Molecular Microbiology (1992) 6(1), 5-14

MicroReview Escherichia coii DNA helicases: mechanisms of DNA unwinding T. M. Lohman Biochemistry and Molecular Biophysics. Washington University Schoot of Medicine, Box 8231. 660 South Euclid Avenue, StLouis. Missouri 63110-1093, USA. Summary DNA helicases are ubiquitous enzymes that catalyse the unwinding of duplex DNA during replication, recombination and repair. These enzymes have been studied extensively; however, the specific details of how any helicase unwinds duplex DNA are unknown. Although it is clear that not all helicases unwind duplex DNA in an identicai way, many helicases possess similar properties, which are thus likely to be of general importance to their mechanism of action. For example, since helicases appear generally to be oligomeric enzymes, the hypothesis is presented in this review that the functionally active forms of DNA helicases are oligomeric. The oligomeric nature of helicases provides them with multiple DNA-binding sites, aliowing the transient formation of ternary structures, such that at an unwinding fork, the helicase can bind either singie-stranded and duplex DNA simultaneously or two strands of singie-stranded DNA. Modulation of the relative affinities of these binding sites for single-stranded versus duplex DNA through ATP binding and hydrolysis would then provide the basis for a cycling mechanism for processive unwinding of DNA by helicases. The properties of the Escherichia coli DNA helicases are reviewed and possible mechanisms by which helicases might unwind duplex DNA are discussed in view of their oiigomeric structures, with emphasis on the E. coii Rep, RecBCD and phage T7 gene 4 helicases.

Introduction Processes such as DNA replication, recombination and repair require the formation of intermediates in which Received 8 August, 1991; revised 26 September, 1991, Tel, (314) 362 4393; Fax (314) 362 7183,

regions of duplex DNA are unwound transiently, yielding partially single-stranded (ss) DNA. The unwinding of duplex DNA during these processes is catalysed by a class of enzymes, DNA helicases. which catalytically destabilize the hydrogen bonds between the base pairs in a reaction that is coupled to the hydrolysis of nucieoside5'-triphosphates. (Note that helicases are distinct from topoisomerases, which are enzymes that cleave and religate the covalent phosphodiester bonds in the DNA backbone.) tn these reactions, an unwinding fork propagates along the DNA from the site of initiation, resulting in a progressive 'unzippering' of the duplex DNA, which can be facilitated by the stcichiometric binding of a single-strand binding (SSB) protein, as depicted schematically in Fig. 1. Some helicases can unwind duplex DNA in vitro at rates of 500-1000 bp s"""; this high rate Increases interest in understanding their mechanisms of action, DNA helicases. first discovered in 1976 (Abdei-Monem ef ai. 1976), appear to be ubiquitous in both prokaryotes and eukaryotes (Geider and Hcffmann-Berling, 1981; Matson and Kaiser-Rogers, 1990; Thommes and Hubscher, 1990). RNA and RNA/DNA helicases have also been identified and these have been proposed to function during transcription (Steinmetz et ai, 1990). translation (Ray etai., 1985) and RNA splicing (Company etai, 1991). An increasing number of putative helicases have also been identified on the basis of comparisons of their primary structures (Hodgman, 1988; Lane, 1988); however, the extent to which such sequence comparisons can identify true helicases is unknown. The molecular details of the mechanism of DNA unwinding are unknown for any helicase. Although it is clear that all helicases do not unwind DNA by the same precise mechanism, various helicases display enough similarities in their properties to suggest that aspects of these mechanisms must be similar. One property that appears to be general is the propensity of helicases to self-assemble into oligcmeric structures; the hypothesis is presented here that the functionally active forms of most, if not all. helicases are oligomeric and that this is centra! to the mechanisms by which these enzymes unwind duplex DNA. The DNA-unwinding mechanisms discussed here, which are based on this hypothesis, are speculative; however, they are presented to stimulate

6

7". M. Lohman

TA.

Rg. 1. A schematic drawing representing the progressive unwinding of duplex DNA by a helicase (triangle), with subsequent binding of the ssDNA by a single-stranded binding (SSB) protein (circles).

thinking and future experiments about these fascinating and important enzymes. Because of space limitations and the focus of this review, a general discussion of the functions of Escherichia coli DNA helicases cannot be presented and only recent references are cited in many cases; however, comprehensive reviews of the properties and functions of DNA helicases are available (Matson and Kaiser-Rogers, 1990; Matson. 1991). Processes involving E. coHDUA helicases Because of the large number of E. coti helicases (see Table 1) and the potential for complementation in vivo, the precise physiological function of some of these helicases remains uncertain. For example, although the E. coli Hep and Helicase II proteins have been implicated in E. coti replication, strains carrying independent mutations in the rep or uvrD genes (encoding Helicase II) are viable (Colasanti and Denhardt, 1987); however, rep/uvrD

double mutants appear to be lethal (Washburn and Kushner, 1991; Taucher-Scholz etai., 1983). Similarly, some rep/rho{ts) double mutants are non-viable at temperatures that are permissive for the rho{Xs) mutant (Fassler et at., 1985). Because of such complementation and the potential existence of suppressor mutations, it is difficult to draw firm conclusions concerning whether a particular helicase is involved in an essential function such as DNA replication, based on genetic data alone. In another example, a priA mutant strain of E. coli remains viable even though the PriA helicase (n', factor Y) is known to function in assembly of the primosome (Lee and Kornberg, 1991; Nurse efa/., 1991). The E. coti DNA helicases are discussed below, according to the process within which each is known or believed to function. In addition to these, the E coti Rho protein is an RNA/DNA helicase that functions in transcription termination (Steinmetz etat., 1990). DNA reptication tn view of the fact that the DnaB helicase is an essential reptication protein, it has been proposed as the primary replicative helicase in E. coti (Lebowltz and McMacken, 1986). However, since other helicases are also likety to function in E. coti DNA reptication, it is not ctear if DnaB is essentiat because of its heticase activity or its rote as part of the primosome. The E. coti Rep helicase, atthough not an essentiat E. coti protein (Cotasanti and Denhardt, 1987), is required for reptication of a number of ssDNA phages, such as OX174 and f i (Lane and Denhardt, 1975). In addition, mutations in the rep gene result in a -twofold stower rate of reptication fork movement in E.

