Functional characterization of UvrD helicases from Haemophilus influenzae and Helicobacter pylori

Functional characterization of UvrD helicases fromHaemophilus influenzae and Helicobacter pyloriRuchika Sharma and Desirazu N. Rao

Department of Biochemistry, Indian Institute of Science, Bangalore, India

Keywords

ATPase; DNA–protein interaction; GTP;

protein oligomerization; UvrD helicase

Correspondence

D. N. Rao, Department of Biochemistry,

Indian Institute of Science, Bangalore 560

012, India

Fax: +91 80 2360 0814

Tel: +91 80 2293 2538

E-mail: [email protected]

(Received 31 January 2012, revised 16

March 2012, accepted 10 April 2012)

doi:10.1111/j.1742-4658.2012.08599.x

Haemophilus influenzae and Helicobacter pylori are major bacterial patho-

gens that face high levels of genotoxic stress within their host. UvrD,

a ubiquitous bacterial helicase that plays important roles in multiple DNA

metabolic pathways, is essential for genome stability and might, therefore,

be crucial in bacterial physiology and pathogenesis. In this study, the func-

tional characterization of UvrD helicase from Haemophilus influenzae and

Helicobacter pylori is reported. UvrD from Haemophilus influenzae

(HiUvrD) and Helicobacter pylori (HpUvrD) exhibit strong single-stranded

DNA-specific ATPase and 3¢–5¢ helicase activities. Mutation of highly con-

served arginine (R288) in HiUvrD and glutamate (E206) in HpUvrD abro-

gated their activities. Both the proteins were able to bind and unwind a

variety of DNA structures including duplexes with strand discontinuities and

branches, three- and four-way junctions that underpin their role in DNA repli-

cation, repair and recombination. HiUvrD required a minimum of 12 nucleo-

tides, whereas HpUvrD preferred 20 or more nucleotides of 3¢-single-strandedDNA tail for efficient unwinding of duplex DNA. Interestingly, HpUvrD was

able to hydrolyze and utilize GTP for its helicase activity although not as

effectively as ATP, which has not been reported to date for UvrD character-

ized from other organisms. HiUvrD and HpUvrD were found to exist pre-

dominantly as monomers in solution together with multimeric forms.

Noticeably, deletion of distal C-terminal 48 amino acid residues disrupted the

oligomerization of HiUvrD, whereas deletion of 63 amino acids from C-ter-

minus of HpUvrD had no effect on its oligomerization. This study presents

the characteristic features and comparative analysis of Haemophilus influen-

zae and Helicobacter pylori UvrD, and constitutes the basis for understand-

ing the role of UvrD in the biology and virulence of these pathogens.

Structured digital abstractl HiUvrD and HiUvrD interact by molecular sieving (View interaction)l HpUvrD and HpUvrD interact by molecular sieving (View interaction)l HiUvrD and HiUvrD interact by cross-linking study (View interaction)l HpUvrD and HpUvrD interact by cross-linking study (View interaction)

Introduction

Helicases are a highly diverse group of enzymes that

couple the energy derived from NTP hydrolysis to

translocate along and catalyze the transient unwinding

of DNA or RNA duplexes [1]. These molecular motors

Abbreviations

dsDNA, double-stranded DNA; EMSA, electrophoretic mobility shift assay; HiUvrD, Haemophilus influenzae UvrD; HpUvrD,

Helicobacter pylori UvrD; ssDNA, single-stranded DNA; SF1, Super-Family 1.

FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 1

http://www.uniprot.org/uniprot/Q02322


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are ubiquitous in all living organisms and play key

roles in almost all aspects of DNA metabolism, includ-

ing replication, repair, recombination, transcription

and RNA processing [2,3]. On the basis of primary

structure analyses and motif arrangement, helicases

have been classified into several superfamilies, of which

Super-Family 1 (SF1) is the best characterized [4].

UvrD or DNA helicase II is a SF1A helicase univer-

sally distributed across Gram-negative bacteria that

can translocate along single-stranded (ss)DNA or

unwind DNA duplex in a 3¢–5¢ direction [5]. UvrD

was first identified by its indispensible role in the

nucleotide excision repair and mismatch repair path-

ways [6,7]. In addition, UvrD plays critical roles in

rolling circle plasmid replication, processing of Oka-

zaki fragments in the absence of DNA polymerase I

and replication fork reversal in Escherichia coli poly-

merase III mutants with multiple functions at inacti-

vated replication forks [8–11]. UvrD can dislodge a

number of noncovalent protein–DNA interactions like

replication terminator Tus from its cognate site Ter

and RecA from ssDNA [12,13]. Recently, UvrD,

together with Rep and DinG helicases, has been found

to be essential for promoting efficient replication

across highly transcribed regions by removing RNA

polymerase from replication forks [14]. UvrD plays a

unique role in the replication of plasmid DNAs con-

taining multiple covalent DNA–protein cross-links,

implicating its function as a sensor of DNA damage

[15]. Expression of uvrD is regulated at the transcrip-

tional level by an SOS-response [16]. Knockout of

uvrD in E. coli remains viable, although it is lethal in

either a polA or rep background, and exhibits sensiti-

vity to UV light, elevated rates of recombination and

mutations [17]. This multitude of functions of UvrD

make it important to all organisms, more so in patho-

genic bacteria or extremophiles surviving under

extreme conditions such as in soil, on ocean floors or

at high temperatures. Pathogenic organisms have to

adapt to the hostile host environment, are exposed to

a high level of DNA-damaging agents like oxidative

radicals and nitrosative species generated as a result of

host immune response, combat antibiotics and acquire

increased virulence.

Several lines of evidence suggest that helicases are

not only crucial to the physiology of a number of

pathogenic organisms, but also contribute to their

virulence. Furthermore, UvrD and its homologues like

Rep and PcrA constitute attractive targets for drug

discovery because their deletion is lethal in some path-

ogenic bacteria such as PcrA in Staphylococcal species

and Bacillus subtilis [18]. In the case of Mycobacte-

rium tuberculosis, uvrD1 gene inactivation reduces its

persistence in a mouse model of tuberculosis infection

[19]. Deletion of uvrD2, a second UvrD homologue, in

Mycobacterium smegmatis and M. tuberculosis is lethal,

indicating that the gene is essential for this pathogen

[20,21]. Similarly, in Helicobacter pylori, a RecB-like

helicase has been reported to be essential for host

colonization [22]. In another study, disruption of

putative recB-like helicase (hp1553), recG (hp1523),

uvrD (hp1478) and ruvB (hp1059) homologues in Heli-

cobacter pylori affected its viability [23]. The Helico-

bacter pylori UvrD homologue has been shown to

partially complement UV sensitivity, but failed to alle-

viate increased mutation rates of E. coli uvrD mutants

[24]. UvrD is crucial in maintaining genomic integrity

and limits homologous intergenomic recombinations

and deletions, as well as DNA damage-induced genomic

arrangements between DNA repeats in Helicobact-

er pylori [24]. Inactivation of uvrD in Neisseria meningit-

ides leads to increases in the frequency of phase

variation within mononucleotide repeat tracts [25]. The

uvrD null mutant of Haemophilus influenzae is sensitive

to UV radiation, exhibits reduced levels of host-cell

reactivation and decreased phage recombination [26]. In

another study, inactivation of Haemophilus influenzae

uvrD led to a two-fold increase in spontaneous mutation

resulting in rifampicin-resistance and a three-fold

increase in rates of pilus phase variation due to the

destabilization of long dinucleotide repeat tracts [27].

Furthermore, Haemophilus influenzae uvrD could com-

plement E. coli uvrD mutants [28]. Hence, UvrD consti-

tutes an attractive drug target for the development of

new antibacterial agents to combat these pathogens.

Whereas the above studies underscore the cellular

functions of UvrD and its importance in bacterial

physiology and genome maintenance, its role in patho-

gens like Haemophilus influenzae and Helicobact-

er pylori has not been studied in detail. Furthermore,

although UvrD has been extensively studied in E. coli,

a growing body of evidence suggests that different

bacteria differ in their basic DNA repair machinery,

and that helicases like UvrD are crucial for the patho-

genesis of bacteria [29]. Therefore, a detailed biochem-

ical analysis of UvrD from Haemophilus influenzae and

Helicobacter pylori is necessary to understand its

cellular roles in these pathogens, as well as in a quest

for new targets for drug development. It is envisaged

that characterization of Haemophilus influenzae and

Helicobacter pylori UvrD would help in comparative

analysis of UvrD across different bacteria. The studies

presented here form the basis to understand the role

of UvrD in genome maintenance, pathogenesis and

virulence of Haemophilus influenzae and Helicobacter

pylori.

Haemophilus influenzae and Helicobacter pylori UvrD R. Sharma and D. N. Rao

2 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

Results and Discussion

Purification of UvrD from Haemophilus influenzae

Rd and Helicobacter pylori strain 26695

UvrD from Helicobacter pylori strain 26695 (HpUvrD)

shares 29% identity with E. coli UvrD and 31% iden-

tity with Haemophilus influenzae UvrD (HiUvrD). To

obtain sufficient quantities of the protein for in vitro

studies, uvrD gene from Helicobacter pylori strain

26695 (hpuvrD) was PCR amplified and cloned into

pET-15b vector under the control of an isopropyl thio-

b-D-galactoside-inducible promoter. Expression of

HpUvrD (� 80 kDa) was carried out at low tempera-

ture (18 �C) because at 37 �C the protein was

expressed in an insoluble form. The protein was heter-

ologously overexpressed in E. coli with a N-terminal

(His)6-tag (Fig. S1A, lane 3). HpUvrD was purified to

at least 95% homogeneity as can be seen from the

SDS ⁄PAGE (Fig. S1A, lane 6).

Purified HpUvrD was subjected to trypsin digestion

followed by MALDI-TOF MS analysis. The peptide

finger map of HpUvrD thus obtained was matched

with the theoretical peptide map. There was good cov-

erage of the amino acid sequence for the protein

because significant numbers of peptide ions belonging

to HpUvrD (Fig. S1B) were obtained, confirming the

authenticity of the protein.

