Helicases: amino acid sequence comparisons and structure-function relationships

Helicases:

amino acid sequence comparisons structure-function relationships

Alexander Russian Academy

E. Corbalenya

of Medical

Sciences,

and

and Eugene V. Koonin

Moscow, Russia, and National Bethesda, USA

Institutes

of Health,

DNA and RNA helicases are ubiquitous enzymes that mediate the nucleoside-triphosphate-dependent unwinding of nucleic acid duplexes, a necessary step in genome replication, expression, recombination and repair. All proteins with demonstrated helicase activity contain the ‘purine nucleoside-triphosphate-binding pattern; subsets of helicases possess additional conserved motifs. Three large super-families and two smaller families of helicases are described. Experimental results support the value of the conserved motifs for prediction of structure and function of the helicases. Some of these motifs can be used as reliable identifiers of the respective groups of helicases in database searches. The two largest helicase superfamilies share similar patterns of seven conserved sequence motifs, some of which are separated by long poorly conserved spacers. Helicase motifs appear to be organized in a core domain which provides the catalytic function, whereas optional inserts and amino- and carboxy-terminal sequences may comprise distinct domains with diverse accessory roles.

Current Opinion

in Structural

Introduction Unwinding of double-stranded (ds) polynucleotides or base-paired regions in single-stranded 6s) polynucleotides is a prerequisite to basic genetic processes including genome replication, repair, recombination, and multiple stages of expression. Several types of proteins with different activities have been implicated in this reaction. Among them, a pivotal role belongs to polynucleotide-dependent nucleoside triphosphate (NTF) phosphatases possessing SSDNA- and/or RNA-displacing activity, called hekases [ 1,2,3*,4*]. The mechanism exploited by these enzymes for coupling hJTP hydrolysis to polynucleotide-strand displacement is poorly understood. Selected structural, biochemical and genetic data on proteins with demonstrated helicase activity are summarized in Table 1. Little is known about the spatial structure of the helicases, and not one helicase crystal structure has been resolved so far. Experiments on limited proteolysis of bacterial helicases DnaB, Rho, UvrB, UvrD and Rep have revealed a complex domain organization, and indications of interactions between distinct domains have been obtained L5-81. The majority of the characterized helicases con-

Biology 1993, 3:419-429

sist of several identical or non-identical subunits ( [3°,40] ; Table 1). Interaction with ATP and with its non-hydrolyzable analog has been shown to induce major conformational changes in the dimeric Rep helicase [ ~1. A large fraction of the available information on the structure and possible functions of the helicases has been obtained by computer-assisted comparative analysis of their amino acid sequences. This approach has led to delineation of motifs and patterns that are conserved in different subsets of the helicases.Xvo of these sequence signatures, the so-called ‘A’ and ‘B’ motifs of the ‘Walker box’ purine NTP-binding pattern, are shared by all helicases and a wide variety of other NTP-utilizing enzymes [10,11,12*]. It has been claimed that additional motifs are unique identifiers of distinct groups of (putative) helicases and are likely to play an essential role in securing the helicase and associated activities. In this review, we briefly summarize the results of comparative analysis of the ammo acid sequences of helicases and related proteins. Our goal was to assess the validity and usefulness of this approach using the relevant experimental data. Such an assessment is critical as the number of computer-predicted ‘helicases’ is growing much faster than the number of helicases actually studied experimentally.

Abbreviations

ds-clouble-stranded;

F-family;

NTP-nucleoside

@ Current

Biology

triphosphate; SF--superfamily; ss--singfe-stranded. Ltd ISSN 0959-440X

419

420

Sequences

rrbk

and topology

I. Selected features of helicases with demonstrated

activity

and known

sequencP.

Polynuclwtide~ duped faknil~

EnzymeC

SOW72

SF1

Tr# Mel It UvrD (Hd 10 Rep

E. mli

SF1 SF1

SF1

RecB+ HelDa IHel M Pif+

SF1

DdaO

SF1 SF1

Oligomer

Bound

Displaced

DNA

DNA

Oligomer

DNA

DNAIRNA

Homodimer

DNA

RecBCD Monomer

DNA DNA

DMA not RNA DNA DNA

t

DNA

DNA

S’+3’

I

DNA

DNA/RNA

5’+3’

Yeast

.

Phage T4

RecD+fi

E. coli

uL5+~

HSV-1 herpesvirus E. coli

RecBCCl uL5. B, 52

1

1

I

DNA

DNA

Y-03’

1

DNA

DNA

?

DNA

DNA

3’-+5’ (5’+3’1 3’+5’

DNA DNA

DNA DNA/RNA

5’+3’ 5’43’

RNA

RNA

3’-+5’

RNA

RNA

?

. SF2

PriAo

E. coli

SF2 SF2

U&f Rad3

E. coli Yeast

SF2

elF4AM

elF4A + 48

Monomer

SF2

p68+

MOUSe, yESt Human Human

SF2

He1.A”

SF2

BR+

SF2

ULQ+

SF2

Cl+

S-3’

E. coli E. coli E. coli

W poxvirus HSV-1 herpesvirus PPV

SF3

T antigen

SF3 F4

h-J+ fNS1) DoafJ

%ZNS papovavirus AAV. MVM pawovirures E. coli

F4

UvrABC ?

