta. The linker peptide is illustrated as a redeosides are depicted as blue sticks. The polymerase andacid residues are indicated for the RB69 DNA polymeraselymerase are in parentheses. (b) Enlargement to show there in the N-terminal domain of the RB69 DNA polymerase,lm domain, and with a conserved proline residue, P381 in4 DNA polymerases are I50, P424, and P378, respectively.
298 The NPL Motif in Family B DNA Pols
NPL core is a major determinant of DNA polymer-ase stability during chromosome replication.
Fig. 2. Structure-based protein sequence alignments ofthe NPL core for several family B DNA polymerases.Structural information was used to align proteinsequences for the DNA polymerases identified by anasterisk (*). Abbreviations used are as follows: T4,enterbacteriophage T4; RB69, enteriobacteriophage RB69;Phi1, enteriobacteriophage phi1; Aeh1, Aeromonas phageAeh1; P-SSM4, Prochlorococcus phage P-SSM4; Syn9,Synechococcus cyanophage sny9; Pfu, Pyrococcus furiosus;9N-7, Thermococcus sp. 9° N-7; Vent, Thermococcus litoralis;DTok, Desulfurococcus Tok; KOD, Pyrococcus kodakaraensis;Sso, Sulfolobus solfataricus; HSV, herpes simples virus 1;EcoII, E. coli DNA pol II; Vac, vaccinia virus; Yα,Saccharomyces cerevisiae DNA pol α; Yδ, S. cerevisiae DNApol δ; Yɛ, S. cerevisiae DNA pol ɛ; Yζ; S. cerevisiae DNA polζ; Hα, human DNA pol α; Hδ, human DNA pol δ; and Hɛ,human DNA pol ɛ. The standard Clustal coloring foramino acid residues is used: GPST, orange; HKR, red;FWY, blue; ILMV, green. A lower case “h” indicates ahydrophobic residue.
Results
Identification of NPL core residues bystructure-based protein sequence alignments
Family B DNA polymerases are composed ofpalm, fingers, thumb, and exonuclease domains andmany have an N-terminal domain as shown forstructural studies of the phage RB69 DNA poly-merase (Fig. 1a),1 for archaeal family B DNApolymerases,25–28 for the herpes viral DNApolymerase,29 for the yeast DNA polymerase δ,30
and for Escherichia coli DNA pol II.31 We observedthat the N-terminal domain of the phage RB69 DNApolymerase interacts with the palm domain viaclose interactions between loops in the N-terminaland palm domains (Fig. 1b), as noted previously forthe archaeal Sso DNA polymerase.27 However,additional interactions were observed betweenresidues in the N-terminal loop with residues in aregion of the polypeptide chain that joins the N-terminal to the palm domain—the linker region(Fig. 1b). These interactions were observed in allstructures of family B DNA polymerases with N-terminal domains.1,25–34Structure-based protein sequence alignments
were performed as described in Materials andMethods to determine if NPL interactions involveprotein sequence motifs that are conserved in familyB DNA polymerases (Fig. 2). Note that a subset offamily B DNA polymerases, which includes theadeno and ϕ29 DNA polymerases, does not have adomain that corresponds to the N-terminal domainobserved for other family B DNA polymerases,35
and this subset of family B DNA polymerases is notdiscussed here. Protein sequence alignments offamily B DNA polymerases based on structure areindicated by an asterisk (*) and were extended toDNA polymerases from other organisms based onsequence similarities. Previously undetected shortmotifs were revealed. Most striking was the pres-ence of a highly conserved proline residue in thelinker region, which is present in all of the family BDNA polymerases examined. The highly conservedproline residue, which is P378 in the T4 DNApolymerase and P381 in the RB69 DNA polymerase(Fig. 1b), is flanked in general by hydrophobicresidues on the amino-terminal side and by polarresidues on the carboxy-terminal side (Fig. 2).The palm loop sequence (Fig. 2) follows the highly
conservedMotif A sequence (residues 411–420 in theRB69 DNA polymerase and 408–417 in the T4 DNApolymerase) in the primary structure, which assistedalignment. Motif A forms part of the polymeraseactive site (note residue L415 in the RB69 DNApolymerase in Fig. 1a, which corresponds to L412 inthe T4 DNA polymerase). A conserved prolineresidue is present in the palm loop of phage and
many archaeal family B DNA polymerases, mostfrequently adjacent to a serine residue (the SPmotif),but the herpes viral DNA polymerase and manyeukaryotic family B DNA polymerases have analternative CF/CY motif at the analogous position.The archaeal Sso DNA polymerase has an apparenthybrid SY motif. Eukaryotic DNA polymerase ɛ hasa P residue, as observed for the phage and archaealDNA polymerases, but in a QP motif; E. coli DNApol II has a DP motif. The palm motif also has apartially conserved N residue on the amino-terminalside of the loop. On the carboxy-terminal side, the
Fig. 3. The T4 I50L- and L412M-DNA polymerases aresensitive to the antiviral drug PAA. Soft agar containinghost bacteria was overlaid on a PAA gradient from 0 to2 mg/ml. Phage were spotted across the gradient.Plaques, which are the circular clearings on the bacteriallawn, were observed from 0 to 2 mg/ml PAA for the wild-type, P424I-DNA, and P378L-DNA polymerases. Smallerand fewer plaques were observed for phage expressingthe L412M- and I50L-DNA polymerases; these phage arePAA-sensitive.