Table 1. DNA unwinding properties of E. coli and phage helicases.

Optimum DNA Substrate for Unwinding

Helicase

unwinding directionality^

stimulated by forked tail?

DnaB Rho T4dtfa T4 gene 41 17 gene 4 Helicase 1 (F factor tra i) Helicase III UvfAB RecBCD (exo 10 Helicase II [uvrtyj Helicase IV (rte/D) Rep PriA (n' protein) RecQ

5' to 3' 5' to 3' 5' to 3' 5' to 3' 5' to 3' 5' to 3' 5' to 3' 5' to 3' Undefined 3" to 5' 3' to 5' 3' to 5' 3' to 5' 3' to 5'

Yes ? ? Yes Yes ? ? ? No 9

7 7 7 "^

optimum length of 3' ss tail

optimum length of 5' ss tail

unwind blunt-end DNA?

-90 nt

7

7 7

?

>29nt >7nt

a72nt 17 nt

7

9

No f ? No No No

7

7

7 7

7 7

No"

No"

7 7

7 7

Yes Yes ?

>4nt ?

? ?

7

9

No

? Yes

a. Defined by whether a 3' or 5' flanking single-stranded DNA is required for initiation of unwinding. b. Inhibited by a 3" or 5' single strand that is >25 nudeotides.

Escherichia coli DNA heticases coti, Indicatitig that it piays some role in £. coli replication (Lane and Denhardt, 1975). Heiicase II has also been implicated in E. co//DNA replication (Klinkert etat.. 1980). On the basis of the fact that a number of heiicases appear to possess different polarities of DNA unwinding (see Table 1), it has been proposed that two (or more) helicases may be active in E. coli DNA replication, with one functioning on the leading strand and the other functioning on the lagging strand (Yarranton ef at., 1979; Taucher-Scholz et at., 1983). In addition to DnaB, possible candidates are Rep, Helicase II, Helicase 111 and PriA (Lee and Marians, 1989; 1990). DNA repair Helicase II (UvrD) is required for methyl-directed mismatch repair and UvrABC excision repair. The ability of DNA helicase II to initiate DNA unwinding from a nick (Runyon et al., 1990) may be important for its function, since both repair pathways go through nicked DNA intermediates. A complex of the UvrA and UvrB proteins, both of which are components of the excision repair pathway, possesses helicase activity (Oh and Grossman, 1989). The Rep protein has also been implicated in DNA repair (Bridges and von Wright. 1981). Recotnbination The E. coli RecBCD enzyme (exoV) is involved in homologous recombination (Taylor, 1988; Smith, 1990). It is believed to function by unwinding duplex DNA through its helicase activity, thus providing single-stranded regions of DNA for use in recombination as a substrate for the RecA protein. In this respect, RecBCD's ability to recognize 'chi sites' and specifically introduce a nick in duplex DNA during its unwinding reaction is also important for recombination (for reviews see Taylor, 1988; Smith, 1990; Kowalczykowski and Roman, 1990). The RecQ protein is involved in the RecF recombination pathway in E. coti and possesses helicase activity (Umezu et at., 1990). Based on its ability to unwind blunt-ended DNA in vitro, RecO may play a role similar to that of the RecBCD helicase (R. Kolodner, personal communication). Conjugal transfer The trat gene of the E, coti F factor encodes Helicase I, the first helicase characterized biochemically (AbdelMonem et at.. 1976), This 192 kDa polypeptide possesses both a highly processive helicase activity (AbdelMonem etat., 1976; Lahue and Matson, 1988) as well as a nicking activity (Traxler and Minckley, 1988; Reygers et at.. 1991; Matson and Morton, 1991), both of which are required for transfer of the F episome during conjugation.

7

Fig, 2. Representation of a DNA substrate designed to investigate the macroscopic 'directionality' or 'polarity' of DNA unwinding by a helicase.

Processivity and directionality of DNA unwinding and translocation A number ol DNA helicases, e.g. RecBCD, Helicase I, DnaB and Rep (in the presence of the *X174 cisA or fi gene II proteins) unwind DNA with high processivity, (The processivity of DNA unwinding by a helicase is a measure of the number of base pairs unwound for each DNA binding event. At a molecular level, this is related to the relative probabilities for unwinding a base pair versus dissociation of the helicase.) Highly processive unwinding of DNA is a property expected for helicases involved in DNA replication and recombination. However, quantitative measurements of processivity have only been reported for the E co//RecBCD helicase; its maximum processivity corresponds to the unwinding of an average of -25000 bp per binding event (Kowalczykowski and Roman. 1990), (These conclusions are based on the assumption that a 1:1:1 RecBCD complex is the active form of the helicase and furthermore that only ~6% of the RecBCD in these studies was active (Roman and Kowalczykowski, 1989),) Clearly, heiicases that unwind DNA processively must translocate along fhe DNA during the course of unwinding. (Translocation is the process by which a helicase moves continuously, without dissociation, along the contour length of the DNA.) On the other hand, high processivity does not appear to be a property of all helicases in vitro. For example, unwinding of duplex DNA by the E, coii Helicase II protein, encoded by the uvrD gene, appears to be distributive and requires protein in quantities proportional to the extent of unwinding (AbdelMonem etai, 1977; Matson and George, 1987; Runyon et at., 1990). Although the degree of processivity observed in vitro may correlate with whether a helicase functions to unwind long or short stretches of DNA, this is difficult to assess since processivity may be modulated by accessory proteins and is also dependent upon solution conditions in vitro. With the exception of the RecBCD enzyme, all helicases display a preference for unwinding dupiex DNA possessing a 3' or 5' flanking ssDNA (see Table 1). In mosf cases, this preference has been investigated using a partially duplex DNA substrate, such as the one depicted in Fig. 2. If a helicase preferentially unwinds duplex A, then it is referred to as unwinding with a '5' to 3" directionality, with respect to fhe polarity of the infernal region of ssDNA. In interpreting resutts from such experiments, the general assumption is that the helicase binds

