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Relaxing and unwinding on Holliday: DNA helicase-mediated branch migration
DNA Repair
ELSEVIER
Mutation Research 337 ( 1995) 149- I59
Minireview
Relaxing and unwinding on Holliday: DNA helicase-mediated branch migration David E. Adams, Stephen C. West
*
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, EN6 3LLA UK
Keywords: Branch migration;
Holliday junction;
DNA recombination;
DNA repair
1. Introduction
In this review, we focus on the basic mechanisms governing homologous strand exchange during general genetic recombination and recombinational repair, especially during the bacterial SOS response to DNA damage. Recent studies have shown that strand exchange is an enzyme-driven process, requiring the co-ordinated activity of a small number of dedicated recombination proteins. These enzymes can be separated into two categories: (1) those that act early in recombination to facilitate homologous DNA pairing and the initiation of strand exchange (e.g. the Escherichia coli RecA protein); and (2) those that act after recombination intermediates have been formed (e.g. the E. coli RuvABC and RecG proteins). This latter group of enzymes bind specifically to the crossover point, or Holliday junction, within the recombination intermediates and process these intermediates either by branch migration or endonucleolytic cleavage. The significance and importance of these studies is not limited to bacterial systems, however, as it has
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become increasing clear from recent work that the basic mechanisms of recombination and DNA repair are likely to have been highly conserved during evolution (Kowalczykowski and Eggleston, 1994; West, 1994). For example, a Succharumyces cereuisiue strand exchange protein, Rad51, has recently been identified which exhibits structural and functional homology to the E. colt’ RecA protein (Aboussekhra et al., 1992; Shinohara et al., 1992; Story et al., 1993). In vitro, yeast Rad51 forms nucleoprotein filaments very similar to those made by RecA (Ogawa et al., 1993) and catalyses an ATP-dependent strand exchange reaction (Sung, 1994). In addition, a human RecA equivalent was recently cloned (Shinohara et al., 1993) and purified (Benson et al., 1994). This protein, hRad5 1, forms nucleoprotein filaments on duplex DNA very similar to those made by E. coli RecA and yeast Rad51 (Benson et al., 1994; Ogawa et al., 1993). In E. coli, there are many gene products involved in promoting efficient homologous recombination. Here we discuss the activities of six of these gene products, namely those encoded by recA, ruuABC, recG, and uvrD. Each of these proteins has been implicated in the formation and processing of strand exchange intermediates during recombination and DNA repair.
IS0
D.E. Adams, S.C. West/Mutation
reverse f migration
Holliday junction
i
forward migration
1
resolution
Fig. 1. Formation and processing of Holliday junctions during recombination.Holliday junctions (2) form as a central intermediate during general genetic recombination. Movement of these junctions along the DNA during strandexchange requirestwisting of the DNA duplexes about their axes (indicated by the arrows), resulting in the generation of heterodupiexDNA. Branch migration in the forward direction (3) leads to the extension of the heteroduplexjoint, while branchmigrationin the reverse direction (1) tends to abort exchange. Another means of terminating ex-
change is to resolve the junction by endonucleolytic cleavage (4). 2. Branch migration of Holliday enzyme-catalysed process?
junctions:
an
During strand exchange a Holliday junction is created that physically links the two recombining DNA molecules (Fig. 1). Rapid movement of this junction along the DNA during strand exchange is thought to require helical rotation of the DNA about its axes (Meselson, 1972) and proceeds via the breakage and reformation of hydrogen bonds. Junction movement continues until (i) the junction reaches a free DNA end; (ii) it runs into sequence heterology
Research 337 (1995) 149-1.59
(i.e. a mismatch, insertion/deletion or DNA lesion); and/or (iii) it is resolved by a structure-specific endonuclease. Forward migration is responsible for the extension of the heteroduplex joint, while reverse migration is anti-recombinogenic. Early theoretical and experimental studies suggested that the rate of spontaneous branch migration, i.e. branch migration in the absence of added proteins, would be a rapid process (Meselson, 1972; Thompson et al., 1976; Warner et al., 1978). Rates as high as thousands of base pairs per second (bp/s) were predicted and initially observed by following the dissociation of dimeric phage DNA molecules containing a Holliday junction, i.e. x-structures (Thompson et al., 1976; Warner et al., 1978). Recently, however, these rate estimates were re-examined, and it is now clear that non-catalysed branch migration is at least 3 orders of magnitude too slow under physiological reaction conditions to account for the rate of branch migration in vivo. The first indication that spontaneous branch migration might be a slow process came from groups studying cruciform transitions in supercoiled plasmid DNA (Courey and Wang, 1983; Gellert et al., 1983). More recently, slow rates for spontaneous branch migration have been measured in experiments using both RecA-made and synthetically-made four-way junctions (Johnson and Symington, 1993; Miller et al., 1992; Panyutin and Hsieh, 1993; Panyutin and Hsieh, 1994). In one study, deproteinised recombination intermediates, isolated from an in vitro strand exchange reaction catalysed by RecA, were observed to slowly dissociate over time, with a step rate on the order of a few tens of bp/s at 37°C (Miller et al., 1992). Remarkably, even at 72”C, _ 30% of the intermediates were still present after 1 h. This slow rate of spontaneous branch migration has been confirmed in experiments using large, plasmid-based X-structures (Johnson and Symington, 1993). In a recent set of experiments, the rate of noncatalysed branch migration was measured under a wide variety of buffer and salt conditions (Panyutin et al., 1995; Panyutin and Hsieh, 1993; Panyutin and Hsieh, 1994). The step rate was found to be a sensitive function of both temperature and divalent metal ion concentration. For example, between 10 and 1 mM MgCl,, the step rate increased about 4-fold at 37”C, and between 0.5 and 0.1 mM MgCl 2,
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the rate increased over 60-fold. To calculate the step rate of branch migration from the half-life of junction survival, crossover movement was modelled as a simple one-dimensional random walk, where the probability of a forward step is equal to that of a backward step. For the most part, theory and experiment correlated perfectly (Panyutin and Hsieh, 1994). The average rate of spontaneous branch migration in 50 mM NaCl was found to be - 3,300 bp/s at 37°C. By contrast, in the presence of 10 mM MgCl, and 50 mM NaCl, the step rate was 1000 times slower, or 3.3 bp/s. This 3 order of magnitude difference in step rate in sodium versus magnesium is likely to be due to structural changes in the Holliday junction under different salt conditions. In the absence of metal ions, the four-way junction adopts an extended square configuration (Fig. 2, centre), in which base pairs at the base of the junction are partially unstacked (Duckett et al., 1988; Duckett et al., 1990; Lilley and Clegg, 1993). If allowed to freely branch migrate, this junction will move along the DNA at speeds up to a few thousand of bp/s. In the presence of polyvalent metal ions, however, the Holliday junction adopts a folded Xstructure, which is much less susceptible to spontaneous branch migration (Lilley and Clegg, 1993; Murchie et al., 1990; Panyutin et al., 1995). The isomeric form of the junction is determined by the stacking interactions at the base of the crossover and thus frequent isomerisation of the junction may occur during branch migration. This probably accounts for why spontaneous branch migration is a slow process under physiological reaction conditions.
isomer I
151
Several E. cd proteins have now been isolated which bind specifically to Holliday junctions and promote their rapid movement along the DNA: the RuvA and RuvB proteins and the RecG protein (Lloyd and Sharples, 1993a; Tsaneva et al., 1992). Here we compare the branch migrational activities of RuvAB and RecG to that of RecA, which was the first E. coli enzyme found to catalyse branch migration in vitro (Cox and Lehman, 1981; DasGupta et al., 1980; West et al., 1981).