299The NPL Motif in Family B DNA Pols
ET/DT sequence is observed for DNA polymeraseswith the SP palm loop motif; an ST or TT is presentfor the DNA polymerases with the CF/CYmotif; DSand SA are observed for the yeast and human ɛDNA polymerases, respectively. The consensusmotif for the palm loop is Nh(SP or CF/CY)(D/ETor S/TT)h, where h indicates a hydrophobic residue.The N-terminal loop interacts with the palm loop
and with the linker region (Fig. 1b), but there isconsiderable variation in the size of the loop infamily B DNA polymerases and little proteinsequence conservation was observed (Fig. 2). Forthese reasons, N-terminal loop alignments arepresented only for phage DNA polymerases andfor family B DNA polymerases for which a structurehas been determined.
Identification of residues in the NPL core bysuppressor analyses
Suppressor analysis is a powerful genetic methodto probe function and amino acid/domain interac-tions within a protein (intragenic suppression) orbetween proteins (intergenic suppression). Mostmutations that suppress optA1 sensitivity are withinthe T4 DNA polymerase gene. The L412M substitu-tion is a global suppressor of optA1 sensitivity sincethe increased stability of polymerase complexesconferred by the L412M substitution suppresses theoptA1 sensitivity produced by amino acid substitu-tions that decrease stability.19 The increased stabilityof polymerase complexes produces PAA sensitivityand the mutator phenotype.4 In the same geneticexperiment that identified L412M,19 the I50L sub-stitution in the NPL core was also selected as aglobal suppressor of optA1 sensitivity (Fig. 1), and
Table 1. Effects of amino acid substitutions in the NPLcore
a optA1 sensitivity is the plating efficiency of T4 strains on themutant optA1 bacterial host that expresses elevated levels of adGTPase compared to the plating efficiency on the isogenic wild-type host. The plating efficiency of the wild-type T4 strain is 0.85;plating efficiencies relative to the wild-type T4 phage strain arereported. A “0” indicates that no plaques were detected on theoptA1 host except for optA1-resistant phage that appeared at afrequency of ∼1 in 106 to 108.
b Replication fidelity was determined by measuring thefrequency of rIIUV199oc revertants as described in Materialsand Methods. The revertant frequencies reported are relative tothe rIIUV199oc+ revertant frequency of wild T4 phage, ∼1×10−6.A revertant frequency N1 indicates a mutator phenotype and afrequency b1 indicates an antimutator phenotype.
like the L412M-DNA polymerase, the I50L-DNApolymerase is sensitive to PAA and also displays amutator phenotype (Table 1; Fig. 3). Thus, the I50Lsubstitution in the NPL core is expected to increasethe stability of polymerase complexes. (Note that I50in the T4 DNA polymerase corresponds to I52 in theRB69 DNA polymerase.) While the L412M substi-tution in the polymerase active center is in positionto affect PPi and PAA binding directly, the I50Lsubstitution in the NPL core must act indirectly toproduce drug-sensitive polymerase complexes.In a new experiment, two amino acid substitutions
were identified in the NPL core that suppress optA1sensitivity; these amino acid substitutions—P378Land P424L (P381 and P427 in the RB69 DNApolymerase)—suppress the optA1 sensitivity of theR335C-DNA polymerase (R338 in the RB69 DNApolymerase) (Table 1; Fig. 1). The R335C amino acidsubstitution was identified as a suppressor of thePAA sensitivity of the tsP36-DNA polymerase,which has a duplication of residue D863 inthe carboxy-terminal region of the T4 DNApolymerase.23 Since amino acid substitutions thatsuppress PAA sensitivity often confer optA1 sensi-tivity, it was not surprising that the R335C-DNApolymerase is optA1-sensitive and displays anantimutator phenotype; both phenotypes are corre-lated with mutant DNA polymerases that formweak polymerase complexes. Although this was asmall study, it is telling that the only amino acidsubstitutions identified that suppressed the optA1sensitivity conferred by the R335C substitution werein the NPL core.T4 strains expressing the singly mutant P378L-
characterized in vivo (Table 1). Amino acid substitu-tions that suppress optA1 sensitivity may be globalsuppressors that generally increase the stability ofpolymerase complexes and confer PAA sensitivityand the mutator phenotype as observed for theL412M substitution, or the substitutions may spe-cifically correct the defect produced by the firstamino acid substitution. The P378L and P424Lsubstitutions do not appear to be global suppres-sors. Only a 5-fold increase in the rII revertantfrequency was observed for the P378L-DNA poly-merase (Table 1), and PAA sensitivity was notdetected (Fig. 3). Furthermore, the P424L-DNApolymerase was phenotypically opposite to thePAA-sensitive/mutator L412M-DNA polymeraseand displayed optA1 sensitivity and a strongantimutator phenotype as the rII+ revertant fre-quency was reduced 50-fold (Table 1). Thus, theP378L and P424L substitutions likely correct thedefect in function produced by the R335C substitu-tion. A series of additional genetic experiments thatprovide insights into how the P378L and P424Lsubstitutions suppress the optA1 sensitivity pro-duced by R335C substitution are described below.These experiments exploit the optA1 sensitivity ofthe P424L-DNA polymerase.