8

T. M. Lohtnan

to the interior ssDNA and translocates unidirectionally along the ssDNA until a duplex region is reached and subsequently unwound. Hence it is often concluded from such experiments that a particular helicase can translocate unidirectionally along ssDNA. However, such experiments do not indicate whether helicases translocate along ssDNA, much less in which direction. This is because any preference for unwinding duplex A or B in Fig. 2 also depends upon whether the DNA conformation existing at fhe ss/dsDNA junction will support initiation of unwinding by the helicase. Direct evidence for translocation along ssDNA has been obtained only for the E coli priA gene product (n' protein or factor Y) (Lee and Marians, 1989; 1990). The nature of the DNA substrate (i.e. whether or not the substrate is forked, and the precise length of the 3' and 5' flanking ssDNA) can affect the ability of some (and possibly all) helicases to initiate an unwinding reaction (see Table 1). For example, the RecBCD enzyme initiates unwinding preferentially at a blunt-ended duplex and initiation is inhibited if a single-stranded region of more fhan ~25 nucleotides exists on either strand (Taylor and Smith, 1985), Other helicases (e.g. DnaB, T4 gene 41 protein, T7 gene 4 protein. SV40 T antigen) show an increased DNA unwinding activity in vitro if the duplex DNA possesses a pre-formed forked structure. This suggests that these helicases interact with both DNA strands, at least transiently, in order to unwind duplex DNA. Although many helicases are able to unwind duplex DNA possessing only one ssDNA tall (either 3' or 5"), mosf have not been tested to determine whether initiation of unwinding is enhanced if the DNA substrate possesses a pre formed forked structure (see Table 1). It has generally been assumed that a replicational helicase displaying a 5' to 3' 'directionality' acts on the lagging strand (translocating 5" to 3'), whereas one displaying a 3' to 5' 'directionality' acts on the leading strand (translocating 3' to 5'), However, such inferences may prove misleading for helicases

which interact with both complementary single strands during unwinding, since a unique directionality cannot be defined in such cases, Oiigomeric helicases possess multiple DNA-binding sites In order for a helicase to catalyse the unwinding of duplex DNA, it Is anticipated that it must cycle, vectorially, through a series of energetic (conformational) stafes, driven by the binding and/or hydrolysis of ATP and subsequent release of products (ADP + P0|") (Yarranton and Gefter, 1979; Hill and Tsuchiya, 1981). Therefore, a molecular understanding of helicase-catalysed DNA unwinding requires information about the coupling of ATP binding and hydrolysis to DNA unwinding as well as the identification of the intermediate helicase-DNA states. It has been proposed that one intermediate state might involve simultaneous binding of the helicase to ss and duplex regions of the DNA (Yarranton and Gefter, 1979) and evidence for such ternary complexes exists for both the E.co/y Rep and DnaB helicases (Araie? a/., 1981a,b;l. Wong and T. Lohman, unpublished). Although it is possible that multiple DNA-binding sites might exist within a single polypeptide, this has not been demonstrated for any helicase. However, an important aspect of helicase structure that has not been considered explicitly in most discussions of DNA unwinding is the oiigomeric state of the helicase, especially when complexed with DNA. Although the self-assembly states and equilibria of many heiicases have not been examined, all helicases for which this has been examined are known to form oiigomeric structures. These oiigomeric structures probably provide a simple mechanism by which helicases acquire multiple DNA-binding sites. Table 2 lists the helicases that are known to form oiigomeric structures. With the apparent exception of the RecBCD enzyme, the oiigomeric state of these helicases

Table 2. Oiigomeric nature of helicases. Helicase

Oiigomeric form

Reference

£. coii DnaB SV40 large T antigen £. coii Rho E. coii RecBCD T4gene 41 protein T7 gene 4 protein £ co/i helicase III E. coii Rep E co//heiicase II

Haxamer Hexamer Hexamer Hetero-trimer (or hexamer) Oligomer (dimer?) (requires GTP) Oligomer (heterodimer?) Dimer Dimer (induced by DNA binding) Dimer (possibly higher oligomers)

HeLa heltcase HSV-1 origin binding protein

Dimer Dimer

Reha-Krantz and Hurwitz (1978) Mastrangelo etai. (1989) Finger and Richardson (1982) Dykstra etai. (1984) LiuandAlbens(1981) Bernstein and Richardson (1988) Yarranton etai (1979) Chao and Lohman (1991) G.T. Runyon and T. M, Lohman (unpublished) Seoe(a/. (1991) Bruckner efa'. (1991)

Escherichia coli DNA heticases appears to be limited to either dimers or hexamers. However, since the oiigomeric states and self-assembly equilibria of most helicases have not been examined, this conclusion may not be genera!. Furthermore, for most of the heticases in Table 2, the oligomerization state of the functionally active form of the helicase is unknown. For example, on the basis of its subunit composition, the minimal oiigomeric form of the RecBCD helicase is a heterotrimer, although its functional oiigomeric state has not been investigated; even the notion that the subunit stoichiometry of the enzyme is 1:1:1 has not been established firmly. There is good evidence that the active forms of af least three helicases are oiigomeric. The E. coti Rep protein is induced to dimerize upon binding ss or dupiex DNA and a chemically cross-linked Rep dimer retains both ssDNAdependent ATPase and DNA helicase activities and fhus appears to be the active form of the helicase (Chao and Lohman, 1991). The ssDNA-dependent ATPase activity of the Rep protein is also stimulated significantly upon dimerization (I. Wong, unpublished), consistent with fhe possibility that the ssDNA-dependent ATPase activity of the Rep protein may require dimerization. In a second case, the SV40 large T antigen forms a double hexamer when bound to its origin of replication in the presence of ATP (Masfrangelo etat.. 1989). Finally, the three-subunit composition of the RecBCD enzyme and the fact that the individual subunits do not support helicase activity also suggest that its active form is oiigomeric. For at least three of the helicases listed in Table 2, oligomerization is modulated by its interaction with another ligand. The E. coti Rep helicase exists as a stable monomer {M, = 72 802) up to at least 8 ^M in the absence of DNA; however, the binding of either ss or duplex DNA induces the Rep protein to form a stable dimer, with a K^ valueof ~10^M"' (Chao and Lohman, 1991; I.Wong, K. Chao and T. Lohman, in preparation). The oligomerization (dimerization?) of the phage T4 gene 41 protein (helicase/primase) is facilitated by the binding of GTP (Liu and Alberts, 1981). In the presence of ATP, the SV40 T antigen forms hexamers when bound to its origin of replication, whereas it forms tetramers in the absence of ATP (fvlastrangeloefa/., 1989).On the basis of these observations, it is possible fhat other helicases that are monomeric under some solution conditions may assemble to active oiigomeric forms upon binding DNA or nucleotide cof actors.