I

7 Homodimer I Homohemmer

of

duplex unwinding’

E. coli

SF1

RecQ+

stnlctud

(-e ’ L:? ‘+ ’ lL5j*, 3’-.5’ 3’+5’

SF1

SF2

Direction

Subunit

Protein

F-plasmid transfer Recombination. replication, repair Recombination, replication, repair Recombination, repair, viability I Mitochondrial DNA replication. recombination Recombination, replication Recombination. repair, viability Replication

Non-essential

n-31 h-31 11-31

UV sensitivity

11,37*.39*1

1 Non-essential

11-31 IBZI

Non-essential

Il.81

Non-essential

t39*.40=1

Essential

1321

Non-essential

1841

Primosome assembly DNA repair DNA repair

Non-essential

11.66.1

UV sensitivity Essential. UV sensitivity Essential

n-31 IBSl

Initiation of translation Cell growth

RNA

RNA/DNA

3’-+5’

RNA

1

DNA

DNA

3’45’

RNA

RNA

3’-+5’

Initiation of replication Replication7

Essential in yeast Nuclear localization Gsential for replication Essential for replication Inclusion bodies Gsential

RNA splicing? Transcription?

DNA/RNA

3’45’

Replication

DNA

DNA

I

DNA

DNA

5’+3’

gp41

Phage T4

Homohexamer Oligomer

DNA

DNA

5’+3’

Replication, transcription Chromosome replication Replication

F4

pp4

Phage T7

Homodimer

DNA

DNA

5’+3’

Replication

F5

Rho

Homohexamer

RNA

RNA/DNA

5’+3’

Termination transcription

E. calf

Essential for transfer UV sensitivity

Referellceh

Recombination

RNA

DNA’IRNA

Phenotype.3

Pmces%

of

f62.1 wJ2*1 1861 187.881 133,341 1311 1891

Essential

124.901

Essential for replication Essential for replication Essential for replication Essential

11.23*1 11.23*1 11.23el Il.811

40r the majority o( the helicases, only limited data are available. ‘JProtein superfamilies BFI and lamilies ~FI have been described in the lollowing studies: SF1 115-171. SF2 Il6-201. SF3 6 Koonin, unpublished data) I211 F4 123-l. FS fEV Koonin, unpublished data) f8ll. F4 and F5 are newly introduced designations, respectively, for the DnaB-like helicase family, end for Rho helicase, which is related to proton-translocating ATPares. c+. Helicase activity predicted prior to actual demonstration: o, sequence became available after the helicase grouping has been intmduced; #, helicase activity detected only in the presence of other proteinlr) [for detail on RecD protein, see tenl. HSV-1, herpes simplex virus, type 1; VV, vaccfnia virus; PPV, plum pox virus: SV40, simian virus 40; AAV, adeno-associated virus; MVM, minute virus of mice. 4Where available, the apparent native subunit structure of the enwe is indicated. %itially duplex polynucleotides are used to assay helicae activity. The strand to which a helicase binds to start its movement is indicated as the bound polynucleotide. The complementary strand is indicated as the displaced polynucleotide:, Site-specific DNA binding observed. ‘The direction of the helicase movement along the bound pOlynuCleotide chain is regarded as the direction of duplex unwinding. In several cases, movement of the enzyme in both directions was observed under different array conditions. The fess favcuxable option is shorn, in parentheres. Sonly selected processes and phenotypical features are indicated. %itations are limited to review and/or the two most recent papers on helicase characterization and/or classification.

Sequence-based classification a critical evaluation . An overview

of the helicase

of the helicases:

groups

The helicasescan be divided into distinct groups of different size and rank based on the results of amino acid sequencecomparisons.The criteria for delineation of the groups and for assignmentof a specific rank to them have not yet been worked out in detail. Here, we use a twolevel classihcationincluding family and superfamily (SF) ranks to distinguish between casesof strong and weak sequence similarity. Three vast superfamilies and two smaller families have been described (Fig. 1). Each of the superfamilieshas a developed internal hierarchy, and each contains severalclearly defined families. In general, sign&xx similarities are revealedby databasesearchus-

ing programs such as FASTA 1131and BLAST [14] only among members of these smaller families, not among all superfamily members. On the other hand, unique signatures can be used to identify at least the majority of the members of the superfamilies (see below). The majority of the (putative) helicasesbelong to the superfamilies SF1 [ 15-171 and SF2 [ 18-201.As the time of writing, SF1includes about 50 and SF2over 100 proteins encoded by prokaryotic, eukaryotic and viral genes (Fig. 1): The proteins of each superfamily contain simiiar sets of seven conserved motifs. These motifs are distributed along sequence regions ranging in length from -200 to -700 amino acid residues (Fig. 2). The similarity in the structure and arrangement of these motifs suggeststhat

Heiicases Gorbalenya

Fig. 1. .The main groups of helicases. The groups of helicases are designated by circles, with their diameters roughly proportional to the number of proteins in each group. The distance between the circles roughly reflects the relationship existing between the respective groups of helicases. The distinct groups of helicases within the three large superfamilies (SFI-SF3) are shown by small circles inside larger circles. Only selected groups, namely those including proteins with demonstrated helicase activity and/or containing numerous members, are shown. Other groups within SF1 and SF2, many of them including a single putative helicase, are shown by empty circles. Cenerally, groups are designated after the prototype (the best characterized) protein. Rho helicase is related not to other helicases but to proton-translocating ATPases. The sequence-based classification of helicases has not yet been developed consistently; hence, the scheme is very tentative and preliminary.