The L412M substitution suppresses the optA1sensitivity of the P424L-DNA polymerase
If the optA1 sensitivity and antimutator pheno-types of the P424L-DNA polymerase are caused byformation of polymerase complexes with lowstability, then the L412M substitution is expectedto be identified as a suppressor. Individual high-titercultures of T4 phage expressing the P424L-DNApolymerase were prepared under permissive condi-tions and then the lysates were plated on therestrictive optA1 host to identify optA1-resistant
phage, which appear at a frequency of about 1 in10 to 100 million. optA1-resistant phage wereselected at 42 and 30 °C; the higher temperature ispredicted to select for the most robust suppressormutations. Wild-type phage were isolated at 42 °Cin which the mutation coding the P424L substitutionreverted back to the wild-type proline codon. TheL412M substitution was also identified at 42 as wellas at 30 °C (Table 2), which indicates that the generalability of this amino acid substitution to suppressoptA1 sensitivity extends to the P424L substitutionin the NPL core. The doubly mutant L412M/P424L-DNA polymerase is not optA1-sensitive, and repli-cation fidelity as measured by reversion of therIIUV199oc allele is between the strong antimutatorphenotype observed for the P424L-DNA polymer-ase and the modest mutator phenotype observed forthe L412M-DNA polymerase (Table 3).
Identification of amino acids within the NPLcore that suppress the optA1 sensitivity of theP424L-DNA polymerase
A pseudorevertant was also selected at 42 °C inwhich the leucine 424 codon was converted to thecodon for isoleucine (ATT) (Table 2). The P424I-DNA polymerase strain is informative. Since thisstrain produces plaques on optA1 bacteria at 42 °Cand displays only a 10-fold antimutator phenotype(Table 1), a proline residue is not essential forfunction at this position.If the optA1 sensitivity of the P424L-DNA poly-
merase is caused by altered interactions within theNPL core, then compensating amino acid substitu-tions in the N-terminal loop and linker region areexpected to restore function. This proposal wasconfirmed by the identification of four optA1-resistant strains at 30 °C that retained the mutationthat encoded the P424L substitution, but hadacquired additional mutations that encoded aminoacid substitutions within the NPL core. Threesubstitutions were located in the N-terminal loop:D49Y, I50T, and a duplication of residue I50, whichwas accompanied by the A580S substitution, whichresides outside of the NPL core. The P378Hsubstitution was identified for the conserved prolineresidue in the linker region (Table 2). The identifi-cation of suppressor amino acid substitutions at theclosest sites for direct physical interaction within theNPL core (Figs. 1 and 4a) provides evidence thatresidues in the NPL core interact and that these
301The NPL Motif in Family B DNA Pols
interactions are important for DNA polymerasefunction.The compensatory amino acid substitutions with-
in the NPL core are predicted to correct the defect infunction caused by the P424L substitution ratherthan to be global suppressors like L412M since theseamino acid substitutions were not identified inprevious experiments and the close proximity ofNPL core residues provides the means for physicalinteraction. This proposal was tested by construct-ing the I50L/P424L-DNA polymerase since I50Lwas not identified as a suppressor of the optA1sensitivity conferred by the P424L substitution eventhough I50L was identified as a global suppressor inprevious experiments.19 The reason for this is clear.While I50T significantly suppressed the optA1sensitivity conferred by the P424L substitution andpartially suppressed the antimutator phenotype, theI50L/P424L-DNA polymerase was still optA1-sensi-tive; the plating efficiency (0.1) is too low to bedetected by genetic selection (Table 1). Thus, whilethe I50L substitution is a global suppressor of optA1sensitivity conferred by amino acid substitutionsoutside of the NPL core, the I50T substitution isrequired to correct the defect produced by P424Lsubstitution within the NPL core.
