Eur. J. Biochem. 271, 1849–1863 (2004)  FEBS 2004

doi:10.1111/j.1432-1033.2004.04094.x

REVIEW ARTICLE

Unraveling DNA helicases
Motif, structure, mechanism and function
Narendra Tuteja and Renu Tuteja
International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India

DNA helicases are molecular motor enzymes that use theenergy of NTP hydrolysis to separate transiently energetically stable duplex DNA into single strands. They are therefore essential in nearly all DNA metabolic transactions.
They act as essential molecular tools for the cellularmachinery. Since the discovery of the ﬁrst DNA helicase in
Escherichia coli in 1976, several have been isolated from bothprokaryotic and eukaryotic systems. DNA helicases generally bind to ssDNA or ssDNA/dsDNA junctions andtranslocate mainly unidirectionally along the bound strand
and disrupt the hydrogen bonds between the duplexes. Most
helicases contain conserved motifs which act as an engine

to drive DNA unwinding. Crystal structures have revealed
an underlying common structural fold for their function.
These structures suggest the role of the helicase motifs incatalytic function and oﬀer clues as to how these proteins cantranslocate and unwind DNA. The genes containing helicase
motifs may have evolved from a common ancestor. In thisreview we cover the conserved motifs, structural information, mechanism of DNA unwinding and translocation, andfunctional aspects of DNA helicases.

DNA helicases are motor proteins that can transiently
catalyze the unwinding of energetically stable duplex DNA
molecules using NTP hydrolysis as the source of energy[1,2]. They are important enzymatic tools for the cellular
DNA machinery. They are known to play essential roles
in nearly all aspects of nucleic acid metabolism, such as
DNA replication, repair, recombination, and transcription.
All helicases share at least three common biochemical
properties: (a) nucleic acid binding; (b) NTP/dNTP binding and hydrolysis; (c) NTP/dNTP hydrolysis-dependent
unwinding of duplex nucleic acids in the 3¢ to 5¢ or 5¢ to 3¢
direction [3]. Therefore, all DNA helicases described to datealso have intrinsic DNA-dependent NTPase activity [4,5].
These enzymes usually act in concert with other enzymes
or proteins in DNA metabolic activity. Multiple DNA
helicases have been isolated from single-cell types because ofdifferent structural requirements of the substrate at various
stages of the DNA transaction. For example, at least 14
different DNA helicases have been isolated from a simple
single-cell organism such as Escherichia coli, six frombacteriophages, 12 from viruses, 15 from yeast, eight fromplants, 11 from calf thymus, and as many as 24 from humancells. These have been described in the preceding review.
Most helicases from many different organisms contain
about nine short conserved amino-acid sequence ﬁngerprints (designated Q, I, Ia, Ib, II, III, IV, V and VI), calledhelicase motifs [6–10]. These motifs are usually clusteredin a region of 200–700 amino acids called the core region.
Because of the sequence of motif II (DEAD or DEAH or
DEXH), the helicase family is also called the DEAD-box
protein family. The crystal structures of some of the DNA
helicases are now available and have revealed a theme andvariations at the structural level [11]. Despite the ubiquitouspresence of DNA helicases, their biological roles have not
all been investigated. In the preceding review, we covered
history, biochemical assay and properties, and prokaryotic
and eukaryotic DNA helicases including Mcm proteins and
RecQ family of helicases. This review focuses on the helicase
motifs, crystal structures, mechanism of DNA unwinding
and translocation, and various functions of the DNA
helicases.

Keywords: crystal structure; DEAD-box protein; DNA
helicase; helicase motifs; unwinding enzyme.

Helicase motifs
Correspondence to N. Tuteja, International Centre for Genetic
Engineering and Biotechnology, Aruna Asaf Ali Marg,New Delhi 110 067, India. Fax: + 91 011 26162316,
Tel.: + 91 011 26195007, E-mail: narendra@icgeb.res.in
Abbreviations: eIF-4A, translation initiation factor 4A; ERCC,
excision repair cross complementing; NER, nucleotide excision repair;
HSV, herpes simplex virus; SF, super family; SSBP, single-stranded
DNA-binding protein; SV40, simian virus 40; TFIIH, transcription
factor IIH; XP, Xeroderma pigmentosum.
(Received 23 January 2004, revised 9 March 2004,
accepted 17 March 2004)

A computer-assisted amino-acid sequence analysis of helicases from many different organisms has revealed seven tonine short, conserved amino-acid sequence ﬁngerprints or
motifs, called helicase signature motifs [6,12]. With this
discovery, the helicases were classiﬁed into three superfamilies (SF), SF1, SF2 and SF3, based on the extent ofsimilarity and organization of these conserved motifs. Thissuggests that the genes containing helicase motifs evolved
from a common ancestor. These motifs can be used
efﬁciently for the detection and prediction of new helicasesin the genome databases. SF1 and SF2 are related, are thelargest superfamilies, and contain at least seven conserved FEBS 2004

1850 N. Tuteja and R. Tuteja (Eur. J. Biochem. 271)

motifs (I, Ia, II–VI), and SF3 has just three motifs (A, B andC) [3]. Motif III differs between SF1 and SF2. In general,SF1 members (Rep, UvrD and PcrA) are considered to be
ssDNA translocases, whereas SF2 members (RecG, PriA
and HSV NS3) are dsDNA translocases. This property may
be the distinction between the SF1 and SF2 family. Anothergroup called family 4 (F4) contains ﬁve motifs (1, 1a, 2–4)
[3]. The seven motifs of SF1 and SF2 are usually clustered ina region of 200–700 amino acids, the core region. These
conserved motifs are separated by stretches of low sequence
but high length conservation. In contrast, the N-terminal
and C-terminal regions of helicases are characterized bya high degree of sequence and length variability. It has beensuggested that the divergent regions are responsible forindividual protein functions, whereas the highly conserved
domains are involved in ATP binding and hydrolysis orbinding and unwinding of nucleic acids.
X-ray crystallographic studies have suggested that theconserved helicase motifs are closely associated in thetertiary structure of the protein and that they may form alarge functional domain [3]. Recently, a new motif, the Q
motif, was discovered [10]. Figure 1 shows the conserved
sequence of all nine motifs (Q, I, Ia, Ib, II, III, IV, V and VI)of the DEAD-box helicase family. The biochemical functions of only some of them (Q, I, Ia, II, III, and VI) havebeen elucidated and are described below.
Q motif (Gly-Phe-X-X-Pro-X-Pro-Ile-Gln)
This is a newly discovered motif, which is unique to andcharacteristic of the DEAD-box family of helicases [10]. It
consists of a 9-amino-acid sequence containing an invariant
glutamine (Q), giving it its name. It is generally present in arange of 15–22 amino acids upstream from motif I. In yeast,
this motif has been shown to be essential for viability andsite-speciﬁc mutagenesis studies and it is involved in ATPbinding and hydrolysis in the yeast eIF4A [10]. It has been
hypothesized that it is also involved in adenine recognition.
Motif I (Ala/Gly-X-X-Gly-X-Glu-Lys-Thr)
It has been described as the A motif of ATPase (Walker
motif A). AXXGXGKT is the consensus sequence for
RNA helicase and it forms the closely related SF1, whereas
GXXXXGKT, which belongs to SF2 is the consensus