3. RecA protein: jack of all trades RecA is the major recombination protein in prokaryotic cells. Most of what is known about homologous DNA pairing and strand exchange comes from studies of the 38 kDa RecA protein (Kowalczykowski et al., 1994; Radding, 1988; West, 1992). Mutants in recA are highly pleiotropic, affecting not only recombination and DNA repair, but also cell division and mutagenesis (Clark and Sandler, 1994). Firstly, RecA acts as a co-protease (Little, 1984; Roberts et al., 1978) to stimulate the selfcleavage of LexA and a number of other transcriptional repressors (phage-encoded). Cleavage of LexA leads to a diverse set of physiological responses known as the SOS responses, which include derepression of an entire set of genes dedicated to DNA repair (Walker, 1984). Secondly, RecA is a DNA-dependent ATPase that catalyses homologous DNA pairing and crossover formation during strand exchange (Kowalczykowski et al., 1994; West, 1992).
unfolded junction
isomer II
Fig. 2. Isomerisation of the Holliday junction. In the absence of junction binding proteins, the Holliday crossover is free to adopt a number of different conformational states, depending on the reaction conditions. The stacked-X configuration (i.e. isomer I and isomer II) is favoured in the presence of polyvalent metal ions, while the unfolded configuration is favoumd in the absence of metal ions. During spontaneous branch migration in vitro, the Holliday junction may need to inter convert frequently between these different conformational isomers, leading to a slower overall rate of branch migration. The most favourable junction configuration for branch migration is the unfolded junction.
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AS a first step during recombination, RecA binds single-stranded DNA to form a long nucleoprotein filament. This filament extends to duplex regions and DNA within this filament is unwound and stretched to approximately 1.5 times its normal length, a feature unique to homologous recombination (Stasiak and Egelman, 1988). Unwinding of the DNA facilitates the next phase of recombination, i.e. the search for DNA homology. This search is made difficult due to the large size of cellular genomes: > lo6 base pairs in E. coli and > lo9 base pairs in many eukaryotes. Following homologous DNA pairing and the initiation of strand exchange, heteroduplex joints are made by RecA (Fig. 3) in which the DNA contains either a four-way or a three-way Holliday junction (Cox and Lehman, 1987; Kowalczykowski et al., 1994; Radding, 1988; West, 1992). These junctions are then further processed, either by branch migration, and/or by nucleolytic cleavage. Early studies showed that RecA catalyses a slow, unidirectional branch migration reaction at a rate of 2-10 bp/s (Radding, 1988). This sluggish rate indicates that RecA is unlikely to be the major Holliday junction branch migration enzyme in vivo, although this conclusion was not obvious at the time. RecA also catalyses a branch migration reaction through regions of DNA damage and DNA heterology, but only small regions are bypassed (Hahn et al., 1988; Livneh and Lehman, 1982; Morel et al., 1994). Thus, despite being a jack of all trades (i.e. co-inducer of the SOS response, homologous DNA pairing protein, to
heteroduplex
initiator of DNA strand exchange, etc.>, RecA may be master of only a few. This observation raises the question - are there other bacterial proteins that make recombination more efficient?
4. RuvAB-catalysed
branch migration
The ruu locus of E. cofi encodes three gene products known to be important for Holliday junction processing. Cells carrying mutations in any of the three ruu genes (muA, ruuB, ruuC) are sensitive to UV-light and ionising radiation, and exhibit reduced recombination frequencies (Benson et al., 1991; Sharples et al., 1990; Shinagawa et al., 1988). All three ruu gene products have been cloned, overexpressed and purified, and in vitro studies have been invaluable in defining their important biological roles (West, 1994). The RuvA and RuvB proteins of E. coli are co-expressed from a common, LexA-regulated operon during the E. coli SOS response (Benson et al., 1988; Shinagawa et al., 19881, and like RecA, are required by cells for the efficient recovery from DNA damage. The 22 kDa RuvA protein acts to target the 37 kDa RuvB protein to the site of the Holliday junction (Hiom and West, 1995; Iwasaki et al., 1992; Parsons et al., 1995a; Parsons et al., 1992; Parsons and West, 1993; Shiba et al., 1991), and stimulates the ATPase activity of RuvB (Mitchell and West, 1994; Shiba et al., 1991). RuvB is the motor that drives branch migration (Adams and West,
multi-stranded DNA
homoduplex
direction of RecA strand exchange Fig. 3. RecA-mediated 4-strand exchange. RecA protein catalyses a 4-strand exchange reaction between two homologous DNA duplexes (i.e. homoduplexes), which become intertwined within the RecA nucleoprotein filament. Pairing results in the formation of multi-stranded DNAs and is followed by strand exchange. RecA protein remains tightly bound to the products of strand exchange (i.e. heteroduplexes) and needs to be recycled at the end of the reaction.