The use of suppressor analysis to identify NPLnetworks; connecting the NPL core to thefingers domain and to residues that bind DNA
Several amino acid substitutions were identifiedoutside of the NPL core that suppress the optA1sensitivity of the P424L-DNA polymerase. The DNApolymerase view in Fig. 4a shows the NPL coreresidues—I50, P378, and P424 in the T4 DNApolymerase (I52, P381, and P427 in the RB69 DNApolymerase) at the hub of interactions between severalprotein domains. Amino acid substitutions wereidentified in the base regions of the N (light green)and P (yellow) helices of the fingers domain—G466Sand L567F, respectively (residues G469 and L570 inthe RB69 DNA polymerase) (Fig. 4a and e). Note thatresidue L567 resides within the conserved Motif Bsequence in family B DNA polymerases (Fig. 5a).Amino acid substitutions were also identified for
residues Y460 and N579 (residues Y463 and N582 inthe RB69 DNA polymerase), which are in position toform H-bonds (Fig. 4a). The H-bond distance forRB69 DNA polymerase residues Y463 and N582 is∼2.6 Å in the closed structure, which increases to∼2.9 Å in the open structure. The Y460F/H andN579L substitutions in the T4 DNA polymerase areexpected to disrupt H-bonding and other interac-tions that may extend to L567, which is discussedabove, and to A580 (A583 in the RB69 DNApolymerase) (Fig. 4a). The A580S substitution wasidentified in combination with a duplication ofresidue I50 in the NPL core (Table 2). While one of
the substitutions alone may be responsible forsuppression, it is likely that the combination ofboth substitutions is required, which will be testedin future experiments. Note, however, that A580 is ahighly conserved residue in helix Q (Fig. 5a). HelixQ (dark green in Fig. 4a) interacts with residues inthe conserved Y/FxGG/AxV motif (red residues inFig. 4a), a sequence that is important for interactionswith DNA in the primer-terminal region.36,37 Thus,the amino acid substitutions for residues at basepositions of helices N and P (G466S, L567F) and thesubstitutions for residues Y460, N582, and A580appear to link P424 in the NPL core to the fingersdomain and to conserved residues that are impor-tant for DNA interactions.
Several amino acid substitutions that counter theoptA1 sensitivity conferred by the P424L substitutionare in position to affect fingers domain movementsin addition to residues in the base regions of helicesN and P of the fingers domain, G466 and L567,which were discussed above. The S369A substitu-tion (S372 in the RB69 DNA polymerase) is inposition to affect interactions with E471 (E474 in theRB69 DNA polymerase) in helix N (Fig. 4b and c).Q478 in helix N (Q481 in the RB69 DNA polymerase)and E98 (E100 in the RB69 DNA polymerase) appearto form an interacting pair of amino acids; threeamino acid substitutions (E98K, Q478R, and Q478H)that could affect such interactions were identified.The F487L substitution in helix N (L490 in the
RB69 DNA polymerase) is at the edge of the onlyregion of significant protein sequence divergencebetween the T4 and RB69 DNA polymerases, but theclosest interacting residues, E716-G717 in the RB69DNA polymerase and E713-D714 in the T4 DNApolymerase, are flanked by conserved residues (Fig.4d and e). Residues E716 and G717 in the RB69 DNApolymerase reside in the loop region of a β-hairpinstructure (Fig. 4e), which connects with a patch ofhighly conserved basic residues (K705, K706, andR707) that interact with the primer template region.Movement of the fingers domain brings residueL490 in the RB69 DNA polymerase to about 6.7 Åfrom residue G717 in the closed structure (Fig. 4e)and to about 13 Å in the open structure (Fig. 4d). Wespeculate that the F487L substitution in the T4 DNApolymerase suppresses the optA1 sensitivity con-ferred by the P424L substitution by a mechanismthat links movements of the fingers domain tointeractions that affect DNA binding by the patch ofconserved basic residues.
Mechanism for suppressing the optA1sensitivity conferred by the R335C aminoacid substitution; regulating movementof the fingers domain
The optA1 sensitivity of the R335C-DNA poly-merase is suppressed by the P424L substitution,
Fig. 5. Amino acids identifiedby suppressor analysis of the T4DNA polymerase compared toother family B DNA polymerases.(a) Protein sequence alignments inMotif B and helix Q. Sites of aminoacid substitutions that suppress theoptA1 sensitivity of the T4 P424L-DNA polymerase are indicated byan asterisk. The alanine residue inhelix Q is conserved, but not theadjacent asparagine. The leucineresidue in Motif B is also notconserved. (b) The Y460F/H andN579L substitutions were identifiedas suppressors of the optA1 sensi-tivity conferred by the P424L sub-stitution in the T4DNApolymerase.
Y460 and N579 are in position to form H-bonds (Fig. 4a). Residues corresponding to Y460 and N579 are not conserved inother family B DNA polymerases.
303The NPL Motif in Family B DNA Pols
which also confers optA1 sensitivity and antimutatorphenotypes. The ability of the R335C and P424Lsubstitutions individually to confer optA1 sensitivityand antimutator phenotypes but together to sup-press both phenotypes suggests that suppression isachieved by a physical link between the two aminoacid substitutions. The fingers domain is the mostreasonable physical connection between residuesR335 and P424 based on structure (Fig. 4b and c).Residue R338 in the RB69 DNA polymerase (R335 inthe T4 DNA polymerase) forms part of a platformthat cradles helix P in the RB69 DNA polymeraseclosed complex (Fig. 4c) but is separated from helixP in the open complex (Fig. 4b). Thus, the fingersdomain moves with respect to R338 during cycles ofnucleotide incorporation. Residue P424 in the NPLcore is located near the base of the fingers domain(Fig. 4d and e). We propose that the R335C andP424L substitutions individually disrupt the normalpositioning and movement of the fingers domain toproduce DNA polymerase complexes with reducedstability, but together both substitutions correctpositioning to restore function. These observationsalong with the identification of several amino acidsubstitutions in the fingers domain that suppress theoptA1 sensitivity conferred by the P424L substitu-tion indicate a functional relationship between NPLcore residues and the fingers domain.