DNA-binding properties of Haemophilus

influenzae and Helicobacter pylori UvrD

Several DNA structures are generated within the cell

during DNA-processing pathways, and are specifically

recognized and processed by helicases. PriA helicase

binds to DNA in a structure-specific manner with the

3¢-terminus of the leading strand at the branch point

of arrested replication forks and displacement loop

(D-loop) structure [30,31]. Staphylococcus aureus PcrA,

a homologue of UvrD, was shown to bind partial

duplex DNA structures containing hairpin structures

adjacent to the 5¢-ssDNA region [32]. Therefore, it was

of interest to study the DNA-binding properties of

HiUvrD and HpUvrD.

The DNA-binding specificity of Haemophilus influen-

zae UvrD (HiUvrD) was assessed by electrophoretic

mobility shift assay (EMSA) using a 63-mer ssDNA

(ODN 1; Table S1) or duplex DNA (structure 1;

Table S2). HiUvrD displayed robust binding to

ssDNA substrate (Fig. 1A) but its interaction with

blunt-ended DNA duplex (Fig. 1B) was much weaker

relative to ssDNA. To gain insight into the role of

HiUvrD in replication, repair, recombination and

transcription, the ability of HiUvrD to bind different

DNA structures resembling intermediates of these

processes (Table S2) was investigated using EMSA.

HiUvrD exhibited relatively high affinities for a number

of branched DNA substrates like splayed-duplex,

3¢-overhang, 3¢-flap, three-way junction and four-way

junction (structures 2–6, Table S2 and Fig. 1C–G).

Complex formation with blunt-ended duplexes that were

either nicked or looped (structures 7 and 8, Table S2)

was relatively poor (Fig. 1H,I) compared with ssDNA

and branched DNA structures. Multiple bands and

smear, rather than a discrete band, observed at lower

HiUvrD concentrations perhaps represent complexes

containing different amounts of HiUvrD bound to

DNA substrates. The observed DNA smears might also

result from dissociation of the protein from DNA

during electrophoresis, perhaps due to the formation of

unstable DNA–protein complexes at low concentrations

of HiUvrD. From analysis of the binding data, it is

evident that HiUvrD can bind to all the tested DNA

structures with different affinities in the order: splayed-

duplex ‡ 3¢-flap ‡ ssDNA > 3¢-overhang > four-way

junction > three-way junction > nicked duplex >

looped duplex ‡ double-stranded (ds)DNA duplex.

Similar to HiUvrD, HpUvrD was able to robustly

interact with ssDNA but exhibited poor interaction

with duplex DNA (Fig. S2A,B). Furthermore,

HpUvrD was able to efficiently bind DNA structures

containing ssDNA regions like splayed-duplex, 3¢-over-hang and 3¢-flap, as well as branched duplex structures

like three-way junction resembling a replication fork,

and four-way junction resembling a Holliday junction

(structures 2–6, Table S2 and Fig. S2C–G). However,

HpUvrD exhibited a weak interaction with nicked

(structure 7, Table S2) or looped (structure 8,

Table S2) DNA duplexes with blunt ends (Fig. S2H,I)

although the binding of HpUvrD to these structures

was comparatively better than HiUvrD. The order of

DNA binding for different DNA structures exhibited

by HpUvrD was: splayed duplex > ssDNA >3¢-flap>

3¢-overhang > three-way junction = four-way junction

> looped duplex >>> nicked duplex ‡ dsDNA

duplex.

The kinetic parameters for interaction of HiUvrD

and HpUvrD with ssDNA and dsDNA substrates

were assessed using SPR that measures the interaction

between two molecules in real time. HiUvrD and

HpUvrD were able to quantitatively bind both ssDNA

(Fig. 2A,B) and dsDNA (Fig. 2C,D) as observed from

a concentration-dependent increase in the resonance

signal. KD values of 5.6 and 10.5 nM were obtained for

the interaction of HiUvrD and HpUvrD with ssDNA,

respectively, whereas KD values of 38.6 and 6.2 lM

R. Sharma and D. N. Rao Haemophilus influenzae and Helicobacter pylori UvrD


were obtained for the interaction of HiUvrD and

HpUvrD with dsDNA, respectively. Therefore, both

the UvrD proteins exhibited � 1000-fold higher affin-

ity for ssDNA than dsDNA, which is consistent with

the EMSA results and indicates that UvrD prefers

ssDNA to load onto its substrates. Moreover, HiUvrD

interacted with ssDNA with a two-fold higher affinity

than HpUvrD (Fig. 2A,B). Overall, the affinities of

both HiUvrD and HpUvrD for ssDNA are compara-

ble with E. coli UvrD [33]. ATP, its nonhydrolysable

analogue AMPPNP, and its hydrolysis product ADP

in the absence of metal cofactor did not affect the

affinity of HiUvrD and HpUvrD for splayed-duplex,

suggesting that ATP and DNA binding by UvrD are

independent of each other (data not shown).

Weak interaction of HiUvrD and HpUvrD with

looped duplex (Figs 1I and S2I) suggests that UvrD

cannot bind internal ssDNA regions and required free

DNA ends to load onto DNA substrates. Interestingly,

both HiUvrD and HpUvrD are able to bind blunt-

ended but branched structures, i.e. a three-way junc-

tion resembling a replication fork (Figs 1F and S2F)

and a four-way junction that resembles a Holliday

junction (Figs 1G and S2G), better than unbranched

duplex DNA (Figs 1B and S2B) while exhibiting high-

est affinity for branched structures with ssDNA ends

like splayed-duplex (Figs 1C and S2C). Holliday junc-

tions, in general, are specifically recognized by RecG

and RuvAB helicases [34]. However, the DNA-binding

data presented here suggests that HiUvrD and

HpUvrD can also recognize and possibly process these

recombination intermediates in the absence of the

recombination helicases. Interestingly, Helicobact-

er pylori lacks helicases like RecBCD [29], which is

involved in recombinational repair of double-strand

breaks and rescue of damaged replication forks [35], as

well as RecQ [29], which is involved in replication fork

reversal, DNA end resection, processing of D-loop,

branch migration and resolution of double Holliday

junctions [36]. Therefore, the ability of HpUvrD to

interact with different DNA structures like 3¢-over-hang, 3¢-flap, three- and four-way junctions might have

important implications in recombination, replication

fork reversal and overall genome stability in Helico-

bacter pylori. This is further supported by an earlier

study in which E. coli UvrD was shown to bind 3¢-flap

Fig. 1. Interaction of HiUvrD with DNA. EMSAs were performed by incubating different monomeric concentrations (0, 0.05, 0.1, 0.2, 0.4,

0.6, 0.8, 1.2, 1.8, 2.4, 3, 6, 9 and 12 lM) of HiUvrD with 1 nM radiolabeled DNA substrates: (A) ssDNA, (B) DNA duplex, (C) splayed-duplex,

(D) 3¢-overhang, (E) 3¢-flap, (F) three-way junction, (G) four-way junction, (H) nicked DNA and (I) looped DNA.



resembling a replication fork with blocked synthesis of

the leading strand, suggesting its function in DNA rep-

lication progression [37]. In addition, E. coli UvrD

also plays critical roles in the processing of Okazaki

fragments in the absence of DNA polymerase I [11]

and replication fork reversal when DNA polymer-

ase III is nonfunctional [9]. Interestingly, the

DNA-binding properties of HiUvrD and HpUvrD are

significantly different from S. aureus PcrA helicase, a

closely related UvrD homologue in Gram-positive

bacteria, which interacts weakly with ssDNA, blunt

duplex, partial forked or bubbled duplex, and 3¢- and5¢-overhangs [32]. Binding of HiUvrD and HpUvrD to

different DNA structures emphasizes their ability to

recognize and process intermediates of DNA replica-

tion, recombination and repair. This is similar to E. coli

UvrD that has been previously reported to unwind

forked DNA structures suggesting its ability to target

replication and recombination DNA intermediates [38].

ATP hydrolysis by HiUvrD and HpUvrD

Multiple sequence alignment of HiUvrD and HpUvrD

with other helicases shows the presence of Walker A

or motif I and Walker B or motif II, defined by

conserved GxGKS ⁄T and DExx (e.g. DEAH or

DEPH) signature sequences, respectively, within the

N-terminal region of the proteins (Fig. 3). These

motifs are characteristic of proteins that bind and

hydrolyze NTP and the ATP hydrolysis activity of

purified HiUvrD and HpUvrD was therefore deter-

mined. The ATPase activity of HiUvrD and HpUvrD

was first checked in the absence or presence of ssDNA

as well as dsDNA. Different concentrations of Hi-

UvrD or HpUvrD were incubated with excess ATP

(2 mM) and [c-32P]ATP was used as a tracer. Hydroly-

sis of ATP by both HiUvrD and HpUvrD was

enhanced substantially by ssDNA (data not shown).

HiUvrD and HpUvrD exhibited � 15% and � 20%,

respectively, ATPase activity in the presence of

dsDNA compared with ssDNA, whereas no apprecia-

ble ATP hydrolysis was observed in the absence of

DNA (data not shown). Therefore, ssDNA acts as an

effector for ATP hydrolysis by HiUvrD and HpUvrD

accounting for their high affinity for ssDNA. ATP

hydrolysis by nearly all helicases is greatly enhanced

upon binding ssDNA, which possibly reflects a confor-

mational change that stabilizes ATP in a conformation

optimal for its rapid hydrolysis [39]. Using pre-steady-

state kinetics, ATP hydrolysis was found to be coupled

Fig. 2. SPR analysis of binding of HiUvrD and HpUvrD with ssDNA and dsDNA. SPR sensorgrams for the interactions of HiUvrD (A) and

HpUvrD (B) with ssDNA or HiUvrD (C) and HpUvrD (D) with dsDNA are shown. 60-mer oligonucleotide (700 response units) or correspond-

ing duplex (680 response units) were immobilized on the surface of streptavidin sensor chip. Varying concentrations of HiUvrD (A,C) or

HpUvrD (B,D) in standard buffer were passed over DNA surfaces and change in resonance signal was observed. A blank surface, which

lacked any immobilized DNA, was used as a reference cell to nullify nonspecific interactions and bulk responses.



to the ability of PcrA [40] as well as UvrD [41] to

translocate along ssDNA.