Modulation of DNA binding affinity by nucleotide cofactors '' Helicases, by definition, require ATP (or another nucleoside triphosphate) for function, and the proposal has been made that binding of ATP, its hydrolysis, and the

9

subsequent release of products (ADP and POf ) cycles the helicase through specific conformational (energetic) states, thus effecting the unwinding reaction (Hill and Tsuchiya, 1981). In fact, nucleotide- and DNA-dependent changes in the conformational states of DnaB (Nakayama etat., 1984), Rho (Bear etat., 1985), Rep and Helicase II (Chao and Lohman, 1990) have been observed. Qualitative studies with Rep (Arai et al., 1981b), DnaB (Nakayama et at., 1984) and Helicase III (Das et al., 1980) indicate thaf the relative affinities of these enzymes for ss and dsDNA are modulated by the nucleotide (ATP or ADP) that is bound to the helicase. In the case of Helicase 111, the unliganded (no nucleotide) protein binds well to both ss and dsDNA; however, upon binding ATP, the affinity of Helicase III for dsDNA decreases significantly, while high affinity is retained for ssDNA (Das etat., 1980). Qualitative binding studies with the Rep helicase indicate that both the affinity and dissociation rate constant for ssDNA are affected differentially by ATP, ADP or nonhydro I ysable ATP analogues (Arai etat.. 1981b). Furthermore, competition studies with polymeric ss and duplex DNA suggest that both Rep and DnaB helicases are able fo bind ss and dsDNA simultaneously to form a ternary complex (Arai etai., 1981b; Arai and Kornberg, 1981), These studies suggest that ATP and its hydrolysis products are allosteric effectors of DNA binding. Recent measurements of the equilibrium constants for binding of ss and ds oligodeoxynucleotides to the Rep helicase (I, Wong, K. Chao, and T. Lohman, in preparation) have led to a more detailed picture of the allosteric effects of nucleotide cofacfors in modulating Rep's affinity for DNA. Binding of either ss or ds oligodeoxynucleofides (16 nucleotides or bp) induces Rep to dimerize and each subunit of the Rep dimer (Pj) can bind one oligodeoxynucleotide fo form either P2S2 or P2D2 at saturation, where S and D represent single strand and duplex oiigodeoxynucleotides, respectively. Furthermore, competition studies with ss and duplex oligodeoxynucleotides indicate thaf both conformations of DNA bind competitively to each Rep subunit and that a hybrid complex, P2SD, can form. Coupled wifh the fact fhat a chemioally cross-linked Rep dimer maintains DNA helicase activity (Chao and Lohman, 1991), these results suggest that at an unwinding fork the Rep dimer might form one or more of the complexes depicted in Fig. 3, In Fig. 3a, the individual Rep subunits each bind one of the two complementary single strands. In Fig, 3b, both subunits of the dimer bind to the same strand of ssDNA. In Fig. 3c, however, one subunit is bound to ssDNA, while the other subunit is bound to duplex DNA. Further studies indicate fhat nucleotide cofactors differentially modulate the affinities of ssDNA or duplex DNA for the second Rep subunit, when the first subunit of the Rep dimer is bound with ssDNA; the species PgSg is favoured in the presence of

10

T. M. Lohman ADP, whereas P2SD is favoured in the presence of the non-hydrolysable ATP analogue, AMPP(NH)P (I. Wong and T. Lohman, unpublished). These allosteric effects of ATP binding and hydrolysis modulate the population distribution of DNA-bound Rep dimer states in a manner that suggests a cycling scheme that might be used to unwind duplex DNA as discussed below (see Fig, 4c).

A

Models for DNA unwinding

B

5',

Fig. 3. Possible modes by which a dimeric heiicase might bind to an unwinding fork.

B

Translocation

Unwinding

In general, models for protein-catalysed DNA unwinding can be either 'passive' or 'active'. In 'active' models, the protein plays a direct role in breaking the hydrogen bonds between the base pairs, whereas in 'passive' models, the profein stabilizes the unwound state by binding to the ssDNA fhat forms transiently because of local internal melting or 'fraying' of the end of the DNA duplex, Qne major difference between these two classes of models is that passive models require the protein to bind only to ssDNA, whereas active models can incorporate direct protein binding to duplex as well as ssDNA, although this is nof a strict requirement. Helix-destabilizing (SSB) proteins (Chase and Williams, 1986; Lohman et at., 1988), which are ATP-independent, are believed to destabilize duplex DNA by a passive mechanism, since they do nof bind duplex DNA with high affinity. However, as discussed above, the few helicases for which the DNA-binding properties have been examined can bind both ss and

Fig. 4. Models (or the DNA unwinding catalysed by a dimeric helicase (triangles), A. Both subunits of the dimeric helicase bind lo each of the complementary single strands, but with opposite polarities, enabling both to translocate in the direction of the unwinding (ork. Each subunit remains bound to the same single strand of DNA throughout the unwinding reaction. This configuration is unlikely for a homo-dimer. B. Botti subunits bind to each of the complementary single strands, but with the same polarities because of the C^ symmetry of the dimer subunits, thus both subunits translocate with the same polarity along ssDNA. resulting in a 'loop-tailed' structure. Each subunit remains bound to the same single strand of DNA throughout the unwinding reaction. C. 'Rolling' model: a dimeric helicase (subunits possessing C2 symmetry) unwinds by interacting directly with both duplex and ssDNA. Each subunit alternates binding to duplex DNA as the dimer translocates when one subunit releases ssDNA and rebinds to duplex DNA. ATP hydrolysis is directly coupled to destabilization ot the base pairs.