Fig. 4. Locations of amino acid substitutions that suppresreplicate DNA when the dGTP pool is reduced (optA1 sensitivRB69 DNA polymerase structures were adapted from Franklininterest are depicted in space fill; T4 DNA polymerase residucolored as in Fig. 1. (a) NPL core residues in the closed structuP378 (T4 residues I50, P424, and P378) are near a cluster of aapparent network of interactions that connect to the Y/FxGG/polymerase are depicted in red space fill) that interacts with threspectively, showingmovement of the fingers domain—helixconformations, respectively, showing the conserved basic resiG717 and L490 in the RB69 DNA polymerase (D714 and F487
Using suppressor analysis to determinehow the bacteriophage T4 DNA polymeraseforms polymerase complexes that remain stablewhen dNTP pools are low; identificationof the NPL core
Suppressor analysis or “forced evolution” is apowerful genetic method to uncover interactionsbetween amino acid residues and structures that areimportant for function; this method is especiallyuseful in revealing interactions that are wellremoved from the active site. We propose thatsuppressor analysis can be used to uncover themechanism used by the phage T4 DNA polymeraseto form stable polymerase complexes that canreplicate long stretches of DNA even when dNTPpools are low. Two T4 DNA polymerase phenotypeswere used: optA1 sensitivity and PAA sensitivity,which are correlated with mutant DNA poly-merases that form polymerase complexes withdecreased or increased stability, respectively. SinceDNA replication by optA1-sensitive T4 DNA poly-merases is severely restricted under conditionswhere the dGTP pool is reduced, complexes formedwith these mutant DNA polymerases dissociate
s the inability of the phage T4 P424L-DNA polymerase toity). The open (PDB ID: 1Q9X) and closed (PDB ID: 1IG9)et al.1 and Freisinger et al.,33 respectively. Amino acids ofes are indicated in parentheses. The protein domains arere of the RB69 DNA polymerase—residues I52, P424, andmino acids identified by suppressor analysis that form anAxVmotif in the linker (residues 391–396 in the RB69 DNAe template strand. Open (b) and closed (c) conformations,N (lime green) and helix P (yellow). Open (d) and closed (e)dues that interact with DNA and the relative positions ofin the T4 DNA polymerase).
prematurely while waiting for the next availabledGTP. Selection for optA1-resistant phage revealssecond-site mutations that encode amino acidsubstitutions that suppress optA1 sensitivity eitherby correcting the defect caused by the first aminoacid substitution or by generally increasing thestability of polymerase complexes, as observed forthe L412M substitution in the polymerase active site.The L412M substitution also confers PAA sensitivity(Fig. 3). Amino acid substitutions that confer optA1and PAA sensitivity were identified in a cluster ofresidues that we have named the NPL core (Fig. 1);we propose that the NPL core functions in regulat-ing the stability of polymerase complexes. Residuesin the tripartite NPL core are partially conserved infamily B DNA polymerases, especially the prolineresidue in the linker region (Fig. 2).We were first drawn to the NPL core because of
identification of the I50L substitution as a globalsuppressor of the optA1 sensitivity of several DNApolymerases.19 The I50L substitution resembles theL412M substitution by conferring sensitivity to theantiviral drug PAA (Fig. 3) and amutator phenotype(Table 1); thus, we predict that the I50L substitutionincreases the stability of polymerase complexes asobserved for the L412M substitution.4,5,13 Addition-al amino acid substitutions in the NPL core wereidentified as suppressors of the optA1 sensitivity ofthe R335C-DNA polymerase—P378L and P424L(Table 1). While a leucine substitution for P378 hadonly a small effect on function, the P424L substitu-tion conferred optA1 sensitivity and an antimutatorphenotype (Table 1). Since optA1 sensitivity andantimutator phenotypes are observed for amino acidsubstitutions that reduce the stability of polymerasecomplexes, as observed for the I417V and A737Vamino substitutions,4–6 we predict that the P424Lsubstitution reduces stability. This proposal issupported by the ability of the L412M substitutionto suppress the optA1 sensitivity conferred by theP424L substitution. Thus, amino acid substitutionsthat apparently increase (I50L) and decrease (P424L)the stability of polymerase complexes were identi-fied in the NPL core.If residues within the NPL core interact during
DNA replication, then substitutions for NPL coreresidues that suppress the optA1 sensitivity con-ferred by the P424L substitution should be identi-fied. This proposal was confirmed. Three“compensating” amino acid substitutions wereidentified in the N-terminal loop—D49Y, I50T, anda duplication of residue I50, which was accompa-nied by the A580S substitution—and P378H wasidentified in the linker region (Tables 1 and 2).Since amino acid substitutions within the NPL
core confer optA1 sensitivity or PAA sensitivity, asobserved for amino acid substitutions within thepolymerase active site, the NPL core may be actingas a second control center in regulating the stabilityof polymerase complexes. As already discussed, theL412M substitution was selected as a strongsuppressor of the optA1 sensitivity conferred bythe P424L substitution (Table 2). But what happens
if the L412M substitution in the polymerase activesite is combined with the I50L substitution in theNPL core? Since individually both amino acidsubstitutions confer PAA sensitivity and mutatorphenotypes, if residues in the polymerase activecenter and the NPL core function independently,then increased PAA sensitivity and more replicationerrors are expected for the double mutant comparedto the single mutants. We attempted to construct thedouble mutant, but synthetic lethality was observed(Table 3). Thus, while amino acid substitutions thatapparently increase (L412M) and decrease (P424L)the stability of polymerase complexes can “balance”each other, the combination of two amino acidsubstitutions that confer the same phenotypesproduces a DNA polymerase that cannot functionin vivo. Lethality may be the consequence of toomany mutations (error catastrophe), or if bothamino acid substitutions act to increase the stabilityof polymerase complexes, then translocation may beimpeded. Both possibilities can be tested in futurein vitro experiments.