sequence for other ATPases, GTPases, and a few DNA
helicases [7]. In the consensus sequence, the X could be any
amino acid. It was shown that the lysine (K) of GKT binds
to b and c phosphates of the ATP molecule, and themutation of K to the uncharged amino acid asparagine
abolished the binding of ATP [13]. The side chain of theinvariant K of motif I occupies the position that will beoccupied by the bound Mg2+ ion when the NTP–Mg2+
complex binds the enzyme. On binding NTP–Mg2+, thelysine side chain contacts the b phosphate of the bound
NTP and may act to stabilize the transition state duringcatalysis. An ATPase-deﬁcient mutant also lacked helicase
activity, demonstrating that ATP hydrolysis was required
for the unwinding of duplex nucleic acid [3].
Motif Ia (Pro-Thr-Arg-Glu-Leu-Ala)
Structural information on SF1 and SF2 helicases indicatethat motif Ia is involved in ssDNA binding. In a recent
study, speciﬁc mutations were designed in motif Ia of UL9
of HSV-1, and these mutants were analyzed genetically andbiochemically [14]. Mutant proteins with residues predicted
to be involved in ssDNA binding (R112A and R113A/
F115A) exhibited wild-type levels of intrinsic ATPase
activity and moderate to severe defects in ssDNA-stimulated ATPase activity and ssDNA binding. All the mutant
proteins lacked helicase activity but were able to dimerize
and bind the HSV-1 origin of replication as well as wild-type
UL9. These results indicate that residues from the Ia motif
contribute to the ssDNA-binding and helicase activities of
UL9 and are essential for viral growth [14].
Motif II (Val-Leu-Asp-Glu-Ala-Asp)
This motif represents a speciﬁc form of the ATPase B(Walker B) motif. The proteins containing this motif arealso called DEAD-box proteins [15]. The DE of the DEAD
sequence is highly conserved and is also present in many
proteins, which play a role in DNA and RNA replication[7]. The residue D of DE has been shown to interact with
Mg2+, which is required for ATP binding. A mutation in Dof DE also affects the ATPase and helicase activities. The
ATPase motif seems to be responsible for coupling of
ATPase and helicase activity [13]. The eukaryotic initiation
factor 4A (elF-4A) is the prototype and the best biochem-

Fig. 1. Schematic diagram representing organization and involvement in the catalytic functions of the best conserved motifs of the DEAD-box helicases.
Open boxes represent the conserved helicase motifs, and the consensus amino-acid sequence of each motif is shown by the single-letter code inside
the boxes (c ¼ D, E, H, K, R; o ¼ S, T; x ¼ any amino acid). Labels above the open boxes (Q, I, Ia, etc.) are the names assigned to the motifs. The
relative positions of the motifs and spacing between motifs are arbitrary. The numbers between the motifs and above the arrows are the typicalrange of amino-acid residues. The deﬁned functions for some of the motifs (Q, I, Ia, II, III and VI) are also given.

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Structure and function of DNA helicases (Eur. J. Biochem. 271) 1851

Fig. 2. Organization and variations in the conserved helicase motifs of DEAD-box proteins of SF2. The DEAD, DEAH, and DEXH box proteinsmainly belong to the family of RNA helicases, but some DNA helicases also contain these motifs. The proteins of the DEAH* subgroup show thegreatest variations in the conserved motifs and are characterized by DNA helicase activity. Numbers above the arrow indicates the range of
distances between the conserved domains and the distances of the two ﬂanking domains of the N-terminus and C-terminus of the diﬀerent proteins.
The Q motif is not shown here.

ically characterized member of this family. With thegrowing number of identiﬁed helicases, some variations inmotif II have been observed, and these are arranged indifferent subgroups of SF2 such as DEAD, DEAH, DEXH
and DEAH* box proteins (Fig. 2). The proteins of the
DEAH* subgroup show the strongest variations in theconserved regions and are characterized by a DNA helicase
activity.
Motif III (Ser-Ala-Thr)This motif of the SF2 family plays an important role inunwinding. In UL5 of HSV-I, a motif III mutant exhibitedapparent uncoupling of ATPase and DNA helicase activities as the mutant enzyme retained ATPase activity butlacked signiﬁcant helicase activity [16]. Mutation in thisdomain (SAT to AAA) leads to loss of RNA helicase
activity in eIF-4A while its ATP binding and hydrolysis as
well as RNA binding activities remained intact [13]. This
result suggests that motif III of SF2 is involved in the
coupling of ATPase to unwinding.
Motif VI (Tyr-Ile-His-Arg-Ile-Gly-Arg)
Several helicases exhibited nucleic acid-binding defects whenthe motif VI residues were altered [13]. Point mutation in
E. coli UvrD DNA helicase (SF1 family) has a negative
effect on a variety of activities including ssDNA binding,
ATP hydrolysis, and ligand-induced conformational changes. An apparent uncoupling of ATPase and helicase
activities was observed with a motif VI mutant of HSV1UL5. Changing the basic residues to H, or R to theuncharged glutamine abolishes RNA binding and reduces
ATP hydrolysis, which also results in reduced helicase
activity. All these suggest that motif VI, which mediates
conformational changes associated with nucleotide bindingby virtue of its close proximity to both the NTP-binding site
and the DNA-binding site is required for the helicase to
move along the DNA substrate.
Motifs I and IV of Rep and PcrA make direct contact
with the nucleotide in the enzyme–ADP binary complexes.
The residues in motifs Ia, III and V of Rep protein makedirect contacts with the bound oligonucleotide [3]. Motifs Ia
and V interact primarily with the sugar–phosphate backbone, whereas motif III is involved in hydrogen-bond andstacking interactions with the DNA bases. Mutation ofglycine in motif V of UL5 is reported to decrease the afﬁnityof the enzyme for ssDNA as well as the rate of hydrolysis[3]. Most of the mutational studies have been performed in
the three members of SF1, E. coli UvrD, HSV-1 UL5, andyeast UpfIP. The UvrD is closely related to the Rep and
PcrA helicases [3]. The presence of the conserved helicase
motifs in a protein is not sufﬁcient for its identiﬁcation asa helicase. For example, despite the presence of these
conserved helicase motifs in the E. coli Mfd protein [17]and the human Rad54 protein [18], these proteins do notcontain helicase activity. Direct biochemical demonstrationof ATP-dependent helicase activity of these proteins isessential before they can be classiﬁed as bonaﬁde helicases.
Only a few DEAD-box proteins have been shown biochemically to contain unwinding activity. For example, pea
DNA helicase 45 [19], yeast Rad3 [20], Dna2 [21], E. coli
RecQ [22], human REQL helicase [23], Plasmodium cynomolgi eIF-4A [24] and ERCC3 [25,26] are DNA helicases(Fig. 3), and human p68, mouse elF-4A, Xenopus An3 andXeroderma pigmentosum (XP) 54, Drosophila VASA, PRH
75, Arabidopsis thaliana DRHl and AtRHI are RNA
helicases [20,27,28] (Fig. 3).