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1995; Miller et al., 1993; Tsaneva et al., 1992; Tsaneva et al., 1993), while RuvA plays an important ancillary role, reducing the requirement for RuvB in branch migration approximately 50-fold. Together RuvA and RuvB form an ATP-dependent DNA helicase that is capable of unwinding a partial duplex up to 558 bp in length with a defined 5’ to 3’ polarity (Tsaneva et al., 1993). The RuvAB DNA helicase activity can be specifically targeted to Holliday junctions, and is stimulated by the presence of the E. cofi SSB protein (Tsaneva and West, 1994). RuvA protein itself stimulates both the rate and extent of RuvB-mediated DNA unwinding (Adams and West, 19951, indicating that RuvA acts as a helicase accessory factor for RuvB. Consistent with this hypothesis is the observation that RuvB protein possesses sequence motifs shared by other E. coli DNA helicases (Lloyd and Sharples, 1993b; Tsaneva et al., 1993). From these observations, it was proposed that RuvAB-catalysed branch migration involves the unwinding and rewinding of duplex DNA near the base of the Holliday junction. At some stage in the reaction, ATP hydrolysis is required. RuvAB form an unusual DNA unwinding machine, however, as ATP hydrolysis is apparently not directly coupled to branch migration (A. Mitchell and S.C.W., unpublished results). Recent experimental studies indicate that RuvAB form a tripartite protein complex on Holliday junctions, as assayed by enzymatic probing and electron microscopy (Hiom and West, 1995; Parsons et al., 1995a). A diagram of this complex is shown in Fig. 4. Consistent with DNase 1 footprinting (Hiom and West, 19951, gel filtration (Mitchell and West, 1994), and electron microscopic studies (Parsons et al., 1995a), tetramers of RuvA protein are depicted as binding to the base of the crossover with the junction arms in a square configuration. Supporting evidence for this unfolded arm geometry has come from band-shift experiments, which showed that RuvA stabilises the unfolded form of a Holliday junction (Parsons et al., 1995a). Also shown in the model is the position of the RuvB protein. Two hexameric rings of RuvB sit on opposite arms of the junction, with duplex DNA passing through the centre of each protein ring. Image analysis and mass measurements have indicated that each RuvB ring contains six protein monomers (Stasiak et al., 1994), leading to
1.53
RuvAB complex
branch migration
Fig. 4. RuvAB-mediated branch migration. RuvA and RuvB form a tripartite protein complex that promotes the branch migration of unfolded Holliday junctions. Two hexameric rings of RuvB protein sit on opposite arms of the junction, with RuvA protein bound at the base. DNA within the cavity of each RuvB ring is thought to be transiently unwound and rewound, leading to twisting and translocation of the DNA in the direction indicated.
the suggestion that hexameric RuvB rings are the active form of the enzyme during branch migration. This suggestion has received indirect experimental support by the finding that RuvAB form stable (RuvA),(RuvB), complexes in solution (Mitchell and West, 1994). To carry out branch migration, RuvAB are thought to catalyse a transient unwinding reaction in the duplex DNA lying within the interior of the RuvB ring, leading to translocation of the DNA through the branch migration complex (Adams and West, 1995; Parsons et al., 1995a; Tsaneva et al., 1993). Repeated cycles of DNA unwinding and rewinding may lead to twisting of the junction arms about their axes, leading to base pair swapping at the crossover. Rapid
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junction movement is also thought to be facilitated by RuvA holding the junction arms in an unfolded configuration (Parsons et al., 1995a). This model for RuvAB action bears striking resemblance to the DNA unwinding reaction catalysed by the SV40 large T-antigen (Dean and Hurwitz, 1991; Mastrangelo et al., 1989). When this ringshaped DNA helicase binds to its cognate replication origin, two protein rings are seen by electron microscopy to sit at the palindromic origin (Wessel et al., 1992). Each belicase ring contains six protein monomers, similar to RuvB. Unwinding and translocation of DNA through the origin unwinding complex results in the generation of single-stranded DNA, which in turn allows for binding of the DNA polymerase apparatus. RuvB and SV40 large T-antigen thus belong to an emerging group of hexameric DNA helicases, which includes the E. cd DnaB protein (Bujalowski et al., 1994) and T7 helicase/primase (Egelman et al., 1995), that serve important functions during DNA replication, recombination and repair.
5. Role of RecA and RuvAB heterologies
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but strand exchange is slow and requires large amounts of ATP (Hahn et al., 1988; Morel et al., 1994; Tsaneva et al., 1992). The situation with the RuvAB complex is entirely different. Both genetic (Lloyd and Buckman, 1995; Matic et al., 1995) and biochemical evidence (Iype et al., 1994; Parsons et al., 1995b; Tsaneva et al., 1992) support the idea that RuvAB have a unique role in recombinational repair. For example, when four-strand exchange substrates are heavily irradiated with UV-light in vitro, RecAmediated strand exchange is severely blocked, whereas addition of RuvAB to the strand exchange reaction stimulates recombinational bypass (Tsaneva et al., 1992). In addition, when RuvAB are added to ongoing three-strand RecA reactions between single-stranded circular and linear duplex DNA, where the linear duplex DNA harbours heterologous inserts ranging in size from 198 to 1037 bp, RuvAB was found to greatly stimulate bypass of the large inserts (Iype et al., 1994). This bypass is likely due to RuvAB itself. Recently, RuvAB were shown to unwind DNA heterologies up to 1800 bp in size in the absence of other proteins (Parsons et al., 1995b).
in bypass of DNA
A critical problem in both general genetic recombination and recombinational repair is the promotion of strand exchange through heterologous and damaged DNA sequences. Heterologous insertions, deletions, mismatches and DNA lesions can all potentially disrupt the normal exchange of DNA strands. The mechanism by which branch migration enzymes like RecA and RuvAB promote the movement of Holliday junctions through damaged DNA and DNA heterologies is of great interest, and it is vital for understanding how species barriers are maintained (Matic et al., 1995) and for understanding what limits recombination between closely related gene families (Bianchi and Radding, 1983). Renewed interest in this problem has arisen with the finding that a single base mismatch can significantly impede spontaneous branch migration (Panyutin and Hsieh, 1993). RecA protein is able to promote four-stranded exchanges through short regions of DNA lesions, mismatches, and heterologous insertions ( < 100 bp),
6. RuvAB-mediated filaments
dissociation
of RecA protein
The E. coli RuvA and RuvB proteins possess another activity that may be important for genetic recombination. Together RuvA and RuvB promote the ATP-dependent dissociation of RecA nucleoprotein filaments from duplex DNA (Adams et al., 1994). This ability is thought to be useful in several regards, both in cleaning up the DNA of the proteins used to create the Holliday junction, and in recycling RecA protein during recombinational repair (Adams et al., 1994; Kuzminov, 1993). The requirement for ATP in the RecA displacement reaction suggests that helicase translocation is required (Adams et al., 1994). Several groups have now obtained evidence for DNA helicases being able to displace tightlybound protein from DNA (Bedinger et al., 1983; Bonne-Andrea et al., 1990; Eggleston et al., 1995; Salinas and Kadadek, 1994; Yancey-Wrona and Matson, 1992). The physical act of threading doublestranded DNA through the cavity of the RuvB ring, coupled with transient DNA unwinding, may provide
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a processive mechanism for RecA displacement. Alternatively, a direct physical interaction between RecA and RuvAB may be required (Adams et al., 1994). At this point in time, the exact mechanism of RuvAB-mediated RecA displacement from duplex DNA is unknown, Nor is it clear whether RuvAB can displace RecA bound to other forms of DNA (Iype et al., 1994). Recently, the T4 dda helicase was shown to dissociate tightly-bound proteins from duplex DNA (Salinas and Kadadek, 1994) and to dissociate synthetic DNA triple helices (Maine and Kodadek, 1994). Perhaps RuvAB can remove RecA from 3- and 4-stranded intermediates and also dissociate the multi-stranded structures made by RecA. The ability of RuvAB to dissociate RecA protein from recombination intermediates may also allow other Holliday junction processing enzymes, like the 19 kDa RuvC protein, to gain access to the Holliday junction, and resolve these junctions by endonucleolytic cleavage (Benson and West, 1994; Dunderdale et al., 1991; Iwasaki et al., 1991). The preference of RuvC for a small 4 bp consensus sequence, S*/$-TI G/C-3’ (B ennett et al., 1993; Shah et al., 1994) may make this enzyme dependent on RuvAB (or another E. c&-encoded branch migration activity) to position the Holliday junction near preferred sites of cleavage (i.e. resolution ‘hotspots’). Recent structural studies of RuvC and RuvC-Holliday junction complexes will lead to rapid progress in our understanding the molecular details of the RuvC junction cleavage reaction (Ariyoshi et al., 1994; Bennett and West, 199Sa; Bennett and West, 1995b). Remarkably, single mutants in ruuA, ruuB, or ruuC are viable, exhibiting only a 2-3-fold deficiency in genetic recombination (Lloyd and Sharples, 1992). These observations imply that there are other pathways for processing of Holliday junctions in E. co/i cells.