Networks of amino acid interactions thatconnect the NPL core to DNA
If the NPL core contributes to determining thestability of replicating complexes, how can this bedone without contacting DNA? Suppressor analysisrevealed apparent networks of interactions thatconnect NPL core residues with DNA in theprimer-terminal region via the conserved Y/FxGG/AxV motif (colored red in Fig. 4a) andpossibly to the conserved patch of basic residues(Fig. 4d and e). Since many of the amino acidsubstitutions that suppress the optA1 sensitivityconferred by the P424L substitution are predicted toaffect movement of the fingers domain (E98K,R335C, S379A, G466S, Q478H/R, F487L, andL567F), we propose that the NPL core is in positionto relay movements of the fingers domain toresidues that interact with DNA in the primer-terminal region.Strong evidence for the link between the NPL core
and fingers domain is provided by the ability of theP424L substitution in the NPL core to suppress theoptA1 sensitivity conferred by the R335C substitu-tion even though the P424L substitution also confersoptA1 sensitivity. Thus, these amino acid substitu-tions are not acting independently but appear to beconnected by the fingers domain. Since helix P of thefingers domain makes close contact with R338 in theRB69 DNA polymerase (corresponds to R335 in theT4 DNA polymerase) in the closed structure (Fig.4c), the R335C substitution in the T4 DNA polymer-ase may disrupt positioning of the fingers domain,which appears to be restored by the P424L substi-tution. The L340P substitution (L343 in the RB69DNA polymerase) provides additional evidence thatpositioning of the fingers domain in the closedstructure affects function (Fig. 4c). The L340Psubstitution confers PAA sensitivity and a strongmutator phenotype,21,38 which are opposite to the
305The NPL Motif in Family B DNA Pols
optA1 sensitivity and antimutator phenotypes con-ferred by the R335C substitution. Thus, amino acidsubstitutions that perturb interactions with helix Pin the fingers domain can either decrease (R335C) orincrease (L340P) the ability of the DNA polymeraseto form stable polymerase complexes when dGTP isin short supply.
Model
We propose the following model to explain theoptA1 sensitivity/antimutator and PAA sensitivity/mutator phenotypes and to provide insights intohow the T4 DNA polymerase forms stable poly-merase complexes that can withstand low dNTPpools but still retain the ability to proofreadmismatches.One of the critical steps in determining the fidelity
of DNA replication occurs just following nucleotideincorporation. If an incorrect nucleotide is incorpo-rated at the primer terminus, then proofreadingensues, which requires separation of the end of theprimer strand from the template and repositioning ofthe primer end within the exonuclease active site;these steps may require enzyme dissociation.2 Incor-poration of correct nucleotides, however, is the norm.DNA polymerases are proposed to form polymerasecomplexes that are in rapid equilibrium between theuntranslocated and translocated positions until thecorrect dNTP is bound in the nucleotide bindingpocket that is formed in the translocated complex.23,24Initial binding of the correct nucleotide is rapid5 and isfollowed by a slower conformational change to formaclosed complex, which provides increased nucleotidediscrimination.39 But what happens if the correctnucleotide is not available? One possibility is proof-reading even though the primer terminus is correct.optA1-sensitive/antimutator T4 DNA polymerasesdegrade correctly replicated DNA even in thepresence of dNTPs and degradation is exacerbatedby low dNTP pools.2,6 In contrast, PAA-sensitive/mutator T4 DNA polymerases proofread fewermismatches and do not degrade correctly replicatedDNA as readily when dNTP pools are low.4 Sincethese mutant DNA polymerases are PAA-sensitiveand PAA is a PPi analog that binds to the untranslo-cated complex, PAA-sensitive DNA polymerases arepredicted to favor formation of untranslocated com-plexes. Thus, enhanced stability of polymerase com-plexes is correlated with formation of PAA-sensitive,untranslocated polymerase complexes.Previous studies have focused on interactions
within the polymerase active site, but studiespresented here indicate that residues in the NPLcore play an important additional role that is linkedto movement of the fingers domain and to con-served residues that bind DNA. DNA polymerasedissociation likely requires release of DNA contactsin the polymerase active center as well as release ofcontacts that are mediated via the NPL core. Theadditive interactions observed for amino acid sub-stitutions in the NPL core and in the polymeraseactive site suggest that both sites function in DNA
binding, but independently of each other. If the NPLcore mediates DNA contacts between the Y/FxGG/AxVmotif and possibly the patch of conserved basicresidues (Fig. 4), then DNA association duringtranslocation may be maintained by alternatingDNA binding with NPL-mediated contacts andcontacts within the polymerase active site.We have made a movie that illustrates the
relationship between the open and closed forms ofthe RB69 DNA polymerase as it cycles betweenrounds of nucleotide incorporation (SupplementalMaterials). While the movie does not show the entirereaction cycle, it clearly demonstrates the spatialarrangements of the amino acid substitutionsdescribed in this article as the fingers domainswings back and forth between both conformations.The relationship between the NPL core and the NPLinteraction network is shown. The movements in theNPL hinge are subtle, but rapid and subtleconformation changes are predicted while thepolymerase translocates along the DNA template.