To establish the relationship between the initial

velocity of the reaction and the enzyme concentration,

the rate of ATP hydrolysis catalyzed by increasing

HiUvrD (0 –150 nM) and HpUvrD (0 –125 nM) con-

centrations in the presence of ssDNA (2 lM) and ATP

(4 mM) was determined. A plot of initial velocity

against the corresponding enzyme concentration

yielded a linear relationship indicating a direct depen-

dence of the initial velocity of the reaction on HiUvrD

and HpUvrD concentration (Fig. S3A,B). When a ser-

ies of similar reactions containing HiUvrD or HpUvrD

(10 nM), ssDNA (2 lM) and different concentrations

Fig. 3. Alignment of amino acid sequences of E. coli UvrD (Eco), Haemophilus influenzae UvrD (Hi), Helicobacter pylori UvrD (Hp), S. aureus

PcrA and E. coli Rep. Helicase motifs are highlighted and labeled as ‘Q to VIa’. Box with dotted line shows the amino acids deleted to gener-

ate HiUvrDDC48. Shaded box shows the amino acid residues deleted to generate HpUvrDDC63.



(0–1 mM) of ATP in a standard reaction buffer con-

taining 5 mM MgCl2 were performed, a conventional

hyperbolic dependence of the rate of reaction on ATP

concentration was obtained, such that the rate of ATP

hydrolysis was initially linear and later saturated with

increasing ATP concentrations that gave best-fit to the

Michaelis–Menten equation (data not shown). Nonlin-

ear regression analysis of this data yielded a Km(ATP)

value of 102.5 ± 8 lM for HiUvrD and 112 ± 6.6 lM

for HpUvrD, whereas the kcat value was 83.5 s)1 for

HiUvrD and � 105 s)1 for HpUvrD (Table 1). These

values are comparable with UvrD proteins from other

organisms like E. coli, M. tuberculosis UvrD1 and

M. smegmatis UvrD2 [19,42–44].

To determine the cofactor requirements of HiUvrD

and HpUvrD, ATPase assays were carried out in the

presence of other metal ions like Mn2+ and Ca2+.

Steady-state kinetic analyses of rates of ATP hydroly-

sis showed that Mn2+ and Ca2+ could partially sup-

port the ATPase activity of HiUvrD with three-fold

and eight-fold reduced rates, respectively, compared

with Mg2+ (Table 1). Similarly, Mn2+ and Ca2+

could support the ATPase activity of HpUvrD with

� 3.5- and 7-fold reduced rates, respectively, compared

with Mg2+ (Table 1). The specific requirement for

Mg2+ is exhibited by most helicases including

M. tuberculosis UvrD1 [19]. The specificity for Mg2+

can be attributed to the conservation of acidic residues

in helicase motif II whose carboxyl oxygens preferen-

tially coordinate Mg2+ [45]. The structure of E. coli

UvrD showed that a single Mg2+ ion is coordinated

directly to conserved aspartate and glutamate residues

of motif II, threonine of motif I, and b and c phos-

phates of ATP [46].

Several enzymes like Thermus thermophilus UvrD

[47], E. coli Rep helicase [48], Helicobacter pylori DnaB

[49] and Type III restriction endonuclease PstII [50]

can hydrolyze and utilize GTP in addition to ATP.

Therefore, the hydrolysis of GTP by HiUvrD and

HpUvrD was also monitored to further characterize

the NTP utilization by these enzymes. GTP was hydro-

lyzed with � 6-fold and � 1.5-fold reduced rates com-

pared with ATP by HiUvrD and HpUvrD,

respectively (Table 1). GTP does not support E. coli

UvrD activities [43]. This suggests that specific selec-

tion of adenine base by motif I and Q-motif within the

ATP-binding site of UvrD, as evident from cocrystal

structure of E. coli UvrD–AMPPNP–DNA [46].

Mutational analysis of HiUvrD and HpUvrD

Analysis of the amino acid sequence of HiUvrD and

HpUvrD revealed the presence of motif I and motif II,

also known as Walker A and B, respectively, which

are highly conserved regions present in ATP-bind-

ing ⁄hydrolyzing enzymes (Fig. 3). Mutational analysis

of amino acid residues within these motifs has estab-

lished their significance in catalysis. The conserved

lysine residue in motif I is essential because it contacts

the phosphates of Mg2+–ATP ⁄Mg2+–ADP, whereas

the following serine ⁄ threonine and aspartic acid resi-

dues in motif II coordinate the Mg2+ ion essential for

ATP hydrolysis [46].

Site-directed mutagenesis was performed to replace

the highly conserved glutamate 226 and glutamate 206

of Walker B motif in HiUvrD and HpUvrD, respec-

tively, with glutamine. Mutation of the invariant gluta-

mate residue E226Q in motif II of HiUvrD exhibited a

retarded growth phenotype, because it was not possible

to recover the transformants when the wild-type E. coli

expression strains BL21(DE3)plysS or BL21(DE3)-

plysE were transformed with pET-14b–hiuvrDE226Q

(data not shown). Similar results were obtained with

E. coli UvrDE221Q [33] and E. coli UvrDK35M [51].

Table 1. Steady-state kinetic parameters for NTP hydrolysis by HiUvrD and HpUvrD. n.d., not determined.

Protein Metal NTP Km (lM) kcat (s)1) kcat ⁄ Km (s)1ÆlM)1)

HiUvrD Mg2+ ATP 102.5 ± 8 83.5 ± 2 0.81 ± 0.09

Mn2+ ATP 134 ± 13 34.9 ± 1.9 0.26 ± 0.02

Ca2+ ATP 153.7 ± 21 18.9 ± 1 0.12 ± 0.02

Mg2+ GTP 506.7 ± 21.9 64 ± 1.4 0.13 ± 0.01

HiUvrDR288A Mg2+ ATP (1.1 ± 0.1) · 103 n.d. n.d.

HiUvrDDC48 Mg2+ ATP 115.3 ± 5 179.1 ± 6.3 1.55 ± 0.03

HpUvrD Mg2+ ATP 112 ± 6.6 105 ± 1.6 0.94 ± 0.08

Mn2+ ATP 105.9 ± 26.6 28.4 ± 2.3 0.27 ± 0.1

Ca2+ ATP 113.4 ± 14.6 14.7 ± 0.6 0.13 ± 0.02

Mg2+ GTP 162.1 ± 12.2 56.4 ± 2.3 0.35 ± 0.02

HpUvrDE206Q Mg2+ ATP 87.6 ± 10.4 4.8 ± 0.2 0.06 ± 0.01

HpUvrDDC63 Mg2+ ATP 102.5 ± 10.9 90.1 ± 1.8 0.88 ± 0.15



Therefore, another mutant was generated in motif IV

of HiUvrD, wherein a conserved arginine residue

(R288) was substituted by alanine. The mutant protein

was purified to homogeneity and showed no apparent

change in mobility as judged from 0.1% SDS ⁄ 10%PAGE analysis (Fig. S4A). The mutant HiUvrD exhib-

ited � 20% ATP hydrolysis compared with the wild-

type protein (Fig. S4B). When ssDNA-dependent ATP

hydrolysis of HiUvrDR288A was monitored as a func-

tion of the ATP concentration under steady-state con-

ditions, a linear relationship between rates of ATP

hydrolysis and ATP concentration was obtained, such

that the maximum velocity could not be attained

(Table 1). Therefore, the Km value was found to be

very high as compared with the wild-type HiUvrD

(Table 1). This suggested that HiUvrDR288A is defec-

tive in nucleotide binding similar to E. coli UvrD-

R284A, which also failed to saturate with ATP [52].

Unlike HiUvrDE226Q, the mutant HpUvrDE206Q

protein could be overexpressed and purified to near

homogeneity, as analyzed on SDS ⁄PAGE (Fig. S4C).

The absence of a growth defect in mutant HpUvrD

might be because of the divergent genomic relationship

of HpUvrD with its E. coli counterpart (the two pro-

teins share 34% identity and 54% similarity at primary

structure level), such that it may not interact with and

sequester UvrD, and other repair proteins in E. coli,

which is otherwise a primary reason for dominant

negative effect that could result in poor growth of the

transformants. HpUvrDE206Q fractionated like the

wild-type protein and exhibited no apparent change in

the electrophoretic mobility (Fig. S4C). HpUvrDE206Q

was found to be catalytically deficient (Fig. S4D) and

exhibited � 17-fold reduced ATPase activity compared

with the wild-type protein (Table 1). Using steady-state

kinetic analysis, the kcat value of HpUvrDE206Q was

found to be 4.785 s)1 (Table 1), which is only � 4.5%

that of the wild-type protein, whereas Km (87.6 ±

10.4 lM, Table 1) remained unchanged.