SF3

SF1and SF2might haveevolved from a common ancestor [191. SF3 initially consisted entirely of (putative) helicases of small DNA and RNAviruses [21]. These proteins contain only three conservedmotifs, including the A and B motifs of the NTP-binding pattern, tightly packed in a domain of - 100 amino acid residues [ 211.It has since been shown that human herpesvirus 6, a large DNA virus, encodes a related protein whose gene was probably derived by recombination with a parvovirus [22]. Very recently, it has been demonstratedthat SF3includes another putative helicase of a large DNA virus, a baculovirus, and at least one cellular protein encoded by the Marcbantia poLymo@ba mitochondrial genome (EV Koonin, unpublished data). Helicases related to bacterial DnaB protein comprise a relatively small compact family of proteins containing, in addition to A and B, three distinct conserved motifs [23*1. All of these (putative) helicases are functionally and physically associatedwith DNA primases, either as two domains of one protein, or as two distinct but interacting proteins. So far, only bacterial and bacteriophage members of the family have been described. Bacterial transcription termination factor Rho, which is a DNA-RNA helicase [24], shows significant sequence similarity to proton-translocating ATPases (EV Koonin, unpublished data) [25], demonstrating the lirst example of apparent evolutionary relationship between a helicase and a group of non-helicaseNTPases(Fig. 1). We are unawareof any proteins that possesshelicase activity but do not belong to one of the above five groups.

and Koonin

Predictive

power of sequence analysis: ‘true’ helicases

The recognition of motifs that are conserved in vast groups of helicases such as SF1 and SF2 has led to the obvious hypothesis that these motifs contain the amino acid residues most important for the helicase functions, specilically those Involved in catalysis and in substrate&and binding. Since the Introduction of the sequence-basedclassification scheme, the sequences of several new helicases with demonstrated activity have become available (Table 1). All of them have been found to fit perfectly in one of the already established (super)familIes, indicating that the putative functionally important motifs have been identied correctly. A particularly good illustration of this is the identi6cation of orthologous and paralogous relatives [26-29,3@] for the yeast helicase Rad3. At the time when SF2 was originally described, Rad3 was one of the two proteins in this superfamily with proven helicase activity [19]. The position of mutations impairing the activity of Rad3 in close proximity of the conserved motifs was invoked as an independent argument in favor of the identiiication of the latter [19]. At that moment, however,‘the very assignment of Rad3 to SF2 could be questioned because of the marginal level of sequence similarity between this protein and the other members of the superfamily, and becauseof deviations in some of the consensuspositions in the Rad3 sequence [19]. The recent comparative sequence analysis of Rad3-relatedproteins has conlirmed convincingly the original identiiication of the seven conserved motifs and grouping of the Rad3-related(putative) helicaseswith SF2 [30*].

421

422

Sequences and topology

wers

(b)

1 GF1 ‘EL’

p25 In5 UvrD

Human RUBV PYX PVX Hsv-1 E.coli

773 7 699 24 91 23

RecQ eIF4AI Fad3 uL9 18R HsdR SM-2 Suv3

E.coll House Yeast Ev-1 w IFIVPl Y!3ST YEA!X

43 70 36 75 179 301 786 233

p166

IRvuNmMGAGm ACVIHGA.#XGKS PLVVHAVAGAGKS VYLIXNAGSGKS NLLVLAGAGSGKT :: . *.*.a DCLWWXWK.9 DVIAQAQSGm NSI~TGKT VTwRAPmw PyvLTGGlWGKT GGYIWTDXGKT NGILADIIMHIGPTnSXT

Ia 10 15 15 11 20 13 19 18 15 30 10 12 5

DLYVCPTN 36 ITWLPTN 32 HTLGVPDK 19 CWl-GATFl 121 IMAVTFTN 152 .:. ... TVWSPLI 68 ALVLAPTR 68 IIYCSrTM 155 VLWSCFIR 57 VIISLPRI 62 WFVVdRK 57 YLVIVPU 58 GYYAGPLR 47

s&A

bds:lbdl6

II IYIDEAF VIFDdYS AILDFfT IVIDKAG ILVDEFQ :::a.. LAVDUH FVlDKAd VIFDwl LVLDKVm LIID!ZVH FIFD!XCH MIIDEGII VVUIEIq

mu

III 17 19 9 33 19 28 24 218 28 23 19 21 37

VICYGDRCQ VILTGESRQ QALFaDPYQ LVCVGsPTQ VnIWXlDDQ :.:.a. . MALTATADD VLBATMPS IITSGTISP IAMATANA FUITATLED FGFICTPIF LILXXPLQ Y KPRMUT

IV 19 27 9 43 25 52 56 72 83 65 32 178 28

ERsRHnm 55 YYLNATHR 51 FfLmsFR 57 AIFINNKR 461 IRLEQNYR 271 ... . . IIYCNSRA 43 VIFINTFIR 43 WFFPSYL 53 CIFSSNS 37 IVFVA!iVA 43 UFAVDYN 170 LIFFUMTQ 43 CVvMslM 39

V IKSVDGFQGRMEAV AYIVREACJGSVGTa TFIYAGCQGL’IKPKV FvkFcQvtGLmKw AMTITR.SQCLsLDKV LMlUSAI[cLEFpQV .

l .

24 21 17 17 15 30

VI VAvrRARR VeLlRASD tALSRATD nAIlRSKVANSIlTS VGVl’lW4Q

16 16 48 23 28 15 16 14

QETGRAGR hRIGFUXR QCLGRVLR QSLGWRt QFlKGRVGR QAFeRTNR QAQdRAHR QIGCRAGR

a:::.:.

VAT’VAFGHCINKPNV IlTDLLARGIDVQQV VArGKVSEGIDFDHQ I YTTWTVGLSF-DP ISlWLE%VTIfWV IVvGMFLTGFlMPTL ISIRAGGUXNLQTA VASDAIGMGLNlSID

. . . .

258 7 502 9 36 112 .