Crystal structure
Because of the presence of conserved motifs, it was
originally proposed that helicases might generally beorganized into a modular structure consisting of a minimumof DNA-binding and NTP-binding domains. The ﬁrst
DNA helicase crystallized was PcrA from a thermophilic
bacterium Bacillus stearothermophilus [29]. X-ray structuresof PcrA, crystallized with or without ADP, were very
similar. In both cases, the enzyme crystallizes as a monomer
which consists of two parallel domains (1 and 2) with a deep
cleft running between them [30]. Each domain contains two
additional subdomains (A and B). Subdomain 1A (which
carries the Walker A and B motifs) and subdomain 2A
structurally resemble the central region of RecA, containing

1852 N. Tuteja and R. Tuteja (Eur. J. Biochem. 271)

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Fig. 3. Alignment of conserved helicase motifs of DNA and RNA helicases of the DEAD/H-box family. The names of the motifs are marked. The
amino acids are shown by single-letter code and the letters in bold indicate highly conserved motifs. The accession numbers of aligned sequences of
DNA helicases are: Pea (PDH45, Y17186), Rad3 (K03293), Rad15 (X6499), ERCC2 (X52470), ERCC3 (M31899) and RECQL (L36140). The
accession numbers of aligned sequences of RNA helicases are: p68T (D16247), A. th (AB010259), Spinach (X99937), p68H (X52104), VASA
(X12945) and yeast (X52469).

both a helicase-like strand-separating activity and strandannealing activity [31]. Both domains contain large insertion
sequences that form subdomain 1B and 2B, respectively.
The ADP moiety is located at the bottom of the cleft
between subdomains 1A and 2A. However, only 1A canbind ADP. This cleft is lined with conserved helicase motifs.
No site-bound Mg2+ has been found in the two crystal
structures. On the basis of structural information for PcrA,
a possible mechanism of translocation and unwinding is
described below under the subheading Mechanism of DNA
unwinding and translocation.
Crystal structures of binary and ternary complexes of theE. coli Rep helicase bound to ssDNA [dT(pT)15] or ssDNA
and ADP were determined to a resolution of 3.0 Å and
3.2 Å, respectively [32]. The asymmetric unit in the crystals
contains two Rep monomers differing from each other by alarge reorientation of one of the domains, corresponding toswivelling of 130  about a hinge region. It was suggested
that ATP binding and hydrolysis result in an alteration in
the conformation of subdomain Ia, thereby altering the
DNA-binding site. Low-resolution structural information isavailable for the following hexameric helicases: simian virus
40 (SV40) large T antigen, papillomavirus E1, bacteriophage T7gp4, T4gp41, E. coli DnaB, RuvB, Rho and RepA
encoded by RSF1010 plasmid ([11] and references therein).
For some of these, high-resolution structural information
has also recently become available. The above helicases act
on DNA, except Rho, a transcription terminator proteinthat acts to separate RNA–DNA hybrids. The E. coli Rep
helicase and NS3 RNA helicases were also crystallized with

ssDNA, giving the ﬁrst glimpse of how the proteins interact
with nucleic acids [32,33]. The structures of T7gp4
and RepA encoded by the RSF1010 plasmid revealed asingle-domain protomer with a parallel a-b core topologythat includes the RecA core fragment, as well as fourb strands extending the b sheet on the carboxy-proximalside, three of which are similar to strands of the RecA
protein [34–36].
The crystal structure of UvrB helicase [37] and transcription factor IIH (TFIIH) from human [38] and yeast [39]have been also resolved. The structural organization of yeast
and human TFIIH showed that they could be separated
into two distinct entities. The human TFIIH contains twocomplexes: (a) the core TFIIH, which contains ﬁve subunits,
XPB, p62, p52, p44 and p34; (b) the CAK component,
which includes cdk7, cyclinH, and MAT1. The XPD
subunit could be associated with either the core TFIIH or
CAK component [38]. In yeast, the core TFIIH contains thehuman counterparts Tfb1 (p62), Tfb2 (p52), and ssl1 (p44),
but also Rad3 (XPD) and Tfb3 (MAT1) which are found,respectively, closely associated with and absent from thehuman core factor. In contrast with the human factor, theyeast ssl2 (XPB) helicase has been reported to be readily
dissociated from the other core subunit [40]. The molecular structure of human TFIIH showed that it is
16 · 12.5 · 7.5 nm in size and is organized into a ring-likestructure from which a large protein domain protrudes.
Within the ring structure, P44 is ﬂanked on either side by the
XPB and XPD helicases. Schultz et al. [38] suggested aquaternary organizational model for human TFIIH.

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Structure and function of DNA helicases (Eur. J. Biochem. 271) 1853

The crystal structure of full-length eIF-4A from yeast has
been reported [41] to be a dumbbell structure consisting of
two compact domains connected by an extended linker.
This model also suggests the involvement of the conserved
helicase motifs of eIF-4A in its functional activities.
Recently the crystal structure of the monomeric-3¢-5¢ RecG
helicase from the thermophilic bacterium Thermotoga
maritima in a complex with its stalled replication fork
substrate (three-way DNA junction, the preferred physiological substrate) has been solved [42]. The study suggested
that RecG has three domains: domain 1 is for helicase
action, and domains 2 and 3 are motor domains. Theirstudy further suggests that RecG is dsDNA translocase.
The crystal structure of RuvB shows that the RuvB
protomer is a three-domain structure, and the aminoproximal ATP-binding domain has a parallel a–b fold[43]. Recently, Bernstein et al. [44] have determined a 1.8 Å
resolution crystal structure of the catalytic core of E. coli
RecQ in its unbound form and a 2.5 Å resolution structureof the core bound to the ATP analog ATPcS.
In another recent study, the X-ray structure of ahexameric SV40 large T antigen with DNA helicase
activity has been presented [45]. The structure identiﬁes
the p53-binding surface and reveals the structural basis ofhexamerization. The hexamer contains a long, positively
charged channel with an unusually large central chamberthat binds both ssDNA and dsDNA. The hexamer
organizes into two tiers which can potentially rotate
relative to each other through connecting a-helices toexpand/constrict the channel, producing an iris effectwhich could be used to distort or melt the origin andunwind DNA at the replication fork [45]. A molecularmodel for the human nucleotide excision repair protein,
XPD, has been developed based on the structural andfunctional relationship of the protein with a bacterial
nucleotide excision repair (NER) protein, UvrB [46]. The
validity of using the crystal structure of UvrB as atemplate for the development of an XPD model wastested by mimicking human disease-causing mutations(XPD: R112H, D234N, R601L) in UvrB (E110R,
D338N, R506A) and by mutating two highly conserved
residues (XPD, His237 and Asp609; UvrB, H341A and
D510A). The XPD structural model can be used tounderstand the molecular mechanism of XPD humandisease-causing mutations. The value of this XPD model
demonstrates the generalized approach for the predictionof the structure of a mammalian protein based on the
crystal structure of a structurally and functionally related
bacterial protein that shares extremely low sequence
identity (<15%) [46].