The loss of ATPase activity of HiUvrDR288A and

HpUvrDE206Q might be due to conformational

changes in the protein structure or altered substrate-

binding properties. However, no gross conformational

changes were detected between wild-type and mutant

HiUvrD and HpUvrD as determined by CD spectro-

scopy (data not shown). Both HiUvrDR288A and

HpUvrDE206Q exhibited affinities for ssDNA

(KD = 7.4 and 6.5 nM, respectively) comparable with

the wild-type proteins, as determined by SPR, suggest-

ing no effect of the mutations on the ability of the pro-

teins to bind ssDNA (Fig. S5A,B). To determine

whether the mutations affected the ability of HiUvrD

and HpUvrD to bind ATP, different concentrations of

wild-type and mutant proteins were subjected to UV

cross-linking with [a-32P] ATP. Wild-type HiUvrD was

able to efficiently bind radiolabeled ATP, whereas Hi-

UvrDR288A exhibited � 80% reduced ATP binding

compared with wild-type (Fig. S5C,D), which indicates

that the conserved arginine residue of HiUvrD has a

role in ATP binding. From the cocrystal structures of

E. coli UvrD with DNA and AMPPNP, the basic

residue R284 (motif IV, corresponding to R288 in

HiUvrD) was found to be coordinated to the c phos-

phate of the triphosphate moiety [46]. By contrast,

both the wild-type and HpUvrDE206Q were able

to bind radiolabeled ATP with the same efficiency

(Fig. S5E,F), suggesting that mutant HpUvrD was

compromised in ATP hydrolysis rather than ATP

binding. In the crystal structure of E. coli UvrD with

AMPPNP, a nonhydrolysable ATP analogue, E221 in

motif II was found to be coordinated with a Mg2+

ion, and was well-positioned to function as a base to

deprotonate water molecule for inline nucleophilic

attack involved in ATP hydrolysis [46].

Generation of C-terminal truncated HiUvrD and

HpUvrD

Multiple sequence alignment of HiUvrD and HpUvrD

amino acid sequences with E. coli UvrD and other

homologues like PcrA and Rep proteins revealed the

presence of a C-terminal region flanking the SF1

family helicase core, which comprises the characteristic

conserved motifs (Fig. 3). The function of the

C-terminus is unknown and this accessory region

might have a role in the modulation of enzyme activ-

ity, oligomerization of the protein or its interaction

with other proteins or specific nucleic acid sequences.

In addition, the C-terminus of these proteins is more

variable than the helicase core (Fig. 3).

In order to determine the function of the C-terminal

end of UvrD proteins, the distal 48 amino acids of

HiUvrD and 63 amino acids of HpUvrD external to the

conserved helicase motifs were deleted (Fig. 3) to gener-

ate HiUvrDDC48 and HpUvrDDC63. The proteins

were purified to homogeneity similar to the wild-type

protein (Fig. S6A,B). CD spectra of HiUvrDDC48 and

HpUvrDDC63 showed that the proteins were folded

but exhibited a relative reduction in the helical content

compared with wild-type protein (data not shown),

which might be because of the deletion of C-terminal

regions of the protein. Moreover, HiUvrDDC48(KD = 31.2 nM) and HpUvrDDC63 (KD = 39.4 nM)

exhibited an approximately six-fold and four-fold

decrease in affinities for ssDNA, respectively, compared

with wild-type proteins, as determined by SPR



(Fig. S6C,D). This is similar to E. coli UvrD in which

deletion of 73 amino acid residues from the C-terminus

resulted in an approximately four-fold reduction in its

affinity for ssDNA [53].

Both HiUvrDDC48 and HpUvrDDC63 exhibited

a ssDNA-dependent ATPase activity. Although the

ATPase activity of HpUvrDDC63 was comparable

with wild-type protein (kcat = 90.12 s)1, Km =

102.5 ± 10.9 lM; Table 1), HiUvrDDC48 was able to

hydrolyze ATP with almost two-fold higher efficiency

(kcat = 179.1 s)1, Km = 115.3 ± 5 lM; Table 1) than

the full-length protein. This is in contrast to E. coli

UvrDDC40 wherein the turnover rates for ATP hydro-

lysis were almost the same as for wild-type protein

[42]. However, E. coli UvrDD73 (UvrD1-647) exhibited

� 1.3-fold higher rates of ATP hydrolysis than the

wild-type protein [53]. HiUvrDDC48 and E. coli

UvrDD73 [53] exhibit an increase in ssDNA-dependent

ATPase activity, but interact with ssDNA with

approximately four- to five-fold reduced affinities.

Reduced affinity for ssDNA probably reflects an abil-

ity of these truncated UvrD proteins to function as

potent ssDNA-specific translocases such that ATP

hydrolysis is coupled to rapid movement along

ssDNA. A high affinity for ssDNA would mean that

the protein is able to strongly bind to DNA, which

would reduce the rate of translocation.

DNA unwinding by HiUvrD and HpUvrD

In order to test the helicase activity of purified

HiUvrD and HpUvrD, unwinding of a partial duplex

DNA (Table S2, structure 2) by the proteins was

monitored in the presence of ATP and Mg2+. Both

HiUvrD and HpUvrD were able to efficiently unwind

the substrate in a concentration-dependent manner

(Fig. 4A,B). No detectable unwinding of the DNA

substrate could be observed at even the highest con-

centration of the HiUvrDR288A and HpUvrDE206Q

(Fig. 4C,D). This suggests that these mutations render

UvrD catalytically inactive as a helicase and that

ATP-hydrolysis by the protein is closely associated

with helicase activity. HiUvrDDC48 exhibited helicase

activity although it was � 1.5-fold less than the

wild-type protein (Fig. 4C) suggesting that the extreme

C-terminus of HiUvrD is not involved in DNA

unwinding like E. coli UvrDDC40 [42]. However,

HpUvrDDC63 was able to unwind the partial duplex

with the same efficiency as the wild-type protein sug-

gesting that deletion of the C-terminus did not alter

the helicase activity of HpUvrD (Fig. 4D).

The helicase assay was carried out in the presence of

different nucleotides and metal ions to determine the

specific cofactor preference of HiUvrD and HpUvrD.

Helicase activity of both HiUvrD and HpUvrD

required the presence of metal cofactor because no

helicase activity was observed without it (Fig. S7A,B,

lane 3). The unwinding of the partial duplex by

HiUvrD was effectively supported by Mg2+ and

Mn2+ and to a lesser extent by Ca2+ (Fig. S7A).

HpUvrD exhibited maximum DNA unwinding activity

in the presence of Mg2+, to a lesser extent with Mn2+

and very weakly with Ca2+ (Fig. S7B). Other metal

ions like Cd2+, Zn2+, Co2+, Cu2+ and Ni2+ did not

support the helicase activity of HiUvrD and HpUvrD

(Fig. S7A,B). In the presence of Co2+, a partial

precipitation of the DNA substrate was observed that

remained in the well (Fig. S7A,B, lane 9), the cause for

which is unclear at present. A DNA unwinding assay

of HiUvrD and HpUvrD in the presence of different

concentrations of divalent metal ions revealed that

HiUvrD helicase activity was best supported by Mg2+,

followed by Mn2+ and very weakly by Ca2+

(Fig. S7C), whereas HpUvrD was most efficient in the

presence of Mg2+ (Fig. S7D). This is in concurrence

with the ATPase activities of HiUvrD and HpUvrD,

which are best supported by Mg2+ (Table 1), again

suggesting that the ATPase and helicase activities of

UvrD are correlated. Earlier, the helicase activity of

M. tuberculosis UvrD1 was reported to be supported

by Mg2+, Mn2+, and to a lesser extent by Co2+,

Ni2+ and Cu2+ [19].

To determine the NTP specificity of HiUvrD and

HpUvrD, helicase assays were carried out in the pres-

ence of different NTPs. The helicase activity of

HiUvrD and HpUvrD was strictly dependent on the

presence of nucleotides (Fig. 5A,B), consistent with

the previous findings that helicase-catalyzed unwinding

is an energy-dependent process [1]. ATP and dATP

supported the helicase activity of HiUvrD (Fig. 5A),

and with almost same efficiency as observed from

HiUvrD helicase activity in the presence of different

concentrations of these two nucleotides (data not

shown). Other NTPs, as well as ADP and nonhydroly-

sable ATP analogues like AMPPNP, did not support

the HiUvrD helicase activity (Fig. 5A). The inability

of GTP to support the DNA unwinding by HiUvrD is

consistent with the lack of GTP hydrolysis by the

protein (Table 1), suggesting that NTP hydrolysis is

closely associated with helicase activity. Intriguingly,

in stark contrast to both HiUvrD and E. coli UvrD

[46], GTP and dGTP supported DNA unwinding by

HpUvrD in addition to ATP and dATP (Fig. 5B) and

to almost the same levels, as determined by carrying

out helicase assays using increasing concentrations of

either ATP or GTP (data not shown). Like HiUvrD,



no significant DNA unwinding by HpUvrD could be

observed in the presence of CTP, dCTP, TTP, dTTP,

UTP, ADP and AMPPNP (Fig. 5B). Earlier, T. ther-

mophilus UvrD helicase activity was shown to be sup-

ported by ATP and dATP, and up to 40–50% by

GTP and dGTP whereas other nucleotides like CTP,

dCTP, UTP, dTTP supported the activity weakly or

not at all [47]. Interestingly, the nucleotide require-

ment of HpUvrD is similar to the homologous

E. coli Rep helicase, which hydrolyzes GTP and

dGTP with one-third efficiency compared with ATP

or dATP, whereas other nucleotides are hydrolyzed

weakly [48]. Bacillus stearothermophilus PcrA could

utilize all nucleotides except dTTP for unwinding

duplex [54]. Another highly conserved helicase in

Helicobacter pylori, the replicative helicase DnaB was

found to be equally supported by ATP, GTP or

UTP, whereas E. coli DnaB was inactive in the pres-

ence of UTP [49]. Therefore, Helicobacter pylori heli-

cases may have acquired a relaxed specificity for

nucleotides as an adaptation to better survival in the

host or under stress conditions.