7. RecG and UvrD-mediated
branch migration
TWO other E. coli proteins are known to be able to catalyse the branch migration of Holliday junctions in vitro: the 76 kDa RecG protein (Lloyd and Sharples, 1993a) and the 82 kDa UvrD protein (Morel et al., 1993). Both proteins are ATP-dependent DNA
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helicases that unwind duplex DNA in the 3’ to 5’ direction (a polarity opposite to that exhibited by RuvAB). In addition, both proteins can catalyse the branch migration of RecA-made recombination intermediates. The role of RecG and UvrD (in particular) in catalysing the branch migration of Holliday junctions in vivo, however, is less clear than it is for RecA and RuvAB. Mutants in recG exhibit a functional overlap with the ruu genes. Recombination is reduced about 500fold in ruv recG strains, but only Z- to 3-fold in either of the single mutants (Lloyd, 1991). This functional overlap does not extent to DNA repair, however, as recG mutants are much less sensitive to UV irradiation than are single mutants in ruv. Unlike RuvAB, the synthesis of RecG is not induced as part of the SOS response. Sequence analysis of the recG gene shows structural motifs that are conserved in the DExH family of DNA and RNA helicases. This observation suggests that RecG, like RecA and RuvAB, uses a DNA unwinding activity to promote Holliday junction branch migration (Lloyd and Sharples, 1993a). Support for this idea has come from biochemical studies indicating that a single Ala to Val substitution in the helicase III motif of RecG, leads to the production of a mutant RecG protein that is unable to branch migrate DNA, despite retaining its ATPase and junction-binding activity (Sharples et al., 1994). Mutations in this motif also inhibit the eIF-4A RNA helicase, which acts during protein translation (Pause and Sonenberg, 1992). When RecG protein is added to ongoing RecAmediated 4-strand exchange reactions in vitro, reverse branch migration of the junction is observed (Whitby et al., 1993). This reverse branch migration activity may serve to resolve junctions in vivo by running the junction points off a free DNA end. Alternatively, it may serve an anti-recombinogenic function. In support of this hypothesis, recent genetic data indicate an important role for RecG in limiting recombination-dependent stable DNA synthesis (Hong et al., 1995). With four E. cofi proteins (i.e. RecA, RuvAB and RecG) already known to catalyse branch migration in vivo, is there any need (or evidence) for more such activities? The E. coli UvrD protein (helicase II) stimulates RecA-mediated strand exchange in vitro
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and it also catalyses branch migration in the absence of RecA (Morel et al., 1993). In agreement with these biochemical observations, genetic data indicate that certain MM-Dmutants are deficient in recombination (Horii and Clark, 1973; Morel et al., 1993). But some are normal or even exhibit elevated frequencies of recombination (Horii and Clark, 1973; Morel et al.. 1993). Furthermore, purified UvrD can reverse RecA-mediated synapsis in vitro and even inhibit RecA-mediated homologous DNA pairing (Morel et al., 1993). Hence the precise role for UvrD in recombination remains unclear. In principal, it is possible that any DNA helicase may be able to catalyse branch migration, since DNA unwinding is involved. The question is one of specificity. The bacteriophage T4 dda helicase, for example, stimulates UvsX-mediated strand exchange in vitro, but does not catalyse branch migration on its own (Kodadek and Albert& 1987). In addition, the T4 gene 41 DNA helicase can also stimulate UvsXmediated strand exchange, provided its helicase accessory factor, gene 59 protein, is present (Salinas and Kodadek, 1995). Together, gene 41 helicase and gene 59 protein promote the branch migration of three-way junctions in the absence of UvsX. The adapter protein gene 59 does not act like RuvA in RuvAB reactions, however, by targeting its cognate helicase directly to Holliday junctions. Instead, it acts hy targeting Ihe gene 41 helicase directly to DNA bound by the T4 single-stranded DNA binding protein, gene 32 protein, through specific proteinprotein interactions. Thus nature has evolved at least two independent means of targeting DNA helicases to recombination intermediates: either through specific protein-DNA interactions (e-g. RuvAB and RecG to Holliday junctions) or through specific protein-protein interactions (e.g. T4 gene 41/59/32 proteins).
8. Concluding
remarks
DNA helicases play diverse roles in genetic recombination and DNA repair, including (i) the generation of recombinogenic single-stranded DNA ends; (ii) the unwinding of DNA damages; (iii) anti-recombinogenic functions; and (iv) the branch migration of Holliday junctions. Holliday junctions are
more stable than was originally thought and it is now known that they do not spontaneously branch migrate under physiological reaction conditions. Current models of helicase-mediated branch migration postulate that helicase activity is used to unwind base pairs at the strand exchange fork, resulting in the swapping of base pairs and heteroduplex DNA formation. DNA helicase activity is likely also to be used to unwind large blocks of DNA heterology and DNA lesions during recombination and DNA repair. Currently, little or nothing is known about the mechanics of Holliday junction branch migration in higher cells. The recent excitement generated by the study of prokaryotic branch migration proteins is likely to spill over soon and lead to the discovery of eukaryotic homologues of these remarkable enzymes.
Acknowledgements We are grateful to Alison Mitchell, Carol Parsons and Andrzej Stasiak for access to recent and unpublished data. Our research is supported entirety by the Imperial Cancer Research Fund.