Implications for other family B DNApolymerases and future directions
Two types of amino acid substitutions in thephage T4 DNA polymerase are presented: substitu-tions that confer optA1 sensitivity and antimutatorphenotypes or PAA sensitivity and mutator pheno-types. These opposite phenotypes, however, repre-sent two sides of the same coin. While enhancedability to form stable polymerase complexes is anadvantage when dNTP pools fluctuate, the down-side is less proofreading andmore replication errors.Conversely, low stability means premature dissoci-ation when dNTPs are in short supply, whichincreases proofreading but also reduces replicationefficiency. We propose that DNA polymerases indifferent organisms have optimized the balancebetween forming stable polymerase complexes andproofreading to meet specific environmental condi-tions. For example, the PAA sensitivity of the wild-type herpes viral DNA polymerase reflects in-creased formation of the untranslocated complex,which may be necessary if dNTP pools fluctuate.Archaeal DNA polymerases that replicate DNA athigh temperature may also favor formation of stablepolymerase complexes. In contrast, the T4 and RB69DNA polymerases replicate DNA under moderatetemperatures and high dNTP concentrations.40
Since dNTPs are consistently available, the T4 andRB69 DNA polymerases may form less stablepolymerase complexes in order to reduce mismatchextension and increase proofreading.Suppressor analysis identified several amino acid
substitutions for conserved residues in family BDNA polymerase, for example, P358 and P424 in theNPL core (Fig. 2), G466 in helix N (Fig. 4a), and A580in helix Q (Fig. 5a), which demonstrates similaritiesamong family B DNA polymerases. Several aminoacid substitutions were also identified for non-conserved amino acids and these may provideinsights into differences in function. For example,
306 The NPL Motif in Family B DNA Pols
three amino acid substitutions that suppressed theoptA1 sensitivity conferred by the P424L substitutionthat affect interaction between residues Y460 andN579 in the T4 DNA polymerase were identified(Fig. 5b). The phenylalanine and histidine substitu-tions for Y460 and the leucine substitution for N579are predicted to disrupt H-bonding interactions.Since the Y460F/H and N579L substitutions counterthe optA1 sensitivity and apparent decreased stabil-ity conferred by the P424L substitution, these aminoacid substitutions are predicted to increase thestability of polymerase complexes. Residuescorresponding to Y460 and N579 in other family BDNA polymerases are replaced by hydrophobicresidues, frequently phenylalanine for Y460 and avariety of amino acids for N579, which may indicatethat polymerase complexes formed with these DNApolymerases are intrinsically more stable. Interest-ingly, E. coli DNA polymerase II, which hasincreased mismatch extension ability,31 has a phe-nylalanine and a leucine for residues that correspondto Y460 and N579 in the T4 DNA polymerase; Y460Fand N579L were identified as optA1 suppressors inthe T4 DNA polymerase (Fig. 5b). However, sincehigh-fidelity DNA polymerases also have a phenyl-alanine instead of the Y460 observed in the T4 DNApolymerase and a hydrophobic amino acid insteadof N579, it is unlikely that the phenylalanine andleucine substitutions in E. coli DNA polymerase IIalone are responsible for translesion synthesisactivity. However, these substitutions may combinewith other subtle differences to allow mismatchextension ability.31 Similarly, subtle differences arepredicted to be the basis for DNA polymerase drugsensitivity or drug resistance.These studies provide evidence that the NPL core
is conserved in family B DNA polymerases and thattheNPL core plays a role in regulating the stability ofpolymerase complexes via interactions that involvethe fingers domain and conserved residues that bindDNA. The networks will have to be expanded infuture experiments to include the thumb domainsince the optA1 sensitivity and antimutator pheno-types of the A737V-DNApolymerase are suppressedby the I50L substitution in the NPL core and by theL412M substitution in the polymerase active site,19
but it is not clear how the A737V substitutionproduces optA1 sensitivity. The collection of aminoacid substitutions reported here can also be used totest and to further develop computational methodsthat have been used to probe the dynamics of DNAand RNA polymerases.41
Materials and Methods
Databases, structure-based sequence alignments,and DNA polymerase dynamics
The protein sequence database at the National Centerfor Biotechnology Information (National Institutes ofHealth) was the source for the family B DNA polymerasesequences discussed here. Family B DNA polymerase
structures were visualized using the applications Swiss-PDB viewer42 and PyMOL.43 Clustal X44 was used to alignprotein sequences; structurally equivalent regions weremanually put into the Clustal program as distinctalignment regions.A movie describing the relationships between open and
closed forms of the RB69 DNA polymerase is provided.The open, binary complex of the RB69 DNA polymerasewith DNA containing a templating abasic site [ProteinData Bank (PDB) ID:1Q9X]33 and the closed, ternarycomplex with DNA containing a templating A and anincoming dTTP (PDB ID: 1IG9)1 were aligned via theirpalm domains (residues 383–468 and 573–729). The morphbetween open and closed forms of the DNA polymerasewas calculated with the program LSQMAN.45 The abasicsite in the open complex was replacedwith an adenine, thedTTP from the ternary complex was removed, andresidues 505–534 of the fingers domain of both complexeswere removed to aid in clarity. Individual movie frameswere rendered in PyMOL43 and assembled in QuickTimePro (Apple Inc., Cupertino, CA).