This ability of Helicobacter pylori UvrD to utilize

GTP together with ATP might reflect a plasticity of its

nucleotide-binding site. Comparison of the HpUvrD

amino acid sequence with other GTP-binding proteins

like E. coli elongation factors EF-Tu and EF-G

(Fig. S8) showed the presence of a highly invariant

G-1 motif in HpUvrD [55]. However, HpUvrD lacks

the other classical motifs characteristic of GTPase

superfamily, i.e. G-2 to G-5 motifs (Fig. S8) of

which G-4 and G-5 regions bind the guanine ring

[55]. In the absence of the motifs involved in GTP

binding, it is possible that GTP binds within the

ATP-binding pocket of HpUvrD. To investigate

this, a helicase assay was performed in the pres-

ence of ATP together with different concentrations

of GMPPNP, which is a nonhydrolysable analogue

of GTP. In the presence of GMPPNP no unwind-

ing of the substrate was observed (Fig. 5C, lane 3),

whereas HpUvrD was able to unwind the substrate

in the presence of ATP (Fig. 5C, lane 4). In the

presence of a fixed concentration of ATP, with

increasing concentrations of GMPPNP, a progressive

Fig. 4. Helicase activity of HiUvrD, HpUvrD and their variants. (A) Radiolabeled splayed-duplex (1 nM) was incubated with different concen-

trations (0, 0.5, 1, 2, 5, 10, 20, 30, 40, 60, 80, 100 and 125 nM) of HiUvrD and its variants at 37 �C for 5 min. Displacement of radiolabeled

DNA strand was monitored by resolution of reaction mixture using 10% nondenaturing PAGE and representative PhosphoImager scans are

shown. ‘D’ represents reaction with heat-denatured HiUvrD. (B) Varying concentrations (0, 2, 5, 10, 20, 30, 40, 50, 60, 80, 100 and 150 nM)

of HpUvrD or its variants were incubated with splayed-duplex (1 nM) at 37 �C for 5 min. ‘D’ represents reaction with heat-denatured HpUvrD.

(C) Quantification of helicase activity of HiUvrD and its variants. The amount of radiolabeled ssDNA displaced by HiUvrD helicase activity

was quantified and the percentage of substrate unwound was calculated for each enzyme concentration. Data represent the average of at

least three independent experiments, and error bars are standard error about the mean. (D) Quantification of helicase activity of HpUvrD and

its variants. The fraction of unwound substrate was calculated and plotted against concentrations of HpUvrD or its mutants. Error bars repre-

sent standard error about the mean of at least three independent experiments.



decrease in DNA unwinding by HpUvrD was

observed (Fig. 5C, lanes 5–15). At a 10-fold higher con-

centration of GMPPNP compared with ATP, � 80%

inhibition of HpUvrD helicase activity was observed,

which suggests that GMPPNP could compete with

ATP for a common binding site within HpUvrD. In a

converse experiment, helicase assays were carried out in

the presence of a fixed concentration of GTP and

increasing concentrations of AMPPNP (Fig. 5D).

However, in this case, as little as 2 mM of AMPPNP

could inhibit the helicase activity of HpUvrD in the

presence of 3 mM GTP (Fig. 5D, lane 7). This clearly

suggests that although GTP can support HpUvrD heli-

case activity, the enzyme exhibits a strong preference

for ATP.

Helicase activity of HiUvrD and HpUvrD on

different DNA structures

In vivo a number of different DNA structures, gener-

ated as intermediates of different DNA metabolic

pathways, could function as substrates for UvrD heli-

case. Therefore, helicase assays were carried out using

fixed concentration (40 nM) of HiUvrD or HpUvrD

and different DNA structures (Table S2, structures

1–9; 1 nM each), which were used in the DNA-binding

studies. HiUvrD was able to unwind all the substrates

tested although with varying efficiencies (Fig. 6). The

order of unwinding of different substrates by HiUvrD

was: 3¢-flap ‡ 3¢-overhang > three-way junction >

splayed-duplex > four-way junction > nicked duplex

Fig. 5. Helicase activity of HiUvrD and HpUvrD in the presence of different NTPs. (A) Dependence of helicase activity of HiUvrD on different

NTPs. The splayed-duplex (1 nM) was incubated in the presence of 4 mM of MgCl2, 40 nM HiUvrD and 1 mM of different NTPs or dNTPs for

5 min at 37 �C. (B) Helicase activity of HpUvrD in the presence of different NTPs. Helicase assays were carried out using radiolabeled

splayed-duplex (1 nM), HpUvrD (40 nM) and standard reaction buffer in the presence of different NTPs or dNTPs (1 mM) at 37 �C for 10 min.

In both (A) and (B) heat-denatured substrate (lane 1), substrate without protein (lane 2) or reaction in the absence of any NTP (lane 3) were

also used as controls. (C,D) Inhibition of HpUvrD helicase activity in the presence of GMPPNP or AMPPNP. (C) Unwinding of radiolabeled

splayed-duplex (1 nM) was monitored using HpUvrD (40 nM) and ATP (3 mM) in standard reaction buffer and in the absence (lane 4) or pres-

ence of different concentrations (5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30 mM) of GMPPNP (lanes 5–15). DNA unwinding by

HpUvrD in the presence of GMPPNP (3 mM) and absence of ATP was also monitored (lane 3). (D) Helicase activity of HpUvrD (40 nM) was

monitored using partial duplex DNA (1 nM) in the presence of GTP (3 mM) and increasing concentrations (0, 0.5, 1, 2, 3, 4, 5 6, 7, 8, 9 and

10 mM) of AMPPNP (lanes 4–15). DNA unwinding by HpUvrD was also monitored in the presence of AMPPNP (3 mM) alone (lane 3). In both

(C) and (D), lane 1 represents heat-denatured substrate and lane 2 represents DNA substrate alone.



> looped duplex = dsDNA duplex >>> 5¢-over-hang. Substrates like 3¢-overhang, splayed-duplex and

3¢-flap, which had a free 3¢-ssDNA tail (Fig. 6A–C)

were unwound very rapidly and robustly. By contrast,

blunt-ended, nicked and looped DNA duplexes

(Fig. 6D–F) were unwound inefficiently, whereas

5¢-overhang (structure 9, Table S2 and Fig. 6G) was

unwound with the least efficiency, suggesting that

DNA duplex unwinding by HiUvrD requires the pres-

ence of 3¢-ssDNA tails. However, HiUvrD was able to

unwind nicked duplex (Fig. 6E) with efficiency slightly

better than blunt-ended duplex (Fig. 6D). In addition,

HiUvrD could efficiently unwind blunt-ended three-

and four-way junctions (Fig. 6H,I) suggesting its

ability to process replication and recombination inter-

mediates.

HpUvrD was active in unwinding partial DNA

duplexes with 3¢-ssDNA regions like 3¢-overhang(Table S2, structure 3 and Fig. S9A), splayed-duplex

(Table S2, structure 2 and Fig. S9B) and 3¢-flap(Table S2, structure 4 and Fig. S9C). However,

HpUvrD could unwind these DNA structures with

3¢-ssDNA regions at a relatively lower rate compared

with HiUvrD (Fig. 6A–C). This could be due to a

lower affinity of HpUvrD compared with HiUvrD for

ssDNA, as determined by SPR (Fig. 2A,B). Blunt-

ended duplex (Table S2, structure 1 and Fig. S9D),

duplexes with nick (structure 7, Table S2 and

Fig. S9E) and loop (structure 8, Table S2 and

Fig. S9F) were unwound poorly. In addition, no

significant unwinding of 5¢-overhang was observed

(structure 9, Table S2 and Fig. S9G) suggesting that

HpUvrD harbors a 3¢–5¢ helicase activity. Three-way

junction, which resembles a replication fork (structure

5, Table S2 and Fig. S9H), and four-way junction

resembling the recombination intermediate Holliday

junction (structure 6, Table S2 and Fig. S9I) were

unwound by HpUvrD but with less efficiency than

structures with free 3¢-ends. Moreover, HpUvrD could

unwind the three-way junction with a lower efficiency

than HiUvrD probably indicating a different mode of

binding and unwinding of this structure by the two

proteins. This is supported by EMSA of binding of

HiUvrD and HpUvrD to the three-way junction

(Figs 1F and S2F) wherein HiUvrD exhibits formation

of two discrete complexes, which is absent in the case

of HpUvrD.

From the DNA-unwinding studies using different

DNA structures it is clear that like E. coli and

M. tuberculosis UvrDs, both HiUvrD and HpUvrD

function as 3¢–5¢ helicases. Helicases belonging to SF1

require ssDNA with a specific orientation to load onto

DNA [1]. Furthermore, E. coli UvrD has been

reported to initiate unwinding from nicks and blunt

ends but at higher enzyme concentrations [56], whereas

M. tuberculosis UvrD1 could unwind nicked DNA

duplex with much higher efficiency than 3¢-tailedduplex [19]. The unwinding of nicked duplex DNA by

HiUvrD and HpUvrD corroborates their roles in mis-

match repair and nucleotide excision repair during

which UvrD initiates unwinding from nicks generated

by MutH and UvrC endonucleases, respectively, albeit

Helicobacter pylori lacks a functional mismatch repair.

In addition, a number of proteins can stimulate the

activity of UvrD on DNA substrates with strand dis-

continuities. The mismatch repair proteins MutS and

MutL stimulate the unwinding of blunt-ended and

nicked duplexes by UvrD in a mismatch-dependent

manner in the mismatch repair pathway [57]. However,

Helicobacter pylori lacks the homologues of MutS and

MutL proteins but does contain a functional nucleo-

tide excision repair pathway [24,29,58]. E. coli UvrA

and UvrB proteins have been reported to stimulate the

helicase activity of UvrD on a number of different dis-

continuous DNA substrates [59]. Therefore, in future,

it would be interesting to study the modulation of

HpUvrD helicase on different DNA structures by

homologous UvrA and UvrB proteins.

Interestingly, forked structures like 3¢-flap (Figs 6C

and S9C) and a blunt-ended three-way junction

(Figs 6H and S9H) were the best substrates for the

helicase activity of HiUvrD and HpUvrD together

with the standard 3¢-overhang (Figs 6A and S9A).