279 41 107 461 178 342 484 236

Fig. 2. Conserved sequence motifs in the helicase superfamilies I and IL (a) A scheme for the organization of the conserved motifs in a typical helicase. The data on (putative) helicases of SF1 and SF2 are summarized above and below, respectively, the black line depicting an ‘average’ helicase. Conserved motifs are boxed, and the range of spacer lengths between the motifs for all or the majority of the (putative) h&cases is indicated. The consensus shows amino acid residues that are conserved in at least 75% of the proteins belonging to the given superfamily; +, a hydrophobic residue; o, a hydrophylic residue; x, any residue. Proteins sharing inserts between certain pairs of motifs are grouped in the appropriate places and shown together with the size range of their inserts. Only selected members of the Snf2-like family are depicted. The proteins whose inserts or amino-terminal domains include (putative) zinc fingers are highlighted in bold type. In Rad16, the insert encompasses two separately located zinc fingers (751. HEL is the putative helicase of corona-like viruses 116,171. fb) Alignment of the conserved motifs in a sampling of proteins belonging to SF1 and SF2. The segments that have been only tentatively identified with the respective motifs italized. For each superfamily, sequences were selected so as to include a helicase with the complete pattern of conserved residues (UvrD and RecQ to SF1 and SF2, respectively) and (putative) helicases with deviations in one or several conserved positions. The distances between the motifs and the distances from protein termini are indicated. The consensus positions are highlighted by upper case bold typing except for deviating residues. For each of the two superfamilies, alignments were generated by computer-assisted methods as previously described I17,191. Superposition of the SF1 and SF2 motifs was done manually by aligning identical or similar conserved residues and (putative) conserved secondary structure elements I19,47J. Asterisks and colons show identities and similarities in the consensus patterns, respectively, and periods show positions where the consensus residue(s) of one of the superfamilies was found in a significant fraction of the protein(s) of the other superfamily. The putative yeast helicase Nam7 is identical to Upfl protein that has been originally erroneously described as a CTP-binding protein, and subsequently identified as a probable helicase [42°,801. RUBV, rubella virus; PVX, potato virus X (encodes two putative helicases).

As a direct result of the sequence-basedpredictions or independently, helicaseactivity has been detected in several proteins belonging to SFl, SF2 and SF3 (Table 1). Of particular importance is the demonstration of the RNA helicase activity of the potyvirus CI protein [31], which is the iirst experimental validation of the predicted helicaseactivity for a large class of positive-strand RNAvirus proteins. Also, the demonstration of the heUcaseactivity of both of the two herpesvirus helicases, ULS [32] arid UL9 [33,34], has been non-trivial because theseproteins contain replacementsof conserved amino acid residues in motifs IV (UL5) or III, IV and VI (UL9) which are not observed in other helicases.Thus, these experiments have shown that helicase activity might be compatible with significantly modified sequencepatterns. For several proteins, the helicase activity could be demonsttated only when assayedin the presence of a specific ‘accessory’protein subunit. This role has been assigned to protein eIF4I3 for the translation initiation factor eIF4A [35], to UvrA protein for the E. cofi helicase UvrB 1361,and to the ULS2 protein for the herpesvirus helicaseUL5 [321.

Of special interest is the functional analysis of the RecBCD complex. Its two subunits, RecB and RecD, belong to SF1 [ 15-171, and hence both could contribute to the documented helicase activity of the complex [l]; indeed, the puriiied RecB protein has recently been demonstrated to possessa helicase activity, albeit very low and non-processive [37.1. On the contrary, no helicase or ATPase activity could be detected for the RecD subunit although it was labeled by 8-aside-ATP [38]. Recently, the properties of two mutant RecBCD complexes reconstructed from individual subunits carrying the substitution of the invariant lysine residue in the probable ATP-binding site of either RecB or RecD have been studied in detail. The RecBCDcomplex with mutated RecBhas been shown to retain a fraction of the DNA-stimulatedATPaseactivity that most probably could .be attributed to RecD [39-l. Also, the processivity of the helicaseactivity of the RecBCDcomplex was significantly diminished in the RecDmutant [40*]. These experiments showed that RecBis definitely the principal helicase subunit of the RecBCDcomplex. It remains unclear whether or not conditions will be found under which RecD behavesasa bona fide helicase,but it seemscertain that this

Helicases

subunit contributes to the helicase activity of the complex in an ATP-dependent fashion. These results depict the apparent evolution of a helicase into an ‘accessory’ protein which does not necessarily possess helicase activity, at least when isolated, a situation which might have parallels in other systems. Taken together, the above data indicate that, at least for SF1 and SF2, there is a good correlation between the presence of the array of the conserved motifs in a protein sequence and the helicase activity. These motifs have been widely used for prediction of new helicases, and not unexpectedly, the pace of experimental work necessary to verify these hypotheses has not yet caught up with the rush of the predictions. Predictive helicases

power

of sequence

analysis:

putative

The putative, ‘computer-predicted’ helicases can be divided into two categories, according to how well their sequences conform to the patterns of conserved amino acid residues in the actual helicases. A large fraction of these putative helicases contain the precise consensus patterns. These include: proteins of the so-called ‘DEAD’ [18] and ‘DEAH’ [41] families (named after the speciiic signatures in motif II), representing two protein families within SF2; a number of prokaryotic and eukaryotic putative helicases of SF1 including the Senl-like family [42*] ; and the majority of the putative helicases of RNA viruses belonging to SFl, SF2 or SF3 (Fig. 1). Many of these proteins not only contain the exact conserved motifs but also show statistically highly sign&ant similarity to some of the proven helicases. For some of the putative helicases, DNA(RNA)-stimulated ATPase activity has already been demonstrated [43-46]. We believe that it is extremely likely that the proteins of this category possess the actual helicase activity. Putative helicases with deviations in the conserved motifs are also numerous. The relevant examples are: the R subunits of bacterial type 1 and type III restriction endonucleases [47], SecA protein [48-j, yeast suv3 gene product [49*] and Y’ protein [ 501; the whole families of the so-called Snf2-related [51-541 and Ercdrelated [55] proteins within SF2; and the group of RNA viral small helicase-like proteins belonging to SF1 (Figs 1 and 2) [ 171. In some of these proteins, certain motifs (particularly motifs III and IV in SF2) could not even be confidently identified. It cannot be ruled out that at least certain alterations of the conserved motifs may be incompatible with the helicase activity such that the respective proteins might lack this activity. The group of RNA viral small helicase-like proteins has been repeatedly regarded as an example of such proteins. The prediction of the helicase activity in these proteins has been criticized on the basis of experiments which showed that they are not required for the replication of viral RNAs, at least in protoplasts [56,57]. We believe that the evidence provided has not been conclusive and these proteins might exert helicase activity involved in viral RNA replication in viva and/or in other processes, including spread out of the viral RNA in plants. The demonstration of the helicase activity of the herPesvirUS UL9 and UL5 proteins (see above) and of the