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D.E. Adams, S.C. West/Mutation Research 337 (1995) 149-159 Bennett, R.J. and SC. West (1995a) RuvC protein resolves Holhday junctions via cleavage of the continuous (non-crossover) strands, Proc. Natl. Acad. Sci. USA, 92, 5635-5639. Bennett, R.J. and S.C. West (1995b) Structural analysis of the RuvC-Holliday junction complex reveals an unfolded junction. J. Mol. Biol., 252, 213-216. Benson, F., S. Collier and R.G. Lloyd (1991) Evidence of abortive recombination in ru~l mutants of Escherichia coli K-12, Mol. Gen. Genet., 225, 266-272. Benson, F.E., G.T. Illing, G.J. Sharples and R.G. Lloyd (1988) Nucleotide sequencing of the ruu region of E. co/i K-12 reveals a LexA regulated operon encoding two genes, Nucleic Acids Res., 16, 1541-1550. Benson, F.E., A. Stasiak and S.C. West (1994) Purification and characterisation of the human Rad51 protein, an analogue of E. coli RecA, EMBO J.. 13, 5764-5771. Benson, F.E. and S.C. West (1994) Substrate specificity of the Escherichia coli RuvC protein: Resolution of 3- and 4-stranded recombination intermediates, J. Biol. Chem., 269, 5 195-5201. Bianchi, M.E. and CM. Radding (1983) Insertions, deletions and mismatches in heteroduplex DNA made by RecA protein, Cell, 35, 51 l-520. Bonne-Andrea, C., M.L. Wong and B.M. Alberts (1990) In vitro replication through nucleosomes without histone displacement, Nature, 343,719-725. Bujalowski, W.. M.M. Klonowska and M.S. Jewewska (1994) Oligomeric structure of Escherichia coli primary helicase DnaB protein, J. Biol. Chem., 269, 31350-31358. Clark, A.J. and S.J. Sandier (1994) Homologous genetic recombination: the pieces begin to fall into place, Crit. Rev. Microbial., 20, 125-142. Courey, A.J. and J.C. Wang (1983) Cruciform formation in a negatively supercoiled DNA may be kinetically forbidden under physiological conditions, Cell, 33, 817-829. Cox. M.M. and I.R. Lehman (1981) RecA protein of E. coli promotes branch migration. a kinetically distinct phase of DNA strand exchange, Proc. Nat]. Acad. Sci. USA, 78, 34333437. Cox, M.M. and I.R. Lehman (I 987) Enzymes of genetic recombination, Ann. Rev. Biochem.. 56, 229-262. DasGupta C., T. Shibata, R.P. Cunningham and CM. Radding (1980) The topology of homologous pairing promoted by RecA protein, Cell, 22, 437-446. Dean, F.B. and J. Hurwitz ( I99 I) Simian virus 40 large T antigen untwists DNA at the origin of DNA replication, J. Biol. Chem.. 266, 5062-507 I, Duckett, D.R., A.I.H. Murchie, S. Diekmann, E. Von Kitzing, B. Kemper and D.M.J. Lilley (1988) The structure of the Holliday junction and its resolution, Cell, 55, 79-89. Duckett, D.R.. A.I.H. Murchie and D.M.J. Lilley (1990) The role of metal ions in the conformation of the four-way junction, EMBO J., 9, 583-590. Dunderdale, H.J., F.E. Benson, C.A. Parsons, G.J. Sharples, R.G. Lloyd and SC. West (1991) Formation and resolution of recombination intermediates by E. coli RecA and RUVC proteins, Nature, 354, 506-5 IO. Egelman, E.H.. X. Yu, R. Wild, M.M. Hingorani
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(1995) Bacteriophage T7 helicase-primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases, Proc. Natl. Acad. Sci. USA, 92. 3869-3873. Eggleston, A.K., T.E. Oneill, E.M. Bradbury and S.C. Kowalczykowski (1995) Unwinding of nucleosomal DNA by a DNA helicase, J. Biol. Chem., 270, 2024-203 1. Gellert, M., M.H. O’Dea and K. Mizuuchi (1983) Slow cruciform transitions in palindromic DNA, Proc. Natl. Acad. Sci. USA, 80, 5545-5549. Hahn, T.R., S.C. West and P. Howard-Flanders (1988) RecAmediated strand exchange reactions between duplex DNA molecules containing damaged bases, deletions and insertions, J. Biol. Chem., 263, 7431-7474. Hiom, K. and S.C. West (1995) The mechanism of branch migration in homologous recombination: Assembly of a RuvABHolliday junction complex in vitro, Cell, 80. 787-793. Hong, X.K.. G.W. Cadwell and T. Kogoma (1995) Escherichia co/i RecG and RecA proteins in R-loop formation, EMBO J., 14, 2385-2392. Horii, 2.1. and A.J. Clark (1973) Genetic analysis of the RecF pathway of genetic recombination in Escherichia cofi K12: isolation and characterisation of mutants, J. Mol. Biol., 80, 327-344. Iwasaki, H., M. Takahagi, A. Nakata and H. Shinagawa (1992) Escherichia co/i RuvA and RuvB proteins specifically interact with Holliday junctions and promote branch migration, Genes Dev., 6, 2214-2220. Iwasaki, H., M. Takahagi, T. Shiba, A. Nakata and H. Shinagawa (199 1J Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure, EMBO J., IO, 4381-4389. lype, L.E.. E.A. Wood, R.B. Inman and M.M. Cox (1994) RuvA and RuvB proteins facilitate the bypass of heterologous DNA insertions during RecA protein-mediated strand exchange, I. Biol. Chem.. 269, 24967-24978. Johnson, R.D. and L.S. Symington (1993) Crossed-stranded DNA structures for investigating the molecular dynamics of the Holliday junction, J. Mol. Biol., 229, 812-820. Kodadek, T. and B.M. Alberts (1987) Stimulation of protein directed strand exchange by a DNA helicase, Nature, 326, 312-314. Kowalczykowski, SC., D.A. Dixon, A.K. Eggleston, SD. Lauder and W.M. Rehrauer (1994) Biochemistry of homologous recombination in Escherichia coli, Microbial. Rev., 58, 4Ol465. Kowalczykowski, S.C. and A.K. Eggleston (1994) Homologous pairing and DNA strand-exchange proteins, Annu. Rev, Biochem., 63, 991-1043. Kuzminov, A. (1993) RuvA, RuvB and RuvC proteins:.,cleaning up after recombinational repairs in Escherichia coli; BioEssays, 15, 355-358. Lilley, D.M.J. and R.M. Clegg (1993) The structure of the fourway junction in DNA, Annu. Rev. Biophys. Biomol. StNCt., 22, 299-328. Little, J.W. (1984) Autodigestion of LexA and phage lambda repressors, Proc. Natl. Acad. Sci. USA, 81, 1375-1379. Livneh, Z. and I.R. Lehman (1982) Recombinational bypass of