Mechanism of DNA unwinding andtranslocation
The detailed molecular mechanism of DNA unwinding byhelicases is still not known. However, there are certain
features of unwinding and translocation that are probably
common to all helicases. Lohman & Bjornson [47] suggested
that the mechanism could be classiﬁed as either passive or
active. In the passive mechanism, the unwinding protein
interacts with ssDNA and translocates unidirectionally.
Active mechanisms are described for the oligomerichelicases, which bind both ssDNA and dsDNA. There are
two popular models for a general mechanism for helicases, described as active rolling and inchworm models(Fig. 4A,B) [47–49].
In the scheme for the active rolling model (Fig. 4A),
initially both the subunits of the dimer are bound to
ssDNA. As a consequence of binding ATP, one of thesubunits releases the ssDNA and binds to the duplex
region at the fork. This is followed by helix destabilizationand the release of one of the DNA strands in a processthat accompanies the hydrolysis of ATP [47]. It was
proposed that the Rep dimer unwinds DNA by an active
rolling mechanism in which the two subunits alternate
in binding dsDNA and 3¢ ssDNA at the ssDNA/dsDNA
junction. In this model, translocation along ssDNA iscoupled to ATP binding, whereas ATP hydrolysis drives
the unwinding of multiple DNA base pairs for eachcatalytic event (Fig. 4A). The Rep protein exists as astable monomer in the absence of DNA. Once the Rep
monomer binds to DNA, it changes its conformation andforms a homodimer, becoming functionally active intranslocation and unwinding [30]. It is proposed that ATPhydrolysis alternates between one subunit and the otheras an integral part of the translocation and unwinding
reactions [50].
For the inchworm model (Fig. 4B) the enzyme is boundto ssDNA and then translocates along the DNA strand to
the fork region, probably on binding ATP. Helix destabilization and release of one of the ssDNA strands takes place
as ATP is hydrolysed [48]. This model is consistent with any
monomeric or oligomeric state of the protein. Based on the
crystal structure of the PcrA protein Velankar et al. [49] hasproposed a reﬁned model for translocation of the PcrA
helicase along ssDNA. This model supports the initial
inchworm model proposed for the Rep helicase and suggests
that, in the initial stage of the translocation cycle, ssDNA isbound to both domains 1A and 2A of the protein. AfterATP binds to the complex, the cleft between domains 1Aand 2A closes. It has been suggested that domain 1A
releases its grip on ssDNA and slides along it, while domain
2A maintains a tight grip on ssDNA. On ATP hydrolysis,
the protein returns to its initial conformation, the interdomain cleft opens, and domain 2A translocates along the
ssDNA.
The distinction between the inchworm and active rollingmodels is that the former involves two binding sites,
an ssDNA-binding site and a dsDNA-binding site, either
belonging to a single monomeric enzyme or present on two
different subunits of a dimeric enzyme, whereas the latter
involves two subunits of a dimeric enzyme which bindalternately to ssDNA and dsDNA. Both mechanisms
require ATP hydrolysis, but it is not certain at which step
this hydrolysis takes place, although ATP binding appears
to be associated with an increased afﬁnity of the enzyme forduplex DNA or RNA.
As Rep protein and UvrD helicases of E. coli share about
40% amino-acid sequence identity and several biochemical
properties (including formation of dimer in the presence of
ssDNA), it has been suggested by Ali & Lohman [51] thatthe two proteins probably unwind duplex DNA by the samerolling mechanism involving a dimeric helicase. However,Mechanic et al. [52] have reported that a mutant lacking the1854 N. Tuteja and R. Tuteja (Eur. J. Biochem. 271)

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Fig. 4. Models for DNA helicase unwinding and translocation. In both the mechanisms, ATP hydrolysis is required and the helicase protein contacts
and translocates on the sugar–phosphate backbone of the strand. (A) Active rolling model. The two subunits of dimeric helicase are shown as anoval shape in which one is black. The dimeric helicase unwinds by interacting directly with both dsDNA and ssDNA. Each subunit alternatesbinding to dsDNA as the dimer translocates when one subunit releases ssDNA and rebinds to dsDNA [5]. In this model, translocation along
ssDNA is coupled to ATP binding, whereas ATP hydrolysis drives the unwinding of multiple DNA base pairs for each catalytic event.
(B) Inchworm model. This model is consistent with the monomeric or oligomeric state of the protein (shown as an oval shape). The enzyme
monomer ﬁrst binds to ssDNA and then translocates along the DNA strand. It then binds to the duplex region at the fork. This is followed byunwinding and release of one of the ssDNA strands [48]. If the enzyme is a hexamer, such as the SV40 large T antigen, then one of the subunits(monomer 1) remains associated with the fork through the unwinding cycle. (C) Modiﬁed inchworm model. This model is proposed for monomeric
UvrD helicase [52,55]. In this model, it is assumed that the monomeric helicase contains two DNA-binding sites: the leading site (L) binds to both
dsDNA and ssDNA, and the trailing site (T) binds to only ssDNA. On ATP binding, the enzyme changes its conformation from an extended to acompact state, in which the T site is shifted forward along the ssDNA towards the L site. On ATP hydrolysis, the enzyme changes its conformationfrom a compact to an extended state. In the extended state the T site is bound to ssDNA, and the L site is extended forward in the duplex region andunwinds the DNA. In all the models the shape of the helicase and location of the DNA are arbitrary.

C-terminal 40 amino acids (UvrDD40C) failed to dimerize
and yet was as active as the wild-type protein in ATPhydrolysis and helicase assays. Their results show that themonomeric form of UvrD (UvrDD40C) is an active helicase
both in vitro and in vivo. Therefore the mechanism ofunwinding by monomeric UvrD helicase may be different.
Matson’s group have proposed a modiﬁed version of the
inchworm model for DNA unwinding by an active monomeric DNA helicase (Fig. 4C). This model assumes at least
two nonequivalent DNA-binding sites on the monomeric
protein. The leading site (L) has an afﬁnity for duplex DNA
and may also bind ssDNA, whereas the trailing site (T) has
an afﬁnity only for ssDNA. A cycle of unwinding begins
with the enzyme in an extended conformation in which theT site is bound to ssDNA, and the L site is extended forwardin the vicinity of the ssDNA/dsDNA junction. Binding of
ATP to the enzyme induces a conformational change in theprotein to a more compact state in which the T site is shifted
forward along the DNA lattice with respect to the L site.
On ATP hydrolysis a distinct number of base pairs are
disrupted at the ssDNA/dsDNA junction, and productrelease is associated with a return of the enzyme to its
original conformation by extension of the L site forwardwith respect to the T site. At this point, the L site is close to
the new ssDNA/dsDNA junction, and the cycle is repeatedto catalyze further unwinding (Fig. 4C) [52]. Recently,
Maluf et al. [53] have shown that initiation of DNA
unwinding in vitro appears to require a dimeric UvrD
complex in which one subunit is bound to the ssDNA/

dsDNA junction, while the second subunit is bound to the3¢ ssDNA tail.
Bianco & Kowalczykowski [54] have suggested anotherinchworm model, the quantum inchworm mechanism fortranslocation and unwinding of duplex DNA by RecBC
DNA helicase. Translocation and unwinding are two
separate and consecutive events in the mechanism and arebrought about by two different domains [leading (L) andtrailing (T)] within the RecBC enzyme. In this model the Ldomain anchors the enzyme to only one strand of duplex
DNA and translocates along it, whereas the T domain is
responsible for unwinding. During the translocation andunwinding reaction, the L domain binds up to 23 nt aheadof the T domain, and the T domain uses energy derived
from ATP binding and hydrolysis to open the duplex[54,55].
Recently, Ha et al. [56] proposed that the Rep monomer
uses ATP hydrolysis to move toward the junction between
ssDNA and dsDNA but then displays conformational
ﬂuctuations that do not lead to DNA unwinding. DNA
unwinding is initiated only if a functional helicase is formed
via additional protein binding. Partial dissociation of thefunctional complex during unwinding results in interruptions (stalls) which lead to either duplex rewinding on
complete dissociation of the complex or re-initiation ofunwinding on re-formation of the functional helicase. These
results suggest that the low unwinding processivity observedin vitro for Rep is due to the relative instability of thefunctional complex.