Gorbalenya and Koonin

DNA-stimulated ATPase activity of two putative helicases of vaccinia virus [58-60] indicates that even highly conserved positions in helicases might have some potential for mutation without abrogating the activity. Significantly, the majority of the observed substitutions in deviant (putative) helicases are conservative and unlikely to result ln major conformation changes. For instance, the conserved glycine residue of the signature OJIxxGRxxR (using the amino acid one-letter code; x represents any amino acid) in motif VI, previously recognized as a hallmark of SF2, was found to be substituted by Ala, Ser, Glu, Asp or Asn in the sequences of several putative helicases described recently [47,50,52]. This range of replacements appears to preserve the p-turn potential in this position. The computer-predicted putative helicases with deviations in the conserved motifs are a special challenge for experimenters as their characterization might be particularly valuable for understanding the structur&unction relationship in this class of enzymes. How to recognize a helicase: limitations in their use

signatures,

patterns

and

In addition to being indicators of functionally important sites, the conserved motifs and patterns can be used for identification of putative new helicases by searching sequence databases and individual new sequences. A and B motifs of the Walker-type NTP-binding pattern are the only sequence elements shared by all known groups of helicases. These motifs are present not only in the hellcases but also in a wide variety of other NTPases. Thus, they are necessary but not suiBcient to identify a helicase, and as long as other universal helicase motifs are not known, a single unique pattern for selective retrieval of all the helicases from a database cannot be derived. To construct identifying patterns for distinct groups of helicases, it is necessary to combine the NTP-binding pattern (or the A motif alone) with at least one of the other conserved motifs. Such patterns have been generated for SF1 and SF2, and for their subdivisions, as well as for the DnaB-like family (Table 2). The patterns for the two large superfamilies were not however all-inclusive, illustrating the limitations of the approach. Further loosening of the patterns led, predictably, to the retrieval of an increasing number of false positives. SF3 is even less amenable to signature/pattern analysis because of the scarcity of the conserved motifs. Only smaller groups within this superfamily could be identified using simple patterns (not shown). The optimal strategy for sequence-based identification of helicases inevitably should be complex, combining various methods for motif detection and multiple alignment generation with detailed database searches for sequence similarity. Structural helicases

and functional

dissection

of

Original hypotheses on the organization of conserved and variable regions in the spatial structure of the hellcases 115-211 can be evaluated in light of the analysis of accumulating new sequences and the results of sitedirected mutagenesis.

423

424

Sequences and topology

rable 2 Identification

of helicases using amino acid sequence patterns. Helicase group

Retrieved

Missed

False positives

?xx&xGKfTSlx2f&Alx, &HH&AIDtDUx,lTSNlx4~QKl~x,~~A~ c,lVTlxRJTClfTSlR tiotifs I, II, V, VI

SF1

53 (putative) helicases

‘Accessory helicases of positive-strand RNA viruses having two putative helicase genes

One SFII helicase (hepatitis C virus)

XKISTlx, &AIf&ACIDEx~DHQk, QHlx,Rx,R Motifs I, II, VI-relaxed

SF2

103 (putative) helicases

Putative helicases of herpesviruses fUL91, SrmB

One SFI helicase (RadH), three nonhelicase NTPases

‘atterna

I

(E. co/r), suv3

pattern

(yeast), y’ (yeast) putative helicase of the fungal doublestranded RNA virus HyAV. GxCKISTlx, [&A[&ACIDExfDHlx, !QHICx2Rx2R Motifs I, II, VI-stringent

SF2

89 (putative) helicases

As for the previous pattern, plus SnfZlike superfamily (13 proteins)

None

DnaB family

10 (putative) helicases

None

None

pattern

LJUx&JGKISTlx,UU&xDfYHIUxJJx2U K,UIKRI[GAI&A Motifs I, II, Ill

The designations of the motifs for SF1 and SF2 are after [I91 and designations for the DnaB family are after [23*1. U indicates a bulky aliphatic amino acid residue (I,LV,M) and & indicates any bulky hydrophoic residue (I,L,V,M,F,Y,W); x indicates any residue; alternative residues are shown in square brackets.

The helicase domain

Most of the helicasemotifs are predicted to havean analogous structure, P-strand-p-turn-(u-helix>,which is similar to the crystallographicallydetermined structure of the A and B motifs in NTP-binding proteins [13,17,19]. The putative active (charged or polar) amino acid residues are typically located in the central turn or loop, and the amino-terminal /3-strandis usually enriched in bulky hydrophobic residues. A degree of sequence similarity has been found among different motifs in SF2helicases and it has been speculated that they might have evolved via consecutive duplications of a single ancestor motif [19]..Motifs V and VI in the helicases of SF1 and SF2 might have a different structure, ar-helix-@urn-p-strand [19]. Based on the results of sequence comparisons, and on the analogy with other NTPaseswhose spatial structure has been resolved, it has been postulated that the helicases contain a conserved core which consists of a p-pleated sheet with intervening loops and helices [17,19]. The possibility has been discussed that motifs V and Vl in the SF1hekases, which are separatedfrom the upstream motifs by a spacer of variable length, might reside in a distinct domain [61]. A dramatic confirmation of this idea might come from the recent observation that mammaliantranscription factor GF-1 appears to contain