Another SF1 helicase and a homologue of Rep and

UvrD from Saccharomyces cerevisiae mitochondria,

Hmi1p was found to specifically unwind 3¢-flap struc-

tures [60]. 3¢-Flap resembles a replication fork having

a nascent lagging strand with blocked synthesis of the

leading strand, whereas the three-way junction can be

envisioned as a stalled replication fork with dsDNA

on both strands. These results reflect the ability of

HiUvrD and HpUvrD to process damaged or stalled

replication forks and specifically target DNA interme-

diates of replication and recombination in the absence

of other helicases like RecBCD and RecQ. For exam-

ple, during replication fork damage, the ability of

UvrD to specifically bind and unwind the lagging rep-

licating fork with blocked leading strand synthesis

enables it to process the lagging strand and ⁄or leading

strand that is essential for fork reversal [9]. In addi-

tion, ssDNA with a free 3¢-end in complex with RecA

is involved in strand invasion during homologous

recombination. The ability of HiUvrD and HpUvrD

to specifically unwind DNA with 3¢-ssDNA tails is

instrumental for their anti-recombination role by

unwinding DNA recombination intermediates as well



Fig. 6. Unwinding of different DNA structures by HiUvrD. Reactions were performed using different DNA structures (1 nM): 3¢-overhang (A),

splayed-duplex (B), 3¢-flap (C), DNA duplex (D), nicked DNA (E), looped DNA (F), 5¢-overhang (G), three-way junction (H) and four-way junction

(I) that were incubated with HiUvrD (40 nM). Reactions were sampled as a function of time (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7 and

8 min) and scored for oligonucleotide displacement.



as by disruption of the RecA–DNA nucleoprotein

complex [13]. In fact, the ability of HiUvrD and

HpUvrD to unwind blunt-ended four-way junction or

Holliday junction (Figs 6I and S9I) with better effi-

ciency than blunt-ended duplex or looped DNA

directly highlights the proficiency of these helicases in

mediating anti-recombination activity and resolving

stalled replication forks by disrupting intermediate

Holliday junction structures.

Because HiUvrD and HpUvrD showed strong pref-

erence for substrates with 3¢-ssDNA regions, it was of

interest to determine the effect of the length of

3¢-ssDNA tails on the activity of these helicases. For

both HiUvrD and HpUvrD, unwinding of the 3¢-over-hangs (structures 10-13, Table S2) progressively

reduced with the decrease in the length of the

3¢-poly(dT) tail (Fig. S10). In the case of HiUvrD,

almost 90% unwinding of the substrates with 20- and

12-nucleotide tails was observed within 5 min, whereas

� 60% and 20% unwinding was observed for 3¢-over-hangs with eight- and four-nucleotide tails, respectively

(Fig. S10A). With HpUvrD, > 90% substrate with a

3¢-ssDNA-tail of 20 nucleotides was unwound within

5 min, whereas substrates with 12-, 8- and 4-mer

ssDNA regions were unwound to � 70, 50 and 20%,

respectively (Fig. S10B). Hence, HiUvrD could effi-

ciently unwind DNA substrates with 3¢-ssDNA tails of

12 nucleotides or more. However, HpUvrD required a

substrate with 20 nucleotides of 3¢-ssDNA region,

whereas substrates with a 3¢-ssDNA overhang of 12

nucleotides or fewer were unwound relatively less effi-

ciently. Hence, the ssDNA-length dependence of

HiUvrD is similar to that of E. coli UvrD [61], but

that of HpUvrD differs from both E. coli UvrD and

HiUvrD. E. coli UvrD has previously been shown to

require ssDNA with more than eight nucleotides (mini-

mum tail length of 12 nucleotides) for efficient unwind-

ing [61]. However, the substrate requirement of

HpUvrD is similar to that of M. tuberculosis UvrD1

protein which could not unwind substrates with a tail

of 12 nucleotides or shorter and required at least 18

nucleotides [19].

Oligomeric nature of Haemophilus influenzae and

Helicobacter pylori UvrD

The assembly state of helicases can influence their dif-

ferent activities, which could be a regulatory mecha-

nism to control enzyme activities in vivo. Monomers of

several SF1 enzymes like E. coli Rep [62], UvrD [41]

and B. stearothermophilus PcrA [63] function as rapid

and processive ssDNA-specific translocases with

limited helicase activities in vitro. Cooperativity of

multiple monomers or their oligomerization can

greatly enhance helicase function [64,65]. By contrast,

M. tuberculosis UvrD1 was found to exist and function

as a monomer without any oligomeric species [19].

To determine the subunit structure of HiUvrD and

HpUvrD, the purified proteins were subjected to ana-

lytical gel-filtration analysis using a Superose 6 column

calibrated with proteins of known molecular masses.

Both HiUvrD and HpUvrD eluted predominantly as a

monomers corresponding to apparent molecular mass

of � 92 and 88 kDa, respectively (Fig. 7A,B), which is

close to their theoretical molecular masses (� 86 kDa

for HiUvrD and 80 kDa for HpUvrD). Noticeably,

elution peaks of both the proteins were consistently

broader and asymmetric with an initial shallow shoul-

der that constituted � 2–4% of the major peak

(Fig. 7A,B). This indicates that both UvrDs exist pre-

dominantly as monomers in solution together with the

presence of small amounts of higher oligomeric forms

corresponding to dimeric, trimeric and tetrameric spe-

cies, as estimated from elution profiles of standard

proteins (Fig. 7). Hence, UvrD has a tendency to exist

as both a monomer and oligomer in solution with the

equilibrium shifted towards the monomer. E. coli

UvrD has previously been reported to self-assemble

into dimers and tetramers in the absence of any cofac-

tors, and the presence of two active and perhaps inter-

acting monomers bound to DNA was found to be

important for DNA unwinding in vitro [61]. Monomers

of E. coli UvrD, however, rapidly, processively and in

an ATP-dependent manner, translocate along ssDNA

without significantly unwinding DNA duplexes [41].

HiUvrDR288A and HpUvrDE206Q showed same elu-

tion profiles as the wild-type proteins, suggesting that

the point mutations do not affect the oligomeric nature

of the proteins (Fig. 7A,B).

To determine the role of C-terminus of UvrD pro-

teins in their oligomerization, HiUvrDDC48 and

HpUvrDDC63 were subjected to gel-filtration analysis

under the same conditions as the wild-type proteins.

HiUvrDDC48 eluted as a single peak corresponding to

monomeric protein (� 80 kDa) lacking the oligomeric

species (Fig. 7A). This implicates the distal C-terminus

of HiUvrD in its oligomerization. However,

HpUvrDDC63 exhibited same elution profile as the

wild-type protein, i.e. it showed the presence of an ini-

tial shallow shoulder followed by the major mono-

meric peak corresponding to an apparent molecular

mass of � 75 kDa (Fig. 7B). This suggested that

unlike E. coli UvrD and HiUvrD, deletion of C-termi-

nus tail in HpUvrD failed to alter its oligomeric status.

To further substantiate this observation, chemical

cross-linking of HiUvrD, HiUvrDDC48, HpUvrD and



HpUvrDDC63 using glutaraldehyde was performed.

Glutaraldehyde forms amidine cross-links between

primary amines of lysine residues hence, covalently

cross-linking protein subunits. A slow migrating species

(� 180 kDa), corresponding to a dimer of HiUvrD, was

observed in the case of the wild-type protein (Fig. 8A,

lanes 3–5), but was absent in HiUvrDDC48 even at the

highest concentration of glutaraldehyde (Fig. 8A, lanes

7–9). By contrast, both glutaraldehyde-treated HpUvrD

and HpUvrDDC63 showed the presence of a slow

migrating species, which corresponded to dimers of both

proteins together with the presence of a monomer

(Fig. 8B, lanes 3–5 and 7–9). In either case, cross-linked

species were not observed in the absence of glutaralde-

hyde (Fig. 8B, lanes 2 and 6). These results clearly sug-

gest that deletion of distal C-terminal residues

abrogated the oligomerization of HiUvrD, but not of

HpUvrD. Interestingly, the lack of any effect of C-ter-

minus deletion on the oligomeric status of HpUvrD is in

contrast to E. coli UvrDDC40 wherein deletion of the

C-terminal 40 amino acid residues abrogated protein

oligomerization and shifted the equilibrium towards

the monomer [42]. However, deletion of HiUvrD

C-terminus showed same effect as E. coli UvrDDC40.This might be because of the high identity between

E. coli UvrD and HiUvrD, but limited identity of

HpUvrD to the two proteins. It can be envisaged that

regions of HpUvrD additional to its C-terminus may be

involved in oligomerization of the protein.

In summary, this study reports the characterization

of UvrD, an essential helicase in Haemophilus influen-

zae and Helicobacter pylori. HiUvrD and HpUvrD can

hydrolyze ATP in a ssDNA-dependent manner and

can unwind DNA with 3¢–5¢ polarity in the presence

of ATP. Interestingly, the helicase activity of HpUvrD

is supported by GTP, unlike UvrD helicases from

E. coli and Haemophilus influenzae. HiUvrD and

HpUvrD are able to bind and unwind a number of

DNA structures suggesting their roles in a number of

DNA-processing pathways in these pathogens. ATPase

Fig. 7. Oligomeric status of HiUvrD and HpUvrD proteins. (A) Elution profiles of HiUvrD, HiUvrDR288A and HiUvrDDC48 (10 lM each) from

Superose 6 gel-filtration column. (B) Wild-type and mutant HpUvrD proteins (10 lM) were subjected to gel-filtration chromatography to deter-

mine their oligomeric status. Elution profiles of HpUvrD, HpUvrDE206Q and HpUvrDDC63 are shown.



and helicase activities of HiUvrDR288A and HpUvr-

DE206Q were abrogated with no affect on ssDNA

binding by these proteins. However, HiUvrDR288A

exhibited a reduced affinity for ATP, which was not

affected in HpUvrDE206Q. Both UvrDs were found

to exist predominantly as monomers in solution,

together with small amounts of dimeric and tetrameric

species. Deletion of the C-terminal amino acid residues

of HpUvrD did not affect its multimerization suggest-

ing that additional regions of the protein were involved

in intermolecular interactions. This is in contrast to

UvrD proteins from E. coli and Haemophilus influenzae

whose C-terminal mediates their oligomerization.