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Structure and function of DNA helicases (Eur. J. Biochem. 271) 1855

Functions of DNA helicases
The ubiquitous presence of helicases in prokaryotes andeukaryotes reﬂect their fundamental importance in DNA
metabolism such as replication, repair, recombinationand transcription [2,4,5,47,57]. In vivo, DNA unwindingis usually coupled to the action of other proteins suchas initiator proteins, primases, ssDNA-binding proteins(SSBPs), polymerases and many more factors depending on
the associated functions of a particular helicase. SSBPs play
many important roles along with helicases such as: (a)increasing the processivity of the helicase; (b) blocking thereannealing of the unwound strands; (c) inhibiting helicase
action by blocking access of the helicase. The various
helicases and their known function are described below.
Replicative helicases
This section deals with the process of DNA replication(initiation, elongation and termination) and properties ofreplicative helicases. The synthesis of all genomic DNA
involves the highly coordinated action of different types of
enzymes including DNA helicases, which melt the DNA
duplex to generate the replication fork. Figure 5A shows theschematic representation of progressive DNA unwindingof duplex DNA by a replicative DNA helicase at the
replication fork. The replicative DNA helicases occupy acentral position at the replicative fork and can unwind
duplex DNA quickly and processively in the presence ofhelicase reporter (trapping agents) such as DNA polymerase
and SSBPs [55]. The process of DNA replication can bedivided into three phases: initiation, elongation, andtermination.

Fig. 5. Models of DNA unwinding. (A) Replicative machinery showing the interaction of
replicative DNA helicase at the replication
fork. This is a schematic representation of thereplication fork showing the unwinding of the
DNA by replicative hexameric helicase duringfork progression. Both strands of DNA are
synthesized in the 5¢ to 3¢ direction. The leading strand is synthesized continuously,
whereas the trailing strand is synthesized in theform of short fragments. (B) Model of DNA
unwinding during NER in mammalian cells.
In this ﬁgure only the unwinding step isshown. The damaged area is unwound by the
XPB helicase (3¢-5¢) and XPD helicase (5¢-3¢)components of TFIIH. (C) Model of DNA
unwinding during recombination. Recombination machinery shows only the steps ofunwinding and branch migration of theHolliday junction by E. coli RuvAB or RecG
helicases.

Initiation of DNA replication. In E. coli, bacteriophage T7,
and SV40 virus, assembly of a preinitiation complex
precedes initiation of DNA synthesis at the replication
origins. The ﬁrst step of the initiation process is the meltingof the two DNA strands by binding of the subunits of thehexameric helicase to the origin DNA. The separated
strands serve as templates for the primase/polymerase for
new strand synthesis. The loading of helicases on the originof replication requires the participation of speciﬁc accessory
proteins. It has been demonstrated that Rep helicase isloaded on to the replication origin via tight binding with the
CisA protein [58]. The T7 gp4 primase/helicase is loaded on
to ssDNA by a facilitated ring-opening mechanism, andhelicase loading is assisted by the presence of the primasedomain [59]. Genetic and biochemical experiments haveidentiﬁed DnaC as the loading factor for DnaB. In thepresence of DNA and ATP, the bacteriophage T4 gp41
hexamer binds six T4 gp59 molecules leading to a 1 : 1
helicase/helicase loader molar ratio [60]. Recent studies haveshown that the hetero association interactions of T4 gp59
with ssDNA, T4 gp32 and T4 gp41 are all essential in theloading reaction [61].
Elongation of DNA. Elongation of the new DNA chainsrequires further separation of the DNA templates. In every
case, the hexameric helicase responsible for the initial
melting of the origin DNA is also the processive helicasethat unwinds the growing fork [62]. Additional key proteins
are required for melting of the duplex DNA downstream of
the replication fork. DNA polymerase on the leading strand
and the ssDNA-binding proteins that bind on the lagging
strand work with the replicative helicases to complete theelongation mode. It has been proposed that the coupling of

1856 N. Tuteja and R. Tuteja (Eur. J. Biochem. 271)

the helicase to the polymerase during the elongation phase
does not require their physical interaction. The helicase isfunctionally coupled to its homologous SSBP [55].
Termination of DNA replication. Termination of replication involves a protein that forms a tight complex with thetermination site (called Ter), acting as a contra-helicase. In
E. coli and Bacillus subtilis, terminators are organized into
two sets of ﬁve or three Ter sites which, when complexedwith terminator proteins, cause replication arrest in a polar
fashion. These replication termination proteins operate byblocking the ATP-dependent unwinding activity of helicases
in a polar fashion [63]. Ter sequences (acting in cis) andterminator proteins (acting in trans) are the two componentsthat dictate replication arrest. It has been postulated that theprotein–Ter complex thus comprises a trap for replication
forks that arrive at the site, and, as a consequence, also
serves to prevent forks from meeting at other template
positions.
Only some of the E. coli, bacteriophage, and viral DNA
helicases are reported to play clear cut roles in DNA
replication. However, owing to the lack of clearly deﬁned
cell-free systems that catalyze chromosomal replication, it is
difﬁcult to deﬁne the role of any cellular replicative helicase.
Nonetheless, the study of viral replicative helicases hashelped to deﬁne the role of helicases in eukaryotic DNA
replication. The best characterized replicative helicases are
DnaB, PriA and Rep proteins from E. coli, T7gp4 andT4gp41 proteins from bacteriophage, and SV40 large T
antigen, polyoma virus T antigen, bovine papillomavirus
E1, herpes simplex virus (HSV) UL5 and UL9, adenoassociated virus (AAV) Rep 68 and Rep 78, and minutevirus of mice (MVM) NS1 proteins from viral sources[4,47,55]. A comparison of the properties of the replicative
DNA helicases is given in Table 1. Indirect evidence alsosuggests that yeast Dna2P, Xenopus laevis FFA-1 andWerner syndrome helicase (WRN) proteins may be candidates for DNA replicative helicases [64–68]. Liao et al. [69]have reported the ﬁrst direct evidence that Xenopus Bloom’s
syndrome protein homolog (XBLM) plays an importantrole in DNA replication, suggesting that Bloom’s syndrome
may be the consequence of defective DNA replication.