only motifs V and VI, and not the helicasemotifs (Fig. 2) [42-l. In the proteins of distinct families within SF2 (DEAD family [62*], Snf2-like family [ 51-541, and Rad3-likefamily [30*]), a few additional conserved segments outside the sevenprincipal motifs have been found. Pronounced sequenceconservation is maintained in helicase families despite the diversity of their physiological functions and the wide range of organisms from which they originate. On the other hand, an example has been described when apparently functionally similar putative helicase domains found in the R-subunits of type I and type III restriction endonucleaseshave little in common in their amino acid sequences,even in some of the conserved motifs [47]. In a number of recent studies, the importance and sometimes the specilic functions of distinct regions of the helicase domain have been tested. Most frequently, point ammo acid substitutions have been introduced into the conserved segment I (the A motif of the Walker box). The effect of substitutions of the invariant lysine residue implicated in interaction with the y-phosphate of NTP [12] was drastic in nearly all of the test systems. Specifically, replacement of this residue, usually by glutamine, abolished the ATPase and helicase activ-

Helicases

ities of the UvrAB [63] and eIF4AB [64**1 complexes, and of the RecB [39], Rad3 [651, and PriA [66*] proteins. Further, such a replacement partially inhibited the labeling of RecD protein by 8-azido-ATP [67], in accordance with the implication of this lysine and motif I as a whole in NTP binding and hydrolysis. Surprisingly, the invariant @sinein motif I of the E. coli Rho helicase was shown to be dispensable for all known activities of this protein [68]. It has been suggestedthat its function could be relegated to another, non-conserved lysine located in the postulated loop’of motif I [68]. Interesting experiments including mutagenesis of motif II (B) have been performed with the T antigen of simian virus 40 [69]. It has been shown that substitution of alanine for both negatively charged amino acid residues in motif II (B) fully suppresses the activity in virus DNA replication but inhibits the helicase and the ATPaseactivity by only -10%. It cannot be ruled out that, in the mutant T antigen, the function of Mg*+ binding might be adopted, albeit inefficiently, by some other properly positioned negatively charged residue(s). The most detailed analyseshave been reported recently for herpesvirus helicases UL5 [70*] and UL9 [71*], and for eIF-4A, the prototypic member of the ‘DEAD’ family of RNA helicases [64**,72*]. In both viral proteins, mutations have been introduced in the six conserved motifs but only their effect on virus reproduction has been tested. In almost all of these mutants, reproduction has been abolished, the only exception being the replacement of the conserved serine by a similar threonine or alanine residue in motif III of the UL9 helicase [71*].

In the thorough study of eIF4A, the effects of mutations in several positions of the conserved motifs I, II, III and VI on ATP binding, ATP hydrolysis and unwinding of duplex RNA have been examined separately [64**]. First, it turned out that some of the mutations in each of the segments had the effect of uncoupling the ATPase and the helicase activities. Interestingly, the ATPase activity was even enhanced in most of these mutants. The most effective uncouplers were mutations in motif III that resulted in a very high ATPase activity but completely abolished the helicase activity [64**]. Secondly, it was shown that mutations in the distal segment VI impaired not only the RNA helicase but in part also the ATPase activity. This is compatible with the prediction that this segment is juxtaposed with the other conserved segmentsbut is less compatible with the hypothesis that it might be involved (solely) in RNAbinding [18,19]. Thirdly, evidence has been obtained in support of interaction between motifs II and VI [64**]. A correlation between the conserved sequencesin these motifs had been noticed previously, in that the proteins having the signature DEAd in segment II had hxzGRxzR (x indicates any residue; residues for which correlated substitutions have been noticed are shown in lower case) in segment VI, and proteins with DExh in segment II had glutamine in place of his&line in motif VI [I9]. The work of Pause and Sonnenberg [64=*] has shown that substitution of glutamine for histidine in motif VI in wild-type (DEAd) background completely abolishes the h&case activity but only partially impairs the ATP-binding

Gorbalenya and Koonin 425

and ATPaseactivities. The same mutation in the mutant. background (DEAh) does not affect the already modiiied levels of the ATP-binding and helicase activities and onIy slightly decreasesthe ATPaseactivity [ 64**]. III several recently sequenced (putative) helicases, the fourth position in the signature DEAx is occupied by residues other than aspartate or histidine whereas the glutamine in the qxzGRx2R signature of motif VI is conserved [49*,52,62*]. This indicates that, despite the correlation between motifs II and VI, a broader range of variability than previously believed might be compatible with the helicase activity. The coordination of replacements in the conserved positions occupied by polar residues might be a general phenomenon for the helicase-specific motifs. The pronounced diversiiication of conserved sequencesin motifs III and VI between the helicasesof SF1 and SF2 (Fig. 2) also could be rationalized within this framework. Only one of the conserved amino residues found in the helicase-speciiic motifs (i.e. excluding motifs I and II), namely arginine in motif VI, is strictly invariant in all (putative) helicases of SF1 and SF2 (Fig. 2). It is tempting to speculate that this residue plays a pivotal role in the helicase function and might be directly involved in the distortion of hydrogen bonds in polynucleotide duplexes. We are unaware of any data showing that any one of the conserved motifs is not important for the helicase activity although the results of some studies [68,69] have revealeddiI%cultiesin assigning specific functions to particular conserved residues. Also, some of the helicases have additional activities that might not be influenced by impairment of the function of the helicase domain. This is illustrated by the recent experiments with the PriA helicase,which showed that a mutation in motif I abolished its ATPaseand helicase activities but not its capability to mediate the primosome assembly [ 66*]. Optional