Escherichia coli DNA heticases duplex DNA, and their relative affinities for ss and duplex DNA are modulated by ATP binding and hydrolysis. It is possibie, therefore, that these helicases unwind DNA actively by interacting with both ss and duplex DNA, at least transiently. Although, in principle, some helicases might unwind DNA by a passive mechanism, using ATP hydrolysis only for fransiocation along ssDNA, this seems unlikely since the rates of unwinding would then be limited by the rates of transient melting of the ends of the duplex DNA. The unwinding models discussed in this section refiect the hypothesis that the functional forms of helicases are oiigomeric and that each helicase subunit can bind DNA. Oniy dimeric helicases are considered explicitly, since this is the simplest oiigomeric form: however, many of the considerations should also apply to higher-order oiigomeric helicases. For exampie, it appears that the E. coti Rho hexamer is functionally dimeric in the sense that, within the hexamer, its subunits appear to exist in two classes (P. H. von Hippel, personal communication). The fact that some helicases are hexameric may refiect constraints related to interactions with other proteins, rather than a strict requirement for the helicase function. The additional subunits available to a hexameric helicase may also enable it to interact simultaneously with both leading and lagging strands in order to constrain the three-dimensional topology of the unwound DNA, which, in turn, may facilitate the interaction of other proteins. Figure 4 illustrates three models for how a dimeric helicase might bind at an unwinding fori^, translocate, and unwind DNA. The models differ according to whether the helicase interacts with only ssDNA (as in Figs 4a and 4b) or whether it interacts with both duplex DNA and ssDNA (as in Fig, 4c). The models in Figs 4a and 4b differ in the symmetry of the dimer, which affects the relative movement of each dimer subunit along the two single strands of DNA. assuming that each subunit binds ssDNA with the same polarity. The molecular details of how unwinding occurs are not indicated in Figs 4a and 4b and since there is no explicit interaction with duplex DNA, these could represent either passive or active mechanisms of unwinding. Active unwinding by the mechanisms in Figs 4a or 4b might be accomplished by a nucleotide-dependent conformational change in the dimer, which could cause a relative rotation of the two subunits that could result In a 'prying apart' of the base pairs. Figure 4c represents an active unwinding model, the details of which are suggested by the DNA-binding data obtained for the E. coti Rep protein discussed above. Although it is not known whether any of these models are correct for any helicase, they represent possibilities for discussion. In the following sections, specific aspects of the DNA-unwinding reactions cataiysed by the E. coti Rep, RecBCD, and the phage T7 gene 4 helicases are discussed, since much of

11

the available information about DNA-unwinding mechanisms and translocation properties stems from studies of these helicases. E, coli Rep As discussed above, dimeric Rep helicase is able to interact with ss and dsDNA simultaneously, forming a species referred to as PgSD (i. Wong, K. Chao. and T, M. Lohman, unpublished). Furthermore, the two most populated species of the doubly liganded (with DNA) Rep dimer are P2S2 (favoured with ADP bound), which has ss DNA bound to both subunits (Fig. 3b). and P2SD (favoured wifh ATP bound), which has ss and duplex DNA bound to each subunit of the dimer (Fig. 3c). Figure 4c depicts a model based on the assumption that these two species reflect important intermediates in Rep-catalysed DNA unwinding. In this model, at least one subunit of the Rep dimer (although not always the same subunit) is always bound to the 3' ssDNA at fhe fork (i.e. the single strand in Fig. 4c with 3' to 5' polarity toward the duplex), while the other subunit is bound either to the same strand of ssDNA or to the dupiex region ahead of the fork. The roie of ATP binding and subsequent hydrolysis is to moduiate the affinities of the second Rep subunit so that binding to dupiex DNA is favoured when Rep is complexed with ATP/Mg^\ whereas binding of the second Rep subunit to ssDNA is favoured when Rep is compiexed with either ADP/Mg^* or Mg^\ The modei suggested in Fig, 4c has the actuai unwinding step coupied to the hydrolysis of one (or more) ATP moiecuie(s), whereas the translocation step Is coupled to ATP binding, although in principle this could be reversed. Furthermore, each individual subunit of the Rep dimer does not remain bound to the DNA at aii times during the unwinding process; rather, each subunit aiternates among three states: (i) bound to ssDNA, (ii) bound to dupiex DNA, or (iii) dissociated from DNA. However, the functionai heiicase dimer aiways remains bound to the 3' ssDNA through at ieasf one subunit. in this model, 'transiocation' occurs by 'roiling' of the dimer aiong the DNA, rather than 'siiding' of each protein subunit aiong the DNA. (Note that the Rep dimer has been drawn with C2 symmetry, refiecting the predominance of this symmetry in the majority of known structures of dimeric proteins, although this aspect of Rep structure is unknown.) It has been estimated that two molecules of ATP are hydrolysed per base pair unwound during the Rep-cataiysed unwinding of dupiex DNA (Kornberg ef at., 1978; Yarranton and Gefter, 1979; Arai and Kornberg. 1981) and it has been proposed that one ATP may be required to unwind a singie base pair, whereas the second ATP hydroiysed may be coupled to transiocation (Yarranton and Gefter, 1979). This stoichiometry may also reflect the