Bacterial and bacteriophage T4 strains
The E. coli strain CR63 (K strain, supD) was used toprepare T4 phage cultures using standard procedures.Determinations of rII+ mutant frequencies were madeusing the CR63 lambda lysogenic strain (CR63λ). TheoptA+ (2395) and optA1 strains (2396)10 were used to detectoptA1 sensitivity. The bacteriophage T4 strains are derivedfrom strain T4D.
Bacteriophage T4 methods
Replication fidelity was determined for wild-type andmutant phageT4 strains bymeasuring revertant frequenciesfor the rII mutant rIIUV199oc to rII+. The total number ofphage per culture was measured by plating on thepermissive host CR63, and the number of rII+ revertantswas determined by plating on the host CR63λ, whichrestricts growth of rII mutant T4 phage. The revertantfrequency is the ratio of rII+ revertants to the total number ofphage. At least eight parallel cultures were tested for eachexperiment; the median revertant frequency is reported.optA1 sensitivity was determined by measuring the
plating efficiency of phage T4 strains on the optA1 host(2396) and the isogenic optA+ host (2395). At least fivecultures were tested; the average plating efficiency isreported.optA1-resistant phage were selected by plating phage
from independent, high-titer cultures (N1011 phage/ml) onthe nonpermissive optA1 bacterial host 2396. The plateswere incubated overnight at 42 °C (most stringentcondition) or 30 °C (less stringent condition). Phageplaques were produced only if the infecting phage couldreplicate genomicDNAandproduce phage progeny underthe low-dGTP condition. optA1-resistant phage weredetected at a frequency of about 1 in 10 to 100 million.Only 1 (for the most part) or 2 optA1-resistant phageisolates were selected from each culture for further study.The T4 DNA polymerase gene (g43) of each isolate was
sequenced to identify the mutation that conferred optA1resistance. Phage T4 genomic DNA was prepared from0.75 ml of a frozen/thawed phage culture (∼1011 phage)using the standard phenol/chloroform extraction method.The T4 DNA polymerase gene was amplified using thefollowing primers: F 5′-GCCTAATAACTCGGGCTA-TAAACTAAGG and R 5′-GGGACCTGGAGGTCCTAG.
307The NPL Motif in Family B DNA Pols
The PCR product was purified by gel electrophoresis andsequenced using the F and R primers plus three additionalprimers that extended in the reverse direction: 5′-GCGATTTGCTGAACCGCATC, 5′-TTCTGGTGTAAT-GAGTCCTAT, and 5′-GGCGCAGGGTCTTGTTCAAC.Mutant T4 DNA polymerase strains were constructed
by recombination between co-infecting mutant phagestrains or between the infecting phage with cloned copiesof mutant DNA polymerase genes carried on plasmidswithin the host bacteria.46 The T4 DNA polymeraseexpression vector47 was mutated using a variety of site-directed mutagenesis procedures. The single mutagenic-primer method48 was used to construct the P424L-DNApolymerase using the primer 5′-CGCCAGGTTAACAT-TAGTCTTGAAACTATTCGTGGTCAG. The mutagenicbase is underlined. The DNA polymerase gene wassequenced to confirm the presence of all mutations.Sensitivity to the antiviral drug PAAwas determined by
spotting phage (10 μl) onto a PAA gradient that wasoverlaid with soft agar containing host bacteria (CR63).The PAA gradient plates were formed by first pouring35 ml of an agar solution containing 2 mg/ml PAA into a100×100 mm plate and then elevating one edge with apencil. After the agar hardened, the plate was placed flaton the bench and 35 ml of drug-free agar solution wasadded. The plates were used the following day. The agarsolution contained 100 ml 1.3 g tryptone, 0.8 g NaCl, 0.1 gNa citrate, 1.2 g noble agar, and 200 mg PAA for PAA-agar and no PAA for drug-free agar.
Acknowledgements
We thank M. Bryman for the initial selection of theR335C/P378L- and R335C/P424L-DNA polymer-ase strains. This work was supported by anoperating grant from the Natural Sciences andEngineering Research Council of Canada to L.R-K.M.H. was supported by Public Health Service grantCA-52040 from the National Cancer Institute. L.R-K.is a Scientist of the Alberta Heritage Foundation forMedical Research.
Supplementary Data
Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2010.05.030
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