domains

In many proteins, the core helicase domain is combined with additional domains. It is striking that such domains may not only Rankthe helicase domain from one or both ends but also may be inserted between the conserved motifs (Fig. 2). The preferred placement for such ‘optional’ domains is between motifs Ia and II, or IV and V in both SF1 and SF2, and additionally between motifs II and III, or III and IV in proteins of SF2. On the contrary, the distances between motifs I and Ia and between motifs V and VI are highly conserved. An example of an insertion domain in helicasesis the (putative). zincfinger domain that has been found in three different locations in PriA [73], RADS [74], and RADl6 [75], all of which belong to SF2. Conceivably, this domain might bind DNA and function in conjunction with the helicase domain. Some of the Ranking domains might also be involved in the same process with the helicase domain (Fig. 2). Relevantexamples are E. coZiTraI protein, NSl proteins of parvoviruses, and AL1 proteins of geminiviruses, in which the helicase is the carboxy-terminal domain and the amino-terminal domain is an endonuckase invohed in the initiation of rolling circle DNA

426

Sequences and topologY

replication [76]. similarly, in the primase-helicaseproteins of bacteriophages ‘I7 and P4, the amino-terminal domains are primasesand the carboxy-terminal domains are D&&related helicases f761. Yet in other helicases, the flanking domains appear to have a function lndependent of the helicase domain. This is exemplified by the NS3 helicase of flaviviruses and nsP2 helicase of alpha&uses, whose amino- and carboxy-terminal domains, respectively,are proteasesinvolved in the processing of the virus-encoded polyprotein [77]. Conclusions

and future

directions

The helicaseshave been described as an important but narrow class of enzymes involved in DNA metabolism. Our current estimateof the fraction of helicasegenes in both prokaryotic and eukaryotic genomes indicates surprisingly that they comprise as much as 1% of the total number of genes (EV Koonin and AE Gorbalenya, unpublished data). Numerous recent reports have shown that helicases are involved in virtually every aspect of genome replication, repair and expression. We believe that the computer-assisted analysis of the amino acid sequencesof helicases has reached a relatively mature stage.The most important sequencemotifs conserved in vast groups of helicases appear to be alreadyknown. These motifs can be used for the purposeful finding of new helicasesbelonging to specific families using the polymerasechain reaction, as demonstrated by recent work on the identilication of putative DEAD helicasesin yeast [78] and in E. colz’[79]. Along with our expanding of knowledge on particular groups within superfamilies, construction of a complete sequence-based classilication of helicasesis becoming a tractable goal. Remarkably, the enormous diversity of the helicases seemsto be achieved by variations of only a few structural themes, with the obvious dominance of the one based on the seven conserved motifs typical of SF1 and SF2. Surprisingly, the conservation of this structural theme does not seem to have any equivalent in the biochemical and biological properties of the helicases (Table 1). We cannot however rule out that such a common denominator actually exists but the currently employed experimental approaches are inadequate for its detection. Determination of the threedimensional structure of helicasesand manipulations with their core and optional domains, concomitant with the monitoring of various biochemical activities, will hopefully result in a solution to this ex@ing problem. Note added in proof

While this paper was being processed for publication, several important Endings were reported that add new dimensions to the area of helicase research.It has been shown that RuvAB protein complex of E. coli is a DNA helicase invokd in the resolution of Holiday junctions during recombination [9l]. One of the subunits of this complex, the RuvB protein, contains the Walker-type NTP-binding pattern but does not belong to any of the

groups of helicasesdescribed here. Rather,RuvBappears to belong to a vast superfamily of (putative) ATPases whose extremely diverse functions include regulation of * ATP-dependent proteolysis (bacterial ClpA-related proteins), membrane biogenesis (yeast PASl, SEC18) and others (AE Gorbalenya, EV Koonin, unpublished data). These results provide a new example (after the case of RI-IO)of sign&ant sequencesimilarity between a helicase and ATPasehaving different functions and show that the structural diversity of helicases is not yet completely explored. very recently, an attempt has been made to delineatesimilar motifs in the ‘ClpA-related’superfamily (however, RuvB has not been included) and the helicase SF2 [92]. Although an ancestralrelationship of this type might be real, we believe that is it manifested at an even much lower level of significant than the relationship between SF1and SF2discussedin this review; the current methodology of sequence analysismay be not quite adequate to assessobjectively similarities of this kind. Two other studies have shown the role of proteins belonging to SF2 in coupling transcription and repair of lesions in DNA One of these proteins, the product of a newly identified E. coli gene (mfd), is related to RecG but fails to show helicase activity when isolated, although it is involved in DNA-RNAhybrid dissociation in the transcription complex containing damaged DNA [93]. An interesting feature of the Mfd protein is that it shares a related domain with UvrB, another protein of SF2; it has been hypothesized that this conserved domain may be involved in the interaction with UvrA protein. It is located differently, however, in the two proteins, namely upstream of motif I in Mfd and between motifs Ia and II in UvrB, demonstrating in a spectacular manner the module organization of helicase-like proteins. The second transcription-repair coupling factor, human protein ERCC3,has been shown to comprise one of the subunits of the basic transcription factor TFIIH and did possesshelicase activity when assayedindividually [941. These studies not only revealed a new important function of helicase-like proteins but also emphasized once more that caution is due in the direct interpretation of the presence of the helicase motifs in a protein sequence as an indication that the protein in question should have activity when puritied and tested in in vivo unwinding assays(see the discussion of the predictive value of the helicase motifs above). Acknowledgements The authors thank A Sankar for helpful discussions, D Landsman for critic-d reading of the manuscript, P Bork and G Darai for communicaring data before publication, and N Altamura for pointing out the identity between the sequences of Nam7 and Upfl. The literature on heiicases is mt and is growing rapidly. We ask all those investigators work could not be cited in this review because of whose important space and/or scope limitations to accept our sincere apologies.