Repair helicases
DNA repair is a fundamental cellular process for protecting
cells against damaged DNA. The helicases are some of theﬁrst proteins that encounter DNA damage and thereby play
an important role in its repair. During DNA repair,especially in NER, the damaged area on the DNA has tounwind ﬁrst in order be repaired, because most of the DNA
repair machinery enters through ssDNA. The unwinding
step during NER in mammalian cells is shown in Fig. 5B.
Examples and some properties of repair helicases aredescribed in Table 2. In E. coli the UvrAB complex andUvrD are the repair DNA helicases involved in NER. The
structure of UvrB suggests that it functions as a helicase
adapted to the unique requirements of DNA repair [37]. It
has been reported that the UvrA-mediated loading of UvrB
is an asymmetric process which starts on the 5¢ side of thelesion, and the strand-separating activity of the UvrAB
complex is used to create an entry site for UvrB at this FEBS 2004

position [70]. In general, the UV-induced lesions or otherhelix-distorting intrastrand adducts are better tolerated by
DNA helicases than those produced by intercalating agents.
However, in the yeast Saccharomyces cerevisiae the Rad3protein, a repair helicase, is absolutely required in NER andis found to be inhibited by UV damage and intrastrand
cisplatin adducts located on the strand along which the
enzyme translocates, whereas lesions on the opposite strand
had no effect [71]. DNA helicases involved in the initialstages of NER are more sensitive to DNA damage (e.g.
UvrAB complex, Rad3 or XPD) than those that are
involved in the subsequent removal of the damaged DNA(e.g. UvrD). ERCC6 (excision repair cross complementing),
which corrects the repair of Cockayne syndrome complementation group B, encodes a protein containing sevenconserved helicase motifs [72]. The repair proteins ERCC3(XPB) and ERCC2 (XPD) contain conserved helicase
motifs and DNA-unwinding activity. The function of thesehelicases is to open up the structure around the damaged site
to enable the structure-speciﬁc nucleases to excise the DNA[73]. The human XRCC5 DNA repair gene has been shownto encode an 80-kDa subunit of the Ku protein [74] andtogether with the 70-kDa subunit possesses ATP-dependent
DNA helicase activity [75].

Recombination and other functionsof the DNA helicases
The properties of recombination helicases and some otherfunctional helicases are summarized in Table 3. During
genetic recombination, the pairing of homologous regionsof DNA requires both the unwinding of duplex DNA byhelicases for strand invasion and the generation of extended
single-stranded segments. Recombination in E. coli requires
the co-ordinated action of at least 25 proteins, which include
RecA, RecBCD, RecF, RecG, RecN, RecO, RecQ, RecR,
RuvAB, RuvC, PriA, SSBP, DNA polymerases, DNA
topoisomerases and DNA ligases [76]. Of these proteins,
only RecBCD, RecG, RecQ, RuvAB, and PriA act as DNA
helicases (Table 3). Homologous recombination can bedivided into four sequential processes: (a) initiation;(b) homologous pairing and strand exchange; (c) DNA
heteroduplex extension or branch migration; (d) resolution.
DNA helicases are centrally involved in initiation andbranch migration. The role of recombination helicases(RuvAB and RecG) in branch migration is shown in
Fig. 5C. The properties of some of the recombination
helicases are brieﬂy described below.
E. coli RecBCD carries a number of diverse enzymic
activities such as ATP-dependent ssDNA endonulcease,
ssDNA exonuclease, dsDNA exonuclease, RecA-loading,DNA-dependent ATPase and ATP-dependent helicase
activities [55,57,77]. It has sets of helicase signature motifs
in both RecB and RecD, two of its three subunits. RecA canserve as a reporter of RecBCD helicase activity [78]. RecB
and RecD contain 3¢ to 5¢ and 5¢ to 3¢ DNA helicase activity,
respectively [79]. These ﬁndings point to a bipolar translocation model for RecBCD in which the two DNA helicases
are complementary, travelling with opposite polarities, but
in the same direction, on each strand of the antiparallel
DNA duplex. Recently, by electron microscopy of individual molecules, it has been shown that RecD is a fast helicase

E. coli
E. coli

Phage

Phage
Phage
Virus

Virus

Virus
Virus
Virus

Virus

Virus
Yeast

Yeast
Xenopus laevis

PriAb
Repc

T7gp4Ad and 4B

T4gp41e
G40Pf
SV40 TAGg

Polyoma TAGhBPV E1i
HSV UL5j
HSV UL9k

AAV Rep68, Rep78l

MVM NS-1m
MCM4/6/7n

Dna2poFFA-1p

2.
3.

4.

5.
6.
7.

8.

9.
10.
11.

12.

13.
14.

15.
16.
172
170

61
71
83
80–100 (each)

69
114
94

88

A, 63
B, 56
59
300
82

81.7
72.8

52.2

Mol. mass(kDa)

ATP ¼ dATP
ATP

ATP > dATP,
ATP ¼ dATP

ATP > CTP > dATP > GTP > UTP

All NTPs and dNTPs
ATP > GTP,CTP,UTP
ATP ¼ dATP > CTP, dCTP, UTP

ATP, dATP > CTP, UTP  GTP

dTTP > ATP,all others
GTP > ATP, dATP, dGTP
ATP > GTP > CTP > UTP
ATP, dATP, dTTP > dCTP, UTP

ATP ¼ dATP
ATP ¼ dATP  GTP, dGTP

ATP > GTP, CTP

Nucleotide requirement[90]; b [91]; c see [4]; d see [4]; e [92]; f [93]; g [94]; h [95]; i [96]; j [97]; k [98]; l [99]; m [100]; n [101]; o [21] p [68].

E. coli

DnaBa

1.

a

SourceName of helicase

S. No.

ndndnd
5¢ and 3¢ tailsnd5¢ ss tail3¢ ss tail5¢ ss tailnr

3¢ ss tail of 29 nts
5¢ ss tail of 25 nts
3¢ ss tail of 5–10 nts

3¢ ss tail of 6–7 nts

nr
3¢ ss tail of > 4 nts

3¢ ss tail of 90 nts

Fork requirement

Stimulated by SSB and DnaG; stimulatesrolling circle replication, and unwinding is
ori-dependent.
Stimulated by primosome assembly site sequence.
Discovered ﬁrst as replicative helicase;required for replication of /X174, fd and M13 phages.
Required for phage DNA replication;interacts speciﬁcally with T7 DNA polymerase.
Stimulated by T4 gp61; also contains primase activity.
Essential for B. subtilis bacteriophage SPP1 replication.
OBP essential for SV40 replication;
contains both DNA and RNA helicase activities.
Similar to SV40 TAG. OBP required for Polyoma
virus DNA replication.
Required for bovine papilloma virus DNA replication.
UL5 and UL52 required for helicase-primase activities.
Stimulated by ICP8 and initiates origin-speciﬁc
DNA replication.
Contains site and strand speciﬁc endonucleaseactivity; required for viral DNA replication.
Homolog of AAV Rep helicase.
Required for initiation and elongation steps ofchromosomal DNA replication.
Indirect evidence shows its role in DNA replication.
Required for the formation of replication foci;homologous to the human WRN.

Remarks

Table 1. Replicative helicases. nd, Not determined; nr, not required; OBP, origin binding protein; AAV, adeno-associated virus; BPV E1, bovine papillomavirus E1; MCM, minichromosome maintenance;
MVM, minute virus of mice; TAG, T antigen; WRN, Werner syndrome helicase.

 FEBS 2004
Structure and function of DNA helicases (Eur. J. Biochem. 271) 1857

 FEBS 2004

1858 N. Tuteja and R. Tuteja (Eur. J. Biochem. 271)
Table 2. DNA repair helicases. nd, Not determined.

S. No

Name of helicase

Mol. mass(kDa)

Nucleotide
requirements

1.
2.
3.
4.