12

T.M. Lohman

fact that the active Rep heiicase is dimerio. On the other hand, such arguments assume fhat coupiing of ATP hydroiysis to unwinding is 100% efficient, whereas if this were not fhe case, fhen it is possible that muitipie base pairs are unwound per ATP hydroiysed. Lower efficiency couid resuit from the mechanism in Fig. 4c since futile ATP hydrolysis might occur whenever either of the Rep subunits is bound to ssDNA. Interestingiy, a similar stoichiometry of two to three ATP moiecuies hydrolysed per base pair unwound has been estimated for the RecBCD heiicase, which may indicate that the mechanism of unwinding by these two heiicases is similar (see beiow). The Rep heiicase is able to unwind OX174 DNA (5386 bp) and fd phage DNA (-6000 bp) with high processivity in the absence of DNA synthesis, after the supercoiled DNA has been nicked by the OX174 cisA or the fd gene il proteins, respectively (Yarranton and Gefter, 1979; Eisenberg et at., 1977). The CIJX174 cisA and fd gene II proteins nick the pius strand of the respective supercoiied RF DNA at the origin of repiication, fhus providing the site for initiation of Rep heiicase action. There is aiso evidence fhat Rep interacts non-covalentiy with the *X174 cisA protein during the unwinding reaction (SumidaYasumoto etat., 1976). Consistent with this view, the -44 carboxyi terminai amino acids of the Rep protein are required for it to unwind either a OX174 RF DNA-cisA profein compiex or a M13 RF DNA-fi gene II protein compiex, aithough the truncated Rep poiypeptide retains its ATPase and heiicase activities (Chao and Lohman, 1990) as weii as its abiiity to dimerize. However, these phage accessory proteins may aiso provide additionai functions, since in their absence Rep unwinds a 343 bp dupiex DNA iess efficientiy than it does a 71 bp dupiex (Smith et at.. 1989), One possibility is that the processivity of Rep unwinding is modulated by the phage accessory proteins, aithough the processivity of Rep (± OX cisA or fi gene Ii proteins) has not yel been measured quantitativeiy.

E. coli RecBCD Several intermediate structures have been observed by eiectron microscopy during RecBCD-catalysed unwinding of T7 DNA in the presence and absence of the E. coti SSB protein (Tayior, 1988; Smith. 1990). In the presence of the SSB protein, the primary intermediate is a 'iooptailed' structure possessing two ssDNA taiis of unequai iengfh, wifh a ssDNA ioop at the junction with dupiex DNA; the shorter taii and the ioop are part of the same 3' DNA strand (Braedf and Smith, 1989), The 1oop-taii' structures formed by RecBCD are simiiar fo the one depicted in Fig, 4b, aithough those formed by RecBCD possess opposite strand polarity. Some 'twin-taiied'

structures have also been observed under some conditions (e.g. see Fig. 4a), aithough these may be degradation products. Both the RecB and RecD polypeptides are predicted to contain ATP-binding sites and bind 8-azido-ATP (Juiin and Lehman, 1987) and it has been estimated that the RecBCD heiicase hydrolyses -2-3 ATP molecules per base pair unwound (in the presence of excess E. co//SSB protein) (Roman and Kowalczykowski, 1990). On this basis, if has been proposed that the helicase activity of the RecBCD enzyme resides in a RecBD heterodimer, interacting with the unwinding fork as in Fig. 4a, with each subunit hydroiysing one ATP for each base pair thaf Is unwound (Roman and Kowalczykowski, 1990), However, as stated above for the Rep heiicase, these arguments assume that the coupling of ATP hydrolysis fo unwinding is 100% efficient, in order for this modei to expiain the observed formation of ssDNA loops on the 3' ssDNA tail, it has been proposed fhat the two subunits that contact each compiementary single strand must fransiocafe with unequal rates (Roman and Kowalczykowski, 1990), These proposais are based on the assumption that the functionai RecBCD helicase is a heterofrimer; however, if it is a iarger oiigomer (e.g. (BCD)2). then it is possibie that the functional helicase is a dimer of RecB or RecD subunits. This modei does not propose a roie in unwinding for the RecC subunit, aithough RecC may possess the active site for the nuciease activity of RecBCD. However, a further possibiiity is that the RecC subunit may moduiate fhe processivity of the RecBCD heiicase, piaying a roie similar to thaf proposed above for the OX174 cisA or f 1 gene i i proteins in the unwinding reaction cataiysed by fhe dimeric Rep heiicase. A modified modei for RecBCD heiicase activity is simiiar fo that depicted in Fig. 4b, although the RecBCD looptatied structure would possess the opposite poiarity (Braedt and Smith, 1989). in this modei, the DNA-binding subunits of the functionai heiicase dimer (RecBD?) are reiated by C2 symmetry, as proposed for the Rep dimer. If binding of each subunit to ssDNA Is poiar, then each subunit wouid translocate aiong each DNA strand with thg same polarity, but in opposite directions with respect to the unwinding fork. This modei wouid explain the formation of loop-tailed' intermediate structures as observed in the RecBCD-catalysed DNA-unwinding reaction, since a ioop in one strand of the DNA is aiways formed independentiy of the relative rates of translocation of the two subunits. However, in the case of the RecBCD enzyme, it appears that both the 3' ssDNA taii and the ssDNA ioop grow continuousiy throughout the unwinding reaction (Tayior and Smith, 1980). whereas this aiternative modei predicts that fhe 3" ssDNA taii wouid shorten while the ioop grows, af ieast at some point in the reaction. However, even if this mechanism is not appropriate for the