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Lloyd, R.G. (1991) Conjugal recombination in resolvase-deficienr ruuC mutants of Escherichia coli K12 depends on recG, J. Bacterial., 173, 5414-5418. Lloyd, R.G. and C. Buckman (1995) Conjugational recombination in Escherichia coli: genetic analysis of recombinant formation in HFr x F(-) crosses, Genetics, 139, 1123-l 148. Lloyd, R.G. and G.J. Sharples (1992) Genetic analysis of recombination in prokaryotes. Curr. Opin. Genet. Devel., 2.683-690. Lloyd, R.G. and G.J. Sharples (1993a) Dissociation of synthetic Holliday junctions by E. coli RecC protein, EMBO J., 12, 17-22. Lloyd. R.G. and G.J. Sharples (1993b) Processing of recombination intermediates by the RecG and RuvAB proteins of Eschmchia co& Nucieic Acids Res., 21, 1719-1725. Maine, I.P. and T. Kodndek (1994) Efficient unwinding of triplex DNA by a DNA helicase. Biochem. Biophys. Res. Comm., 204, 1119-1124. Mastrangelo, LA., P.V C. Hough, J.S. Wall, M. Dobson, F.B. Dean and J. Hurwitz (1989) ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication, Nature, 338, 658-662. Matic. I., C. Rayssiguier and M. Radman (1995) Interspecies gene exchange in bacteria: the role of SOS and mismatch repair systems in evolution of species, Cell, 80, 507-515. Meselson, M. (1972) Formation of hybrid DNA by rotary diffusion during genetic recombination, J. Mol. Biol., 71, 795-798. Mitchell, A.H. and S.C. West (1994) Hexameric rings of Escherichia coli RuvB protein: Cooperative assembly, processivity, and ATPase activity, J. Mol. Biol., 243, 208-215. Morel, P., J.A. Hejna, S.D. Ehrlich and E. Cassuto (1993) Antipairing and strand transferase activities of Escherichia coli helicase II (UvrD), Nucleic Acids Res., 21. 3205-3209. Morel, P., A. Stasiak, SD. Ehrlich and E. Cassuto (1994) Effect of length and location of heterologous sequences on RecAmediated strand exchange, J. Biol. Chem., 269, 19830-19835. Miiller, B., 1. Burdett and S.C. West (1992) Unusual stability of recombination intermediates made by Escherichia coli RecA protein, EMBO J.. I I, 2685-2693. Miller. B., I.R. Tsaneva and S.C. West (1993) Branch migration of Holliday junctions promoted by the Escherichia coli RuvA and RuvB proteins: I. Comparison of the RuvAB- and RuvBmediated reactions, J. Biol. Chem., 268, 17179-17184. Murchie, A.I.H., W.A. Carter, J. Portugal and D.M.J. Lilley (1990) The tertiary structure of the four-way DNA junction affords protection against DNase I cleavage, Nucleic Acids Res., 18, 2599-2606. Ogawa, T., X. Yu, A. Shinohara and E.H. Egelman (1993) Similarity of the yeast RadSl filament to the bacterial RecA filament, Science, 2.59. I896- 1899. Panyutin, I.G., I. Biswas and P. Hsieh (1995) A pivotal role for the structure of the Holliday junction in DNA branch migratton, EMBO J., 14, 1819-1826. Panyutin, 1.G. and P. Hsieh 11993) Formation of a single base mismatch impedes spontaneous DNA branch migration, J. Mol. Biol., 230, 4 13-424.
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Panyutin, I.G. and P. Hsieh (1994) The kinetics of spontaneous DNA branch migration, Proc. Natl. Acad. Sci. USA, 91, 202 I-2025. Parsons, CA., A. Stasiak, R.J. Bennett and S.C. West (1995a) Structure of a multisubunit complex that promotes DNA branch migration, Nature, 374, 375-378. Parsons, CA., A. Stasiak and S.C. West (1995b) The E. cob RuvAB proteins branch migrate Holliday junctions through heterologous DNA sequences in a reaction facilitated by SSB, EMBO J., in press. Parsons, CA., 1. Tsaneva, R.G. Lloyd and S.C. West (1992) Interaction of Escherichiu coli RuvA and RuvB proteins with synthetic Holliday junctions, Proc. Natl. Acad. Sci. USA, 89, 5452-5456. Parsons, C.A. and S.C. West (1993) Formation of a RuvAB-Holliday junction complex in vitro, 3. Mol. Biol., 232, 397-405. Pause, A. and N. Sonenberg (1992) Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A, EMBO J., 11, 2643-2654. Radding, CM. (1988) Homologous pairing and strand exchange promoted by Escherichia coli RecA protein, in: R. Kucherlapati and G.R. Smith (Eds.), Genetic Recombination. American Society for Microbiology, Washington, pp. 193-230. Roberts, J.W., C.W. Roberts and N.L. Craig (1978) Escherichiu RecA protein inactivates phage A repressor, Proc. Natl. Acad. Sci. USA, 75, 4714-4718. Salinas, F. and T. Kadadek (1994) Strand exchange through a DNA-protein complex requires a DNA helicase, Biochem. Biophys. Res. Comm., 205. SOO41009. Salinas, F. and T. Kodadek (1995) Phage T4 homologous strand exchange: A DNA helicase, not the strand transferase, drives polar branch migration, Cell, 82, I 1 l- 119. Shah, R., R.J. Bennett and SC. West (1994) Genetic recombinacoli
tion in E. cob: RuvC protein cleaves Holliday junctions at resolution hotspots in vitro., Cell, 79, 853-864. Sharples, G-J., F.E. Benson, G.T. llling and R.G. Lloyd (1990) Molecular and functional analysis of the ruu region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination, Mol. Gen. Genet., 221, 219-226. Sharples, G.J., M.C. Whitby, L. Ryder and R.G. Lloyd (1994) A mutation in helicase motif-LB of Escherichia coli RecG protein abolishes branch migration of Holiiday junctions, Nucieic Acids Res.. 22. 308-313. Shiba, T., H. Iwasaki, A. Nakata and H. Shinagawa (1991) SOS-inducible DNA repair proteins, RuvA and RuvB, of Escherichia coli: Functional interactions between RuvA and RuvB for ATP hydrolysis and renaturation of the cruciform structure in supercoiled DNA. Proc. Natl. Acad. Sci. USA, 88, 84458449. Shinagawa, H., K. Makino, M. Amemura, S. Kimura, H. Iwasaki and A. Nakata (1988) Structure and regulation of the Escherichia coli rw operon involved in DNA repair and recombination, J. Bacterial., 170, 4322-4329. Shinohara, A., H. Ogawa, Y. Matsuda, N. Ushio, K. lkeo and T. Ogawa (1993) Cloning of human, mouse and fission yeast recombination genes homologous to RADSZ and recA, Nature Genet., 4, 239-243.