Nevertheless, the studies presented here form the basis

for elucidating the cellular functions of UvrD in

Haemophilus influenzae and Helicobacter pylori, as well

as its importance in physiology of these organisms.

Materials and methods

Bacterial strains and plasmids

Genomic DNA of Helicobacter pylori strain 26695 (cagA+

iceA1 vacAs1am1) was obtained as a kind gift from New

England Biolabs (Ipswich, MA, USA). All plasmid DNAs

were propagated and prepared using E. coli strain DH5a[F’ end A1 hsd R17 (rk

) mk)) glnV44 thi1 recA1 gyrA

(NalR) relA1 D (lacIZYA – argF) U169 deoR {F80dlac D(lacZ) M15}] as host using previously described protocols

[66]. DNA constructs derived from pET-14b and pET-15b

(Novagen, Madison, WI, USA) were used for the overex-

pression of HiUvrD and HpUvrD, respectively. HiUvrD

and its variants were expressed in E. coli BL21(DE3)pLysS

[F) ompT hsdSB (rB) mB

)) gal dcm k(DE3) pLysS (CamR)]

cells by transformation with appropriate plasmid constructs

using a standard protocol [66]. HpUvrD and its mutants

were expressed in E. coli Rosetta(DE3)pLysS [F) ompT

hsdSB(rB) mB

)) gal dcm k(DE3) [lacI lacUV5-T7 gene 1

ind1 sam7 nin5]) pLysSRARE (CamR)].

Chemicals

Restriction endonucleases, T4 DNA ligase, T4 polynucleo-

tide kinase and T4 gene 32 protein were obtained from

New England Biolabs. Ampicillin, Coomassie Brilliant Blue

R-250, proteinase K, Hepes, Heparin–Sepharose, Q-Sepha-

rose, protease inhibitor cocktail, phenylmethanesulfonyl fluo-

ride and isopropyl thio-b-D-galactoside were procured from

Sigma Chemical Company (St Loius, MO, USA). Ni2+–

NTA agarose was obtained from Novagen. [c-32P] ATP

(3500 Ci.mmol)1) and [a-32P] ATP (3000 Ci.mmol)1) were

obtained from BRIT (Hyderabad, India). All other reagents

used were of analytical or ultrapure grade.

Oligonucleotides and radiolabeling

The oligonucleotides used in this study (Table S1) were syn-

thesized by Sigma Genosys (Bengaluru, India). Oligonucle-

otides were labeled at the 5¢-end with [c-32P]ATP (30 lCi)using T4 polynucleotide kinase and purified by Qiagen

nucleotide removal kit (Qiagen GmbH, Hilden, Germany).

Amplification and cloning of Helicobacter

pylori uvrD

The Helicobacter pylori uvrD gene (2049 bp) was

amplified using Helicobacter pylori strain 26695 genomic

DNA as template by PCR with Phusion DNA

Fig. 8. Glutaraldehyde cross-linking of HiUvrD or HiUvrDDC48, and

HpUvrD or HpUvrDDC63. (A) Chemical cross-linking of HiUvrD and

HiUvrDDC48. Wild-type or truncated HiUvrD proteins were treated

with increasing concentrations of glutaraldehyde (0, 0.01, 0.025 and

0.05%) and products analyzed as described in Materials and meth-

ods. Lane 1, protein molecular mass marker; lanes 2–5, HiUvrD

(2.5 lM); lanes 6–9, HiUvrDDC48 (5 lM). (B) HpUvrD or

HpUvrDDC63 (5 lM) were incubated with different concentrations

of glutaraldehyde (0, 0.01, 0.025 and 0.05%) and the reactions

were analyzed using 0.1% SDS ⁄ 6% PAGE. Lane 1, protein molecu-

lar mass marker; lanes 2–5, HpUvrD; lanes 6–9, HpUvrDDC63.



polymerase (Finnzymes Oy, Espoo, Finland). Appropriate

restriction enzyme sites were introduced [a XhoI site in

the forward primer HpuvrDF (Table S1) and a BamHI

site in the reverse primer HpuvrDR (Table S1)]. The

amplified PCR fragment was ligated into the bacterial

expression vector pET-15b under the control of induc-

ible T7 promoter such that (His)6-tag was introduced

at the N-terminus of the HpUvrD protein. pET-15b-

hpuvrD gene was confirmed by restriction digestion and

sequencing.

Overexpression and purification of

Haemophilus influenzae Rd and

Helicobacter pylori UvrD

For expression of HpUvrD, E. coli Rosetta(DE3)pLysS

cells harboring the pET-15b-hpuvrD construct were grown

at 37 �C in Luria–Bertani broth containing 100 lgÆmL)1

ampicillin and 35 lgÆmL)1 chloramphenicol to A600 � 0.6.

The culture was chilled on ice for 45 min. Expression of

(His)6–HpUvrD was induced by addition of 1 mM isopro-

pyl thio-b-D-galactoside and bacterial cells were further

incubated at 18 �C for 10 h to enhance the solubility of the

protein following which they were harvested by centrifuga-

tion at 5000 g for 10 min. Expression of HpUvrD was

checked by resuspension and lysis of bacterial cell pellets in

sample loading buffer (125 mM Tris ⁄HCl pH 6.8, 4% SDS,

10% v ⁄ v glycerol, 0.06% bromophenol blue and 25 mM

b-mercaptoethanol), and subjecting them to 0.1%

SDS ⁄ 10% PAGE [66] followed by staining with Coomassie

Brilliant Blue R-250.

For purification of HpUvrD, bacterial cells were resus-

pended and lysed by sonication in buffer A (25 mM

Tris ⁄HCl buffer pH 7.9 containing 500 mM NaCl, 2 mM

imidazole, 13% v ⁄ v glycerol, 2 mM b-mercaptoethanol and

1 mM phenylmethanesulfonyl fluoride). Cell lysate was frac-

tionated through Ni2+-NTA agarose column and the pro-

tein, thus obtained was loaded on to Heparin–Sepharose

column. Prior to loading on to the Heparin–Sepharose col-

umn, HpUvrD was freshly diluted using 20 mM Tris buffer

pH 8 containing 5 mM b-mercaptoethanol and 10% v ⁄ vglycerol (buffer B) to a final NaCl concentration of 100 mM

because dialysis of HpUvrD in low salt buffer led to its

precipitation. After washing with buffer B containing

100 mM NaCl, HpUvrD were eluted using a linear gradient

of 100–800 mM NaCl. Fractions containing HpUvrD were

pooled and again freshly diluted in buffer B to a final NaCl

concentration of 50 mM prior to loading on to Q-Sepharose

column. The column was washed with buffer B containing

50 mM NaCl and the protein was eluted with a linear gradi-

ent of 50–500 mM NaCl. Fractions containing pure

HpUvrD were pooled, dialyzed in 20 mM Tris buffer pH 8

containing 500 mM NaCl, 10 mM b-mercaptoethanol and

50% (v ⁄ v) glycerol, and stored at )70 �C. HpUvrD concen-

trations were estimated using molar extinction coefficient

38 280 M)1Æcm)1.

HiUvrD was overexpressed and purified using the same

protocol as HpUvrD (Sharma and Rao, unpublished).

MALDI-MS analysis of HpUvrD

Peptide mass fingerprint analysis of trypsin-treated

HpUvrD was carried out as described earlier [67].

Site-directed mutagenesis of HiUvrD and HpUvrD

Site-directed mutagenesis was performed to replace highly

conserved glutamate 226 and arginine 288 in HiUvrD, and

glutamate 206 in HpUvrD, as described earlier [68]. A pair

of sense (HiuvrDE226QF, HiuvrDR288AF and Hpuvr-

DE206QF; Table S1) and antisense (HiuvrDE226QR,

HiuvrDR288AR and HpuvrDE206QR; Table S1) primers,

Phusion polymerase and pET-14b–hiuvrD or pET-15b–

hpuvrD as templates were used in the PCR. The mutagenic

primers were designed to introduce an appropriate restric-

tion enzyme recognition sequence at the site of mutation.

The mutations were confirmed by DNA sequencing.

Mutant proteins were expressed and purified as described

for the wild-type proteins.

The pET-14b–hiuvrD and pET-15b–hpuvrD constructs

were used as templates for the generation of the

HiUvrDDC48 and HpUvrDDC63, respectively, by site-

directed mutagenesis [68]. A stop codon was introduced

in the ORF of hiuvrD and hpuvrD such that the encoded

protein would lack the distal C-terminal 48 amino acid

residues in HiUvrD and 63 amino acids in HpUvrD.

PCR was carried out using specific primers (HiuvrDDCFand HiuvrDDCR or HpuvrDDCF and HpuvrDDCR;

Table S1) that were designed based on multiple sequence

alignment of UvrD proteins such that regions external to

the conserved SF1 motifs were deleted. A restriction site

(PsiI) was introduced at the site of mutagenesis to score

for mutants. The constructs containing the gene encoding

truncated HiUvrD and HpUvrD were confirmed by

sequencing.

ATPase assay

ATP hydrolysis by HiUvrD and HpUvrD was assessed in a

10 lL reaction mixture containing 25 mM Tris ⁄HCl pH 8,

10 mM MgCl2, 20 mM NaCl, 1 mM dithiothreitol, 4 mM

ATP and [c-32P]ATP as a tracer. When present 2 lM of cir-

cular double-stranded or circular single-stranded M13mp18

DNA was added to the reactions. Results were visualized

using PhosphorImager (Fuji FLA-9000) and the amount of

Pi formed after ATP hydrolysis was quantified densitomet-

rically using IMAGEGAUGE V2.54 software (Fujifilm, Tokyo,

Japan). The Km and kcat values were determined by



nonlinear regression analysis of the plot of rate of reaction

versus the ATP concentration using GRAPHPAD PRISM 5 soft-

ware (GraphPad Software Inc., San Diego, CA, USA).