E. coli helicase II (UvrD)aE. coli UvrAB complexb
Yeast Rad3c
Calf thymus helicase Ed

82
180
90
104

ATP > dATP
ATP ¼ dATP
ATP ¼ dATP
ATP, dATP

5.

Mammalian ERCC2e (XPD)

87

ATP, dATP

6.

Mammalian ERCC3f (XPB)

89

ATP

7.

Mammalian ERCC6g(putative helicase)
Human HDH II/Kuh (XRCC5)

168

nd

70/80

ATP ¼ dATP

8.

aRemarks
Involved in methyl-directed mismatch repair.
Involved in NER. UvrB is the helicase component.
Absolutely required for NER, inhibited by DNA damage.
Can unwind DNA from nicks and has been predicted
to play a role in DNA repair.
Component of transcription factor (TFIIH). Unwinds
DNA in 5¢ to 3¢ direction and involved in NER.
Component of TFIIH, unwinds DNA in 3¢ to 5¢
direction and involved in NER.
Contains helicase motifs involved in DNA repair of
Cockayne’s syndrome complementation group B.
Involved in double-strand break repair and V(D) J
recombination of immunoglobulins.

[57]; b [102]; c [103]; d [104]; e [105]; f [106]; g [72]; h [75].

acting on the 5¢-ended strand and RecB is a slow helicase
acting on the 3¢-ended strand on which the single-stranded
loop accumulates [80].
RecG helicase translocates on duplex DNA, driving awedge through the branch point of a fork or Holliday
junction structure, thus catalysing both strand unwinding
and strand annealing (Fig. 5C). RuvAB carries out a similar
reaction. The RecG helicase is necessary for efﬁcient
recombination and repair of DNA in vivo and is also called
as junction-speciﬁc DNA helicase [81]. RecG protein differsfrom other helicases analysed in that it mediates strand
separation via translocation on dsDNA rather than ssDNA.
The RecG helicase of E. coli has been postulated to act onstalled replication forks to promote replication restart via
the formation of a four-stranded (Holliday) junction.
McGlynn & Lloyd [82] have shown that RecG can actively
unwind the leading and lagging strand arms of model
replication fork structures in vitro. Thus, RecG translocatessimultaneously along two DNA strands, one with 5¢ to 3¢
and the other with 3¢ to 5¢ polarity. The unwinding of bothnascent strands at a damaged fork, and their subsequentannealing to form a Holliday junction, may explain theability of RecG to promote replication restart.
E. coli RuvAB helicase is an ATP-driven translocase(pump) that promotes branch migration of the Holliday
junction [83]. These are four-way branched DNA structures
formed during recombination, replication and repair andare processed in E. coli by the RuvA, RuvB and RuvC
proteins. RuvA targets the junction and facilitates loadingof RuvB helicase and RuvC endonuclease to form complexes that catalyse junction branch migration (RuvAB)
and resolution (RuvABC resolvasome) [84]. RuvAB-catalyzed branch migration involves the unwinding and rewinding of duplex DNA near the base of the Holliday junction,with the transient unwinding reaction taking place within
the interior of the RuvB ring. RuvA plays a scaffoldingrole in preparing the homoduplex DNA strand for strand
separation and the recombining strands of the heteroduplex
for reannealing [85]. Recently, Kaplan & O’Donnell [86]demonstrated that DnaB can drive branch migration anddislodges proteins while encircling two DNA strands, itsin vivo role in DNA recombination. The other helicases thatplay a role in recombination are helicase II (UvrD), RecQ
from E. coli, bacteriophage T4 UvsW protein, Ku autoantigen, and WRN helicase (Table 3). Some other functionsof DNA helicases are also described in Table 3.

Role of DNA helicases in transcription
DNA helicases are known to alter the DNA or DNA/RNA
structures, remodeling chromatin and modulating access to
the DNA template by transcriptional machinery. The RNA
polymerases that catalyze the template-dependent elongation of RNA transcripts can be considered to be helicasesthat unwind the dsDNA genome in order to expose thessDNA strand that will serve as the template for RNA
synthesis by the transcription complex. Generally for RNA
polymerases in the elongation-competent state, these helicase and synthesis activities are tightly coupled with theextension of the RNA transcript by one nucleotide involving the consumption of one molecule of NTP and theconcomitant unwinding of a single base pair at thedownstream end of the transcription bubble [55]. DNA
helicases thus play an important role as part of thetranscription complex in most if not all transcriptional
processes including activation (e.g. TFIIH), initiation (e.g.
TFIIH, SNF2), maintenance (e.g. SW1), DNA repair (e.g.
TFIIH, ERCC6/RAD26) and termination (e.g. Factor 2,Rho) [72,73,87–89]. Transcription activation involves theformation of a preinitiation complex, its association with
ssDNA activation, and promoter clearance. TFIIH is an
RNA polymerase II transcription factor that performs
ATP-dependent functions in both transcription initiation,
where it catalyzes formation of the open complex, and inpromoter escape, where it suppresses arrest of the early
elongation complex at promoter-proximal sites. TFIIH
possesses three known ATP-dependent activities: a 3¢ to 5¢
DNA helicase activity catalyzed by its XPB (ERCC3,
RAD25) subunit, a 5¢ to 3¢ DNA helicase activity catalyzed
by its XPD (ERCC2, RAD3) subunit, and a kinase activity
speciﬁc for the C-terminal domain of RNA polymerase II[87,88]. Moreland et al. [88] reported that the TFIIH XPB

 FEBS 2004

Structure and function of DNA helicases (Eur. J. Biochem. 271) 1859

Table 3. Recombination and other functional helicases. HDH, Human DNA helicase; PDH65, pea DNA helicase 65; nd, not determined; RPA,
replication protein A; WRN, Werner syndrome helicase.

S. No.

Name of helicase
aMol. mass(kDa)

Nucleotide
requirement

Remarks

1.

E. coli helicase II (UvrD)

82

ATP > dATP

2.

E. coli Rec BCDb

330

ATP, dATP

3.

E. coli RecQc

74

ATP ¼ dATP

4.

E. coli RuvABd

22

ATP

5.

E. coli RecGe

37
76

ATP, dATP

6.

Bacteriophage T4f
UvsW protein

nd

ATP

7.

HDH II/Kug70

ATP ¼ dATP8.

WRN helicaseh

80
163

9.

HDH IV/nucleolini

100

ATP, dATP

10.

E. coli helicase 1 (Tra I)j

192

11.

E. coli Rhok

50

ATP, dATP >all others
ATP > NTPs

12.

Yeast MER3l

130

ATP

13.

PDH65m65

ATP > dATPa

Indirect evidence suggests its role in recombination
as an antirecombinase; also unwinds bluntend duplex DNA and nicked circle DNA
substrates.
Involved in recombination; generates ssDNA
that can be utilized by RecA protein inhomologous strand exchanges.
Involved in RecF recombination pathway;
functions as suppressor of illegitimate
recombination; also unwinds bluntend duplex DNA.
Required for recombination and repair; helps inbranch migration of a Holliday junction.
Required for normal levels of recombination
and repair; junction speciﬁc helicase and involvedin branch migration of Holliday junction.
Involved in phage recombination and is functional
analog of RecG helicase. Catalyzes branchmigration and also dissociation of RNA–DNA
hybrids.
Involved in V(D)J recombination of
immunoglobulins and also in dsDNA break repair.