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Shinohara, A., H. Ogawa and T. Ogawa (1992) Rad51 protein involved in repair and recombination in Saccharomyces cereoisiae is a RecA-like protein, Cell, 69, 457-470. Stasiak, A. and E.H. Egelman (1988) Visualization of recombination reactions, in: R. Kucherlapati and G.R. Smith (Eds.), Genetic Recombination, American Society for Microbiology, Washington, pp. 265-308. Stasiak, A.. 1.R. Tsaneva, S.C. West, C.J.B. Benson, X. Yu and E.H. Egelman (1994) The Escherichia co/i RuvB branch migration protein forms double hexameric rings around DNA, Proc. Natl. Acad. Sci. USA, 91, 7618-7622. Story, R.M., D.K. Bishop, N. Kleckner and T.A. Steitz (1993) Structural relationship of bacterial RecA proteins to recombination proteins from bacteriophage T4 and yeast, Science, 259, 1892-1896. Sung, P. (1994) Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast Rad5I protein, Science, 265, 1241-1243. Thompson, B.J., M.N. Camien and R.C. Warner (1976) Kinetics of branch migration in double-stranded DNA, Proc. Natl. Acad. Sci. USA. 73, 2299-2303. Tsaneva, I.R., B. Miller and SC. West (1992) ATP-dependent branch migration of Holliday junctions promoted by the RuvA and RuvB proteins of E. coli, Cell, 69, 1171-l 180. Tsaneva, I.R., B. Miller and SC. West (1993) The RuvA and RuvB proteins of Escherichia co/i exhibit DNA helicase activity in vitro, Proc. Natl. Acad. Sci. USA, 90, 1315-1319. Tsaneva. I.R. and S.C. West (1994) Targeted versus non-targeted
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ELSEVIER
Mutation Research 337 ( 1995) 149- I59
Minireview
Relaxing and unwinding on Holliday: DNA helicase-mediated branch migration David E. Adams, Stephen C. West
*
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, EN6 3LLA UK
Keywords: Branch migration;
Holliday junction;
DNA recombination;
DNA repair
1. Introduction
In this review, we focus on the basic mechanisms governing homologous strand exchange during general genetic recombination and recombinational repair, especially during the bacterial SOS response to DNA damage. Recent studies have shown that strand exchange is an enzyme-driven process, requiring the co-ordinated activity of a small number of dedicated recombination proteins. These enzymes can be separated into two categories: (1) those that act early in recombination to facilitate homologous DNA pairing and the initiation of strand exchange (e.g. the Escherichia coli RecA protein); and (2) those that act after recombination intermediates have been formed (e.g. the E. coli RuvABC and RecG proteins). This latter group of enzymes bind specifically to the crossover point, or Holliday junction, within the recombination intermediates and process these intermediates either by branch migration or endonucleolytic cleavage. The significance and importance of these studies is not limited to bacterial systems, however, as it has
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become increasing clear from recent work that the basic mechanisms of recombination and DNA repair are likely to have been highly conserved during evolution (Kowalczykowski and Eggleston, 1994; West, 1994). For example, a Succharumyces cereuisiue strand exchange protein, Rad51, has recently been identified which exhibits structural and functional homology to the E. colt’ RecA protein (Aboussekhra et al., 1992; Shinohara et al., 1992; Story et al., 1993). In vitro, yeast Rad51 forms nucleoprotein filaments very similar to those made by RecA (Ogawa et al., 1993) and catalyses an ATP-dependent strand exchange reaction (Sung, 1994). In addition, a human RecA equivalent was recently cloned (Shinohara et al., 1993) and purified (Benson et al., 1994). This protein, hRad5 1, forms nucleoprotein filaments on duplex DNA very similar to those made by E. coli RecA and yeast Rad51 (Benson et al., 1994; Ogawa et al., 1993). In E. coli, there are many gene products involved in promoting efficient homologous recombination. Here we discuss the activities of six of these gene products, namely those encoded by recA, ruuABC, recG, and uvrD. Each of these proteins has been implicated in the formation and processing of strand exchange intermediates during recombination and DNA repair.
IS0
D.E. Adams, S.C. West/Mutation
reverse f migration
Holliday junction
i
forward migration
1
resolution
Fig. 1. Formation and processing of Holliday junctions during recombination.Holliday junctions (2) form as a central intermediate during general genetic recombination. Movement of these junctions along the DNA during strandexchange requirestwisting of the DNA duplexes about their axes (indicated by the arrows), resulting in the generation of heterodupiexDNA. Branch migration in the forward direction (3) leads to the extension of the heteroduplexjoint, while branchmigrationin the reverse direction (1) tends to abort exchange. Another means of terminating ex-
change is to resolve the junction by endonucleolytic cleavage (4). 2. Branch migration of Holliday enzyme-catalysed process?