Unless otherwise indicated, the enzyme activity data were

plotted as the mean of at least triplicate determinations,

with error bars representing standard error about the mean.

ATP-binding assay by UV-mediated cross-linking

Different concentrations of HiUvrD, HiUvrDR288A,

HpUvrD or HpUvrDE206Q were incubated with 10 lCi of[a-32P]ATP in 10 mM Tris buffer, pH 7.5, containing

50 mM NaCl, 1 mM EDTA, 5% (v ⁄ v) glycerol and 0.1 mM

dithiothreitol, in a final volume of 20 lL. After incubation

on ice for 10 min, the reaction mixtures were subjected to

UV (254 nm) irradiation at a dose of 5.4 JÆcm)2 and a dis-

tance of 5 cm for 30 min on ice. Following this, 20 lg of

BSA and 5 mM of ATP were added to the reaction, and

proteins were precipitated with trichloroacetic acid and

washed three times with chilled acetone. The protein pellet

was resuspended in SDS sample buffer, fractionated by

0.1% SDS ⁄ 10% PAGE, and visualized by Coomassie stain-

ing followed by PhosphorImager (Fuji FLA-9000).

Helicase assay

For helicase assays, different radiolabeled DNA structures

(Table S2) were generated by annealing stoichiometric con-

centrations of different oligonucleotides (Table S1) as

described earlier [69].

The helicase activity of HiUvrD, HpUvrD and their vari-

ants was initially assayed using 1 nM radiolabeled splayed-

duplex (structure 2, Table S2) that was incubated in 25 mM

Tris ⁄HCl pH 8, containing 20 mM NaCl, 4 mM MgCl2,

3 mM ATP, 40 lgÆmL)1 BSA and 1 mM dithiothreitol.

Reactions were initiated by addition of different concentra-

tions of UvrD proteins. The reactions were incubated at

37 �C for 5 min and stopped by the addition of 25 mM

EDTA, 20-fold excess of unlabeled ODN1, 0.5% SDS and

proteinase K (0.02 lgÆmL)1) followed by further incubation

at 37 �C for 15 min. The excess of cold oligonucleotide was

added in the stop buffer to avoid reannealing of the

unwound complementary strands. The samples were elec-

trophoresed through 10% polyacrylamide gel containing

45 mM Tris–borate ⁄ 1 mM EDTA buffer (pH 8.3) at 100 V

for 5 h. The gels were visualized by PhosphorImager (Fuji

FLA-9000), and the percentage of unwinding of substrate

was determined from the densitometric quantification of

amount of remaining DNA substrates as well as displaced

radiolabeled DNA product using IMAGEGAUGE v. 2.54 soft-

ware. Unwinding percentage was calculated from the ratio

of amount of ssDNA product formed by the sum of

amount of substrate remaining and amount of product

formed. Unless otherwise indicated, the enzyme activity

data were plotted in GraphPad PRISM5 as the mean of at

least triplicate determinations, with error bars representing

standard error about the mean.

For helicase assays with different DNA structures, the

radiolabeled DNA substrates (1 nM) were incubated in the

reaction buffer and reaction were initiated by addition of

HiUvrD or HpUvrD (40 nM each). The reactions were

incubated at 30 �C and equal aliquots (20 ll) were with-

drawn at different time points. Reactions were stopped and

processed as mentioned above. These DNA structures were

generated by annealing oligonucleotides containing the

same core sequence represented by ODN1 (or ODN4 in

case of structures 10-13), such that any differences in effi-

ciency of unwinding due to sequence variation between sub-

strates could be eliminated.

EMSAs

EMSAs for the interaction of HiUvrD and HpUvrD with

different DNA structures (1 nM each) were performed in

10 lL reactions containing 25 mM Hepes (pH 8.0), 20 mM

NaCl and 0.1 mM EDTA. Different radiolabeled DNA

structures (1 nM; Table S2) were incubated with increasing

concentrations of HiUvrD or HpUvrD on ice for 30 min.

The reaction mixtures were electrophoresed through 10%

nondenaturing PAGE in 25 mM Tris ⁄HCl buffer (pH 8.0)

containing 200 mM glycine at 4 �C. The gels were visualized

using Fuji FLA-9000 PhosphorImager and quantified using

IMAGEGAUGE v. 2.54 densitometry software.

CD spectral analysis

CD measurements were recorded on a Jasco J-810 spectro-

polarimeter between 200 and 300 nm in a 2-mm path-length

quartz cuvette. All experiments were performed at 25 �C in

10 mM potassium phosphate buffer (pH 8.0). We incubated

1 lM of the HiUvrD, HpUvrD or their mutants for 5 min

in a final volume of 400 lL prior to recording of the spec-

trum. Each experimental spectrum represents the average of

at least 10 determinations.

Gel-filtration analysis

Native molecular masses of wild-type and variant

HiUvrD or HpUvrD were determined by gel-filtration

chromatography using a Superose 6 HR 10 ⁄ 30 column

(GE Healthcare Lifescience, Uppsala, Sweden) connected

with the AKTA basic 10 liquid chromatography system

(GE Healthcare). Chromatography was carried out in

25 mM Tris buffer pH 8 containing 300 mM NaCl, 5 mM

b-mercaptoethanol and 10% (v ⁄ v) glycerol at a flow

rate of 0.3 mLÆmin)1. The void volume (Vo) was

determined using Blue-dextran, and the column was

equilibrated using standard molecular mass markers

(Bio-Rad, Hercules, CA, USA): thyroglobulin (670 kDa),



c-globulin (158 kDa), ovalbumin (44 kDa), myoglobulin

(17 kDa) and vitamin B12 (1.35 kDa). The elution volume

(Ve) for each protein was determined by monitoring

absorption at 280 nm. The molecular masses of wild-type

and variant HiUvrD or HpUvrD were calculated

from the standard curve obtained by plotting molecular

mass of standards (in logarithmic scale) against the

elution volumes.

Chemical cross-linking

The oligomeric nature of HiUvrD, HiUvrDDC48, HpUvrD

and HpUvrDDC63 was determined by chemical cross-link-

ing of these proteins with glutaraldehyde. Each of the pro-

teins was incubated on ice for 5 min with different

concentrations of glutaraldehyde (0.01–0.05%). The reac-

tions were stopped by adding SDS sample buffer and were

fractionated on 0.1% SDS ⁄ 6% PAGE. The protein bands

were visualized by silver staining.

SPR studies

The binding kinetics of HiUvrD and HpUvrD as well as

their mutants with DNA were determined by SPR spectros-

copy using the BIAcore3000 optical biosensor (GE Health-

care) as described earlier [69]. Briefly, 5¢-end-biotinylated60-mer ssDNA (SPR1; Table S1) or dsDNA (obtained by

annealing SPR1 and SPR2; Table S1) were immobilized

on the surface of a streptavidin-coated sensor chip (GE

Healthcare). Different concentrations of HiUvrD or

HpUvrD in the standard buffer containing 10 mM Hepes

buffer (pH 7.4) 150 mM NaCl, 1 mM EDTA and 0.05%

surfactant P-20 were injected onto the DNA surfaces for

120 s at a flow rate of 20 lLÆmin)1 at 25 �C, followed by

a dissociation period of 120 s. The proteins were simulta-

neously injected on one of the four surfaces lacking the

biotinylated oligonucleotide that was used as reference

channel to determine background nonspecific binding and

the bulk concentration of the proteins. The surfaces were

regenerated by using 0.1% SDS. Each experiment was

repeated at least three times to ensure reproducibility of

results. The affinity and kinetic parameters were deter-

mined by analysis of association and dissociation phases

using BIAEVALUATION software v. 3.0 using a 1:1 Langmuir

binding model.

Acknowledgements

Sreelatha is acknowledged for assistance in the SPR

experiments. Arathi is thanked for providing technical

assistance. R. Srinivasan is acknowledged for help dur-

ing initial stages of this work. DNR acknowledges the

JC Bose fellowship and a grant from DST, Govern-

ment of India, which supported this work. RS is a reci-

pient of Senior Research Fellowship from the

University Grants Commission.

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Supporting information

The following supplementary material is available:

Fig. S1. (A) Overexpression and purification of

HpUvrD. (B) MALDI-TOF MS analysis of tryptic-

digest of HpUvrD.

Fig. S2. DNA-binding properties of HpUvrD. Electro-

phoretic mobility shift assays for binding of HpUvrD

with different DNA structures.

Fig. S3. ATPase activity of HiUvrD and HpUvrD as a

function of enzyme concentration.

Fig. S4. Characterization of HiUvrDR288A and

HpUvrDE206Q.

Fig. S5. Interaction of HiUvrDR288A and HpUvr-

DE206Q with ssDNA and ATP.

Fig. S6. Purification of HiUvrDDC48 and HpUvrD-

DC63, and their interaction with ssDNA.

Fig. S7. Dependence of helicase activity of HiUvrD

and HpUvrD on metal ion cofactors.



Fig. S8. Multiple sequence alignment of HpUvrD with

E. coli EF-Tu (EcEFTu) and EF-G (EcEFG).

Fig. S9. Helicase activity of HpUvrD on different

DNA structures.

Fig. S10. Effect of length of the 3¢-ssDNA tails on Hi-

UvrD and HpUvrD helicase activity.

Table S1. Sequences of the oligonucleotides (ODNs)

used in this study.

Table S2. DNA structures used in this study.

This supplementary material can be found in the

online version of this article.

Please note: As a service to our authors and readers,

this journal provides supporting information supplied

by the authors. Such materials are peer-reviewed and

may be reorganized for online delivery, but are not

copy-edited or typeset. Technical support issues arising

from supporting information (other than missing files)

should be addressed to the authors.



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Functional characterization of UvrD helicases from Haemophilus influenzae and Helicobacter pylori

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