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and recommend&d

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the annual

period

of

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IAIN S, R~ECHMANN JL MARTINMT, GARCIAJA Homologous Potyvirus and Flavivirus Proteins Belonging to a SuperfamBy of Helicase-Like Proteins. Gene 1989, 82:357-362.

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WUSONGM, J~NDALHK, YEUNG DE, CHEN W, &ZEU CR: Expression of Minute Viis of Mice Major Nonstt~~tural Protein in Insect Cells: Puritication and IdentiBcation of ATPase and Helicase Activities. Pimlogy 1991, 185:90-98.

CAPONPR, GROSSMAN L Potential Role of Proteolysis in the Control of UvrABC Incision. Nucleic AciCCFRes 1988, 16:1090310912.

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REYNOLDS PR, BIGCARS, P~AKASH L.,PRAKASH S: The Scbizosaccbaromyces pombe rhp3+ Gene Required for DNA Repair and CeR Viability Is FunctionaLly interchangeable with the RAD3 Gene of Saccbaromyces cereuisiae. Nucleic Acids Res

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MURRAY JM, DOE CL, SCZHENK P, CARRAM, LEHMANN AR, WAT~X FZ: Cloning and Characterization of the X pombe fad15 Gene, a Homologue to the S. cereuisiae RAD3 and Human ERCCZ Genes. Nucleic Aciak Res 1992, 20:2674-2678.

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WONGI, LOH~ TM: Auosteric Effects of Nucleotide Cofactom on Escbericbia coli Rep Helicase-DNA Biding. Science 1992, 256:350-355. A thorough study of the interaction of the dimeric Rep helicase with ATP and its non-hyrolyzable analog. It is proposed that the translocation of the helicax along DNA is coupled to ATP binding whereas the energy of ATP hydrolysis might be utilized directly to unwind multiple base pairs in each catalytic act.

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9.

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and Koonin

Whuett JE, SARAS~F M, RUNSW~CK MJ, GAY NJ: Distantly Related Sequences in the n- and j3-Subunits of ATP Synthase, Myosin, Kinases and Other ATP-Requiring Enzymes and a Common Nucleotlde Biding Fold. EMBOJ 1982, 13945-951. GORBALENYA AE, K~NIN EV Viis Proteins Containing the Purine NTP-Binding Pattern. Nucleic Acids Res 1989, 1784138440.

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ScHUtZGE: Binding of Nucleotides by Proteins. Curr Opin Struct Biol 1992, 2:61-67. ;“ne latest concise review of the sequence and kucture conservation in NIP-binding proteins. 13.

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KOONLNEV: Escbericbia CONdinG Gene Encodes a Putative DNA Helicase Related to a Group of Eukaryotic Hehcases Including Rad3 Protein. Nucleic Acids Res 1993, 21:1497. Characterization of the sequence conservation in the family of Rx& related (putative) helicases. 30. .

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IAIN S, R~ECHMANN JL, GARCLIJA RNA Helicase: a Novel Activity Associated with a Protein Encoded by a Positive Strand RNA Vii. N@eiC Acids Res 1990, 18:70037006.

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DODSONMS, LEHMANIR: Association of DNA H&case and Primase Activities with a Subassembly of the Herpes Simplex Viis 1 HeEcase Primase Composed of the UL5 and UL52 Gene Products. Proc Nat1 Acud Sci USA 1991, 88:110~1109.

HODGMANTC: A New Superhmify of Repkative Proteins. Nature 1988, 33322-23. [published erratum appears in Nature 1988, 333:579.1

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F~ERER DS, CHALBERG MD: Purification and Characterization of UL9, the Herpes Simplex Virus Type 1 Origin-Binding Protein. J ViroI 1991, 66:3986-3995.

G~RBAIXNYA AE, KOON~NEv, DONCHENKO AP, BLINOVVM: A Novel Superfsmily of Nucleoside Triphosphate Binding Motif-Containing Proteins which Are Probably Involved in Duplex Unwinding in DNA and RNA Replication and Recombination. FEE&SLett 1988, 2391624.

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PE, E~~MERSON PT: The RecB Subunit of the Es cbetfcbiu coli RecBCD Enzyme Couples ATP Hydrolysis to DNA Unwinding. J Biol Cbem 1992, 267~4981-4987. The first demonstration of helicaseactivity in isolated RecB protein.

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TI~OELSTRA C, VANGoon A, DE Wrr J, VERMEULEN W, BOTTSMA D, HOE~JMAKE~~S JHJ: ERCC6, a Member of a SubfamIIy of Putative Helicases, Is Involved in Cockayne’s Syndrome and Preferential Repair of Active Genes. Cell 1992, 71:939-953.

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Jti~w DA, LEHMANII& Photoaffinity Labeling of the RecBCD Enzyme of Escbericbiu calf with 8Azidoadenosine 5’Triphosphate. J Biol C&em 1987, 26239~9051.

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42.

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44.

N~HI K, MOREL-DavtusF, HERSHEY JWB, LEIGHTON T, SCHNIER J: An eIF4ALike Protein Is a Supressor of an Eschericbiu eolf Mutant Defective in 50s Ribosomai Subunit Assembly. Nature 1988, 336:496-498. WENG~RG, WENGLER G: The Carhoxy-Terminal Part of the NS3 Protein of the West Nile Aavivirus Can Be Isolated as a Soluble Protein after Proteolytic Cleavage and Represents an RNA-Stimulated NTPase. Virology 1991, 184:707-715.

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63.

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64. ..

48. .

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49. .

50.

Louts EJ, HABERJE: The Structure and Evolution of Subtelomeric Y’ Repeats in Saccbaromyces cerevfsfae. Genetics 1992, 133:559-574.

51.

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66. .

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