junctions:
an
During strand exchange a Holliday junction is created that physically links the two recombining DNA molecules (Fig. 1). Rapid movement of this junction along the DNA during strand exchange is thought to require helical rotation of the DNA about its axes (Meselson, 1972) and proceeds via the breakage and reformation of hydrogen bonds. Junction movement continues until (i) the junction reaches a free DNA end; (ii) it runs into sequence heterology
Research 337 (1995) 149-1.59
(i.e. a mismatch, insertion/deletion or DNA lesion); and/or (iii) it is resolved by a structure-specific endonuclease. Forward migration is responsible for the extension of the heteroduplex joint, while reverse migration is anti-recombinogenic. Early theoretical and experimental studies suggested that the rate of spontaneous branch migration, i.e. branch migration in the absence of added proteins, would be a rapid process (Meselson, 1972; Thompson et al., 1976; Warner et al., 1978). Rates as high as thousands of base pairs per second (bp/s) were predicted and initially observed by following the dissociation of dimeric phage DNA molecules containing a Holliday junction, i.e. x-structures (Thompson et al., 1976; Warner et al., 1978). Recently, however, these rate estimates were re-examined, and it is now clear that non-catalysed branch migration is at least 3 orders of magnitude too slow under physiological reaction conditions to account for the rate of branch migration in vivo. The first indication that spontaneous branch migration might be a slow process came from groups studying cruciform transitions in supercoiled plasmid DNA (Courey and Wang, 1983; Gellert et al., 1983). More recently, slow rates for spontaneous branch migration have been measured in experiments using both RecA-made and synthetically-made four-way junctions (Johnson and Symington, 1993; Miller et al., 1992; Panyutin and Hsieh, 1993; Panyutin and Hsieh, 1994). In one study, deproteinised recombination intermediates, isolated from an in vitro strand exchange reaction catalysed by RecA, were observed to slowly dissociate over time, with a step rate on the order of a few tens of bp/s at 37°C (Miller et al., 1992). Remarkably, even at 72”C, _ 30% of the intermediates were still present after 1 h. This slow rate of spontaneous branch migration has been confirmed in experiments using large, plasmid-based X-structures (Johnson and Symington, 1993). In a recent set of experiments, the rate of noncatalysed branch migration was measured under a wide variety of buffer and salt conditions (Panyutin et al., 1995; Panyutin and Hsieh, 1993; Panyutin and Hsieh, 1994). The step rate was found to be a sensitive function of both temperature and divalent metal ion concentration. For example, between 10 and 1 mM MgCl,, the step rate increased about 4-fold at 37”C, and between 0.5 and 0.1 mM MgCl 2,
D.E. Adams, XC. West/Mutation Research 337 (1995) 149-159
the rate increased over 60-fold. To calculate the step rate of branch migration from the half-life of junction survival, crossover movement was modelled as a simple one-dimensional random walk, where the probability of a forward step is equal to that of a backward step. For the most part, theory and experiment correlated perfectly (Panyutin and Hsieh, 1994). The average rate of spontaneous branch migration in 50 mM NaCl was found to be - 3,300 bp/s at 37°C. By contrast, in the presence of 10 mM MgCl, and 50 mM NaCl, the step rate was 1000 times slower, or 3.3 bp/s. This 3 order of magnitude difference in step rate in sodium versus magnesium is likely to be due to structural changes in the Holliday junction under different salt conditions. In the absence of metal ions, the four-way junction adopts an extended square configuration (Fig. 2, centre), in which base pairs at the base of the junction are partially unstacked (Duckett et al., 1988; Duckett et al., 1990; Lilley and Clegg, 1993). If allowed to freely branch migrate, this junction will move along the DNA at speeds up to a few thousand of bp/s. In the presence of polyvalent metal ions, however, the Holliday junction adopts a folded Xstructure, which is much less susceptible to spontaneous branch migration (Lilley and Clegg, 1993; Murchie et al., 1990; Panyutin et al., 1995). The isomeric form of the junction is determined by the stacking interactions at the base of the crossover and thus frequent isomerisation of the junction may occur during branch migration. This probably accounts for why spontaneous branch migration is a slow process under physiological reaction conditions.
isomer I
151
Several E. cd proteins have now been isolated which bind specifically to Holliday junctions and promote their rapid movement along the DNA: the RuvA and RuvB proteins and the RecG protein (Lloyd and Sharples, 1993a; Tsaneva et al., 1992). Here we compare the branch migrational activities of RuvAB and RecG to that of RecA, which was the first E. coli enzyme found to catalyse branch migration in vitro (Cox and Lehman, 1981; DasGupta et al., 1980; West et al., 1981).
3. RecA protein: jack of all trades RecA is the major recombination protein in prokaryotic cells. Most of what is known about homologous DNA pairing and strand exchange comes from studies of the 38 kDa RecA protein (Kowalczykowski et al., 1994; Radding, 1988; West, 1992). Mutants in recA are highly pleiotropic, affecting not only recombination and DNA repair, but also cell division and mutagenesis (Clark and Sandler, 1994). Firstly, RecA acts as a co-protease (Little, 1984; Roberts et al., 1978) to stimulate the selfcleavage of LexA and a number of other transcriptional repressors (phage-encoded). Cleavage of LexA leads to a diverse set of physiological responses known as the SOS responses, which include derepression of an entire set of genes dedicated to DNA repair (Walker, 1984). Secondly, RecA is a DNA-dependent ATPase that catalyses homologous DNA pairing and crossover formation during strand exchange (Kowalczykowski et al., 1994; West, 1992).
unfolded junction
isomer II
Fig. 2. Isomerisation of the Holliday junction. In the absence of junction binding proteins, the Holliday crossover is free to adopt a number of different conformational states, depending on the reaction conditions. The stacked-X configuration (i.e. isomer I and isomer II) is favoured in the presence of polyvalent metal ions, while the unfolded configuration is favoumd in the absence of metal ions. During spontaneous branch migration in vitro, the Holliday junction may need to inter convert frequently between these different conformational isomers, leading to a slower overall rate of branch migration. The most favourable junction configuration for branch migration is the unfolded junction.
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AS a first step during recombination, RecA binds single-stranded DNA to form a long nucleoprotein filament. This filament extends to duplex regions and DNA within this filament is unwound and stretched to approximately 1.5 times its normal length, a feature unique to homologous recombination (Stasiak and Egelman, 1988). Unwinding of the DNA facilitates the next phase of recombination, i.e. the search for DNA homology. This search is made difficult due to the large size of cellular genomes: > lo6 base pairs in E. coli and > lo9 base pairs in many eukaryotes. Following homologous DNA pairing and the initiation of strand exchange, heteroduplex joints are made by RecA (Fig. 3) in which the DNA contains either a four-way or a three-way Holliday junction (Cox and Lehman, 1987; Kowalczykowski et al., 1994; Radding, 1988; West, 1992). These junctions are then further processed, either by branch migration, and/or by nucleolytic cleavage. Early studies showed that RecA catalyses a slow, unidirectional branch migration reaction at a rate of 2-10 bp/s (Radding, 1988). This sluggish rate indicates that RecA is unlikely to be the major Holliday junction branch migration enzyme in vivo, although this conclusion was not obvious at the time. RecA also catalyses a branch migration reaction through regions of DNA damage and DNA heterology, but only small regions are bypassed (Hahn et al., 1988; Livneh and Lehman, 1982; Morel et al., 1994). Thus, despite being a jack of all trades (i.e. co-inducer of the SOS response, homologous DNA pairing protein, to
heteroduplex
initiator of DNA strand exchange, etc.>, RecA may be master of only a few. This observation raises the question - are there other bacterial proteins that make recombination more efficient?
4. RuvAB-catalysed
branch migration
The ruu locus of E. cofi encodes three gene products known to be important for Holliday junction processing. Cells carrying mutations in any of the three ruu genes (muA, ruuB, ruuC) are sensitive to UV-light and ionising radiation, and exhibit reduced recombination frequencies (Benson et al., 1991; Sharples et al., 1990; Shinagawa et al., 1988). All three ruu gene products have been cloned, overexpressed and purified, and in vitro studies have been invaluable in defining their important biological roles (West, 1994). The RuvA and RuvB proteins of E. coli are co-expressed from a common, LexA-regulated operon during the E. coli SOS response (Benson et al., 1988; Shinagawa et al., 19881, and like RecA, are required by cells for the efficient recovery from DNA damage. The 22 kDa RuvA protein acts to target the 37 kDa RuvB protein to the site of the Holliday junction (Hiom and West, 1995; Iwasaki et al., 1992; Parsons et al., 1995a; Parsons et al., 1992; Parsons and