ATP ¼ dATP 
dCTP, CTP

A member of RecQ family of DNA helicases;
may be involved in DNA recombination,
replication and repair. WRN helicase
also accelerates the transcription of ribosomal RNA.
Can unwind DNA–DNA, RNA–RNA and RNA–DNA
hybrids. Involved in preribosomal assembly andribosome biogenesis.
Required for DNA transfer during conjugation. Also
contains nicking activity.
Speciﬁc for RNA–DNA hybrids functions in transcription
termination at speciﬁc sites; contains ssRNA-dependent
NTPase activity.
Involved in meiotic crossing over and stimulated
by yeast RPA.
Homologous to HDHI, may be involved in
rDNA transcription.

[57]; b [107]; c [22]; d [83]; e [81,108]; f [109]; g [75], see [110]; h [65]; i [111], see [110]; j [57]; k [57]; l [112]; m [113].

DNA helicase was primarily responsible for preventing
premature arrest of early elongation intermediates during
the exit of the polymerase from the promoter. TFIIH is also
required for transcription-coupled nucleotide excision repair[87,88]. Finally in E. coli, the Rho protein, a RNA/DNA
helicase, and in Drosophila, Factor 2 are required fortranscription termination [57,87].

Concluding remarks
DNA helicases are involved in almost every aspect of
nucleic acid metabolism, which makes them very importantcomponents of the cell. High sequence conservation has
been maintained in the large group of helicases, suggestingthat all helicase genes evolved from a common ancestor.
The DEAD-box helicases contain common conserved
helicase signature sequences but they differ mainly in theirN-terminal and C-terminal sequences, which contain different targeting signals. Different regulatory mechanisms at
the level of both expression and post-transcription explain
the wide spectrum of functions involving DEAD-box
helicases. The crystal structures of SF1 and SF2 helicases
have shown that the DNA helicase motifs are clustered
together in the tertiary structure, forming an NTP-binding
pocket and a portion of the nucleic acid-binding site.
According to Hall & Matson [3], the conserved helicase
motifs can be envisioned as the engine, generating energy by
the consumption of fuel (NTPs) which is used to perform FEBS 2004

1860 N. Tuteja and R. Tuteja (Eur. J. Biochem. 271)

work. The nonconserved portions of helicase structure may
contain speciﬁc domains such as protein–protein interaction
domains, cellular localization signals, site-speciﬁc DNArecognition domains, and oligomerization interfaces, which
may be unique to individual helicases. The crystal structuresof a few helicases have been resolved, which will greatly help
us to understand the mechanism by which ATP hydrolysis
and conformational changes are coupled to allow these
proteins to carry out their varied enzymatic activities.
Despite the available crystallographic data, many questions
remain unanswered. For example, the mechanism of strand
separation remains a mystery. It is also unclear why
hexameric helicases generally prefer substrates with two
tails (i.e. a real fork) rather than a simple tailed duplex,
which is sufﬁcient for other enzymes such as PcrA and Rep.
These matters should be resolved by future structurestudies, and the structure of helicases complexed withother modulator proteins should help us to understandother details of their mechanism of action.
Although several DNA helicases have now been isolated
from different sources, the role of only a few have been
elucidated to date. As the basics of DNA metabolism arevery similar in all eukaryotes from yeast to mammals, thecombination of genetic studies in yeasts, in vitro systems,
and biochemical characterization should help us to clarify
the roles of the various DNA helicases in vivo. It now
appears that oligomeric helicases (mainly hexameric) areoften integral components of larger protein complexes, andtheir role within the complex is to provide molecular motor
function. Therefore, it would perhaps be naı̈ve to perceive
helicases merely as nucleic acid-unwinding enzymes. To gain
a complete picture of helicase function, it is important tostudy these enzymes in situ as components of the macromolecular machines in which they have a central function.
It will be interesting to see how they are integrated into theoverall processes in which they function. These studies will
provide insight into the mechanisms of action of helicases
themselves, and also into the overall biological systems
within which they operate.
During the elongation phase of DNA replication the basepairs of dsDNA at the replication fork must be opened
continuously and sequentially to expose the ssDNA
templates, which will be copied by the DNA polymerase.
However, DNA at replication forks is synthesized at ratesof  500 bpÆs)1. This means that helicases at these forks
must be opening dsDNA at this rate or faster. It has
been suggested that enhanced thermal ﬂuctuation may be
responsible for sequential base-pair opening by replicative
helicases at the replication fork. This also suggests that
replicative helicases may advance by a passive unwinding
mechanism rather than by some mechanical application ofenergy from NTP hydrolysis. In such a case, ATP hydrolysis
may serve only to generate the ADP-bound form of thehelicase and to initiate the release of protein from thessDNA product in order to complete the ATP-binding andhydrolysis cycle.
A detailed view of the energetics of the translocation
process and the mechanism of unwinding is also lacking.
Low-resolution structural analysis, by electron microscopy,
is beginning to provide clues to some of the functions andmechanisms of action of these remarkable proteins inprokaryotes. On the basis of electron-microscopy studies, adual helicase model has recently been proposed for the
E. coli RecBCD enzyme complex. It will be interesting to
see whether such bipolar, nonidentical helicase ‘‘motors’’
are used by other DNA-processing enzymes. We still needto understand the molecular details of how other proteinsmodulate the activity of helicases or how helicases function
within larger macromolecular complexes such as theprimosome. Structures of helicases complexed with othermodulator proteins will help us to understand in greater
detail their mechanism of action. A better understanding ofthese proteins in plant will have to await breakthroughs on
their high-resolution structures using X-ray crystallography.
Furthermore, in plants, the RNAi approach and/or transgenic antisense/sense plant technology will be very helpful inunderstanding the detailed role of helicases in plant growthand development and therefore will have importantbiotechnological applications.
Overall this ﬁeld is now progressing fast, and accordingly the number of isolated DNA helicases in bothprokaryotic and eukaryotic systems are increasing. Thishas created the problem of a bewildering array of different
names and their classiﬁcation. Therefore it is importantthat a clear, scientiﬁc system for nomenclature andclassiﬁcation of helicases is formulated. This is a complicated problem and does not seem to have a straightforward
solution. However, there may be several possible ways ofclassifying DNA helicases, which may not be convenient.
For example, they can be classiﬁed on the basis of the kindof duplex-strand unwinding (e.g. DNA or RNA or RNA/DNA or DNA/RNA helicase), source of identiﬁcation (e.g.
prokaryotic or eukaryotic DNA helicases), oligomericnature of protein (e.g. monomeric or oligometic DNA
helicases), polarity of translocation (e.g. 3¢)5¢ or 5¢)3¢
DNA helicases), associated functions (e.g. replicative,
repair, recombination, or other functions) or on the basisof helicase motifs present in the superfamily (e.g. SF1, SF2,SF4 and F4). We believe that this class of proteins does
not, at this point, lend itself to a simple scheme ofclassiﬁcation. Overall, the progress in the DNA helicase
ﬁeld is further extending our understanding of DNA
metabolism and is also providing us with a dizzying arrayof molecular tools for cellular machinery.

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