Escherichia ccli DNA helicases RecBCD helicase, it is one that should be considered for ether dimeric helicases. Heiicase/Primase The bacteriophage T7 gene 4 protein is essential for T7 replication (Bernstein and Richardson, 1989), Two forms cf the protein are expressed in vivo, differing only in their A/-termini, because of the presence of two ribosome-binding sites within the gene 4 transcript. The full-length 63 kDa polypeptide possesses both helicase and primase activities, whereas the truncated 56 kDa polypeptide possesses oniy helicase activity (Bernstein and Richardson, 1989). The dependence of its DNA-dependent TTPase activity en gene 4 protein concentration suggests that the 56 kDa protein ottgomerizes. possibly forming a dimer (Nakai and Richardson, 1988), Based on an analysis of the relative use cf priming sites on fl>X174 DNA, it was concluded that the T7 gene 4 protein translocates 5' to 3' along ssDNA (Tabor and Richardson, 1981). Because of this and its role in priming, it has been proposed that the gene 4 protein moves 5' to 3' along the lagging strand during replication, although this translocation polarity would require the protein to stop at each priming site and reverse its direction along the ssDNA in order to synthesize an RNA primer. Similar conclusions have been reached for the T4 gene 41 helicase, which also oligomerizes. although the primase activity exists on a separate polypeptide encoded by gene 61 (Richardson ef ai, 1990). Nakai and Richardson (1988) have proposed that a heterodimer, consisting of the 56 and 63 kDa gene 4 pclypeptides, may be the functional helicase/primase. In this mcdel. the heterodimer is proposed tc interact with the lagging strand, with the 56 kDa pclypeptide translocating with 5' to 3' polarity and functioning as the helicase. Upcn encountering a primer reccgnitlcn site on the lagging strand, the 63 kDa polypeptide is postulated to dissociate from the 56 kDa polypeptide in order to synthesize the RNA primer (moving in the opposite directicn with 3' to 5' polarity along the template tagging strand), whiie the 56 kDa polypeptide continues unwinding the duplex DNA in the 5' to 3' direction. After primer synthesis is complete, the 63 kDa polypeptide dissociates frcm the lagging strand DNA and rebinds at the unwinding fork. This model is consistent with the observation that leading strand synthesis has considerably higher processivity than does lagging strand synthesis in vitro, although the lower processivity fcr lagging strand synthesis in vitro may reflect dissociation cf either the primase or the DNA polymerase. However, a distributive model for primase activity does not address how leading- and lagging strand syntheses are co-ordinated in vivo. In this context, it is not known whether the T7 DNA polymerase functions as a dimeric replisome as a means of co-ordinating leading- and

13

lagging strand synthesis, as has been proposed for other DNA polymerases (McHenry, 1988). Alternative models can be proposed in whioh the functional gene 4 dimer (homo- or heterodimer) remains intact throughout the replication process. In one case, the gene 4 prctein dimer interacts with both leading- and lagging strands, with each subunit translocating 3' to 5' along each ccmplementary strand as depicted in Fig. 4b. This would allow processive helicase activity en the leading strand as well as processive primase activity on the lagging strand, although the directionality of translccation along ssDNA is opposite to that proposed for this helicase. The further advantage of this mechanism would seem to be that translccaticn on the lagging strand is in the same direction as required for RNA primer synthesis. Another alternative model is similar to the 'rolling' model depicted in Fig. 4c, although the gene 4 dimer would bind tc the 5' flanking ssDNA (i.e. the lagging strand) rather than the leading strand. In this model, synthesis of the RNA primer is performed by the gene 4 subunit that happens to be bound to the 5' end of the ssDNA when a primer site is encountered. However, both models predict that gene 4 primase activity is as processive as gene 4 helicase activity, which is not consistent with observations in vitro (Nakai and Richardson, 1988). Conclusions Although it is clear that different mechanisms are used by the many helicases that unwind DNA, many helicases aIsc share features that are likely to be important for their function. This review has presented the hypothesis that the functionally active forms cf helicases are cligcmeric and that such structures provide helicases with multiple DNA-binding sites, which can facilitate the transient formation of ternary structures with ssDNA and/or duplex DNA, Modulation of the relative affinities of these binding sites for ss versus duplex DNA through ATP binding and hydrolysis would then provide the basis for cycling mechanisms that are essential for processive unwinding of DNA, Such ternary structures might also play a rcle in coordinating activity on the leading- and lagging strands during DNA replication. It is clear that helicase-catalysed unwinding of duplex DNA requires further study of the coupled interactions of helicases with ss and duplex DNA, nucleotide cofactors and other ligands (salts, protons, etc.) that modulate these interactions, and that all these interactions are almost certainly modulated by the assembly state of the helicase itself. Acknowledgements I thank H. Hoffmann-Barling, R. Kolodner, S. Kowalczykowski. S. Kushner, S. Matson, N. Nossal, T. Ptatt. C Richardson, and G. Smith for supplying reprints and preprints. W. Bujalowski, K. Chao, G. Runyon and I, Wong for their contributions to our studies cf helicases, I, Wong for hetpfut discussions, D. Bear,

14

T. M. Lohman

S. Matson and P. Otivo for comments on this review, and Lisa Lohman for art work. This work was supported by NtH grants GM30498, GM45948 and the American Cancer Society (Faculty Research Award 303; NP-756).

References Abdet-Monem, M. etai (1976) Eur J Biochem 65: 441-449. Abdet-Monem, M. etai (1977) Eur J Biochem 79: 39-45. Arai, N,. and Kornberg, A. (1981) J Biol Chem 256: 5294-

5298, Arai, K.t. etai (^ 98^ a) J Biol Chem 256: 5247-5252. Arai. N. ef ai (1981 b) J Biol Chem 256: 5287-5293, Bear, D,G, ef ai (1985) Proc NatI Acad Sd USA 82: 19111915. Bernstein. J,A., and Richardson, CC. (1988) JS/o/Chem 263: 14891-14899. Bernstein, JA., and Richardson, CC. (^ 989) J Bioi Chem 264: 13066-13073. Braedt. G., and Smith, G.R, (1989) Proc Natt Acad Sci USA 86: 871-875. Bridges, B.A,. and von Wright, A, (1981) Mutation Res 82:

229-238. Bruckner, R.C. et at. (1991) J Btot Chem 266: 2669-2674. Chao, K.. and Lohman, T.M. (1990) J Biot Chem 265: 1067-1076. Chao, K,, and Lohman, T.M, (1991) J Mot Biot 221: 11651181, Chase. J.W.. and Wittiams, K.R. (1986) Annu Rev Biochem 55: 103-136, Coiasanti, J,, and Denhardt. D.T. (1987) Mot Gen Genaf 209:

382-390, Company, M, etat. (1991) A/afure349:487-493, Das, R,H, etai (^980) J Bioi Chem 255: 8069-8073, Dykstra. C C . ef at. (1984) Cotd Spring Harbor Symp Quant Bioi49: 463-467. Eisenberg, S. etai (1977) Proc NatI Acad Sci USA 74: 3^ 983202, Fasster, J.S. etai (1985) JSacfer/o/161: 609-614. Finger. L.R., and Richardson, J,P, (1982) J/Wo/S/o/156: 203219, Geider, K., and Hoffmann-Berting, H.(1981) Annu Rev Btochem 50: 233-260. Hitt.T,L,,andTsuchiya. T, (1981) Proc Natt Acad Sci USA 7Q:

4796-4800, Hodgman, T.C, (1988) NatureZZZ: 22-23, Julin. D.A., and Lehman, t.R. (1987) J Btot Chem 262:

9044^9051, Klinkert. M, etat. {^ 980) J Biot Chem 255:9746-9752. Kornberg, A, efa/. (1978) JS/o/C/iem253: 3298-3304, Kowalczykowski, S,C. and Roman. L,J. (1990) In Motecuiar Mechanisms in DNA Reptication and Recombination. Richardson, C . and Lehman, t,R, (eds). UCLA Symposium on Moiecuiar and Cellular Bioiogy. New York: Atan R. Liss, pp. 357-373, Lahue. E.E., and Matson, S,W, (1988) J Btoi Chem 263:

3208-3215, Lane. D. (1988) /Vafure 334: 478. Lane, H.E.D,. and Denhardt, D.T, (1975) J Moi Biot 97: 9 9 112. Lebowitz, J.H,, and McMacken, R.M. (1986) JS/o/Cf7em 261: 4738-4748, Lee. E.H,, and Kornberg. A. (1991) Proc A/af//^cac/Sc/L/S/l 88:

3029-3032,

Lee, M,S,, and Marians, K.J. (1989) JS/o/C/iem 264: 1453114542, Lee. M,S,. and Marians, K J . (1990) JS/o/C/iem 265: 17078-

17083, Liu, CC, and Alberts. B, (1981) JS/o/C/7em 256: 2813-2820, Lohman. T,M. etat., (1988) Trends Btochem Sc/13:250-255. McHenry, C.S, (1988) Annu Rev Btochem 57: 519-550. Mastrangeto. I,A. etat. (1989) /Vafufe338: 658-662, Matson, S.W, (1991) Prog Nuct Acid Res 40: 289-326. Matson, S.W.. and George, J,W, (1987) J Biot Chem 262: 2066-2076, Matson, S,W., and Kaiser-Rogers, K.A, (1990) Annu Rev Btochem59: 289-329. Matson, S,W,, and Morton, B,S. (1991) J Btot Chem 266: 16232-16237, Nakai. H,, and Richardson, C C . (1988) J Biot Chem 263:

9818-9830. Nakayama, N, etai (A 984) J Btoi Chem 259: 88-96, Nurse, P. etai {^99^) J Bacterioi A73:6686-^693, Oh, E,Y.. and Grossman, L. (1989) J Biot Chem 264: 1336-

1343. Ray, B,K. etat. (1985) JS/o/Cftem260: 7651-7658, Reha-Krantz, L,J., and Hurwitz, J, (1978) J Biot Chem 253: 4043-^050. Reygers efa/. (1991) E/weOJ10: 2689-2694, Richardson, R,W, et at. (1990) tn Molecular Mechanisms tn DNA Reptication and Recombinatton. Richardson. C , and Lehman, t.R, (eds). UCLA Symposium on Motecutar and Ceiiutar Bioiogy. New York: Atan R, Liss, pp, 247-259. Roman, L.J.. and Kowatczykowski, S.C, (1990) Biochem 28: 2873-2881. Runyon, G,T. ef ai. (1990) Proc Nati Acad Sci USA 87: 6383-6387. Seo. Y.-S., Lee, S.-H, and Hunwitz,J. (1991) J a/0/Chem 266: 13161-13170. Smith, G,R. (1990) tn Nucteic Acids and Moiecutar Biotogy. Vol. 4. Eckstein, F,. and Liltey, D,M.J. (eds), Bertin: Springer Vertag, pp, 78-98, Smith. K.R, etai (1989) J Biot Chem 264: 6119-6126, Steinmetz, E.J. ef ai (1990) J Btot Chem 265:18408-18413. Sumida-Yasumoto, C. ef ai (1978) Cotd Spring Harbor Symp Quant Biot 43: 3^^-329. Tabor, S,, and Richardson. CC. (1981) Proc Nati Acad Sci USA78: 205-209. Taucher-Schoiz, G, etal. (1983) tn Mechanisms of DNA Reptication and Recombinatton. Vot. 10, UCLA Symposium on Moiecutar and Cettutar S/ofogy, Cozzaretti, N, (ed,). NewYork: Atan R, Liss, pp. 65-76, Taytor, A,F. (1988) In Genetic Recombination. Kucherlapati, R., and Smith, G,R, (eds). Washington, D,C,: American Society for Microbiology, pp. 231 -263. Taylor, A., and Smith, G,R, (1980) Ce//22: 447-457, Taylor, A.F., and Smith, G,R. (1985) J Mol Biot A85: 431-443. Thommes, P., and Hubscher, U, (1990) FEBS Letters 268: 325-328, Traxler, B.A., and Minkley, E.G. (1988) J Mot Biol 204: 205209. Umezu, K. et ai (1990) Proc NatI Acad Sci USA 87: 53635367. Washburn. B,K,, and Kushner, S.R. (1991) J Bacterioi 173: 2569-2575. Yarranton, G.T,, and Gefter, M.L. (1979) Proc NatI Acad Sci USA 76:: 658-: 662. Yarranton, G.T. etai (^979) J Biol Chem 254:12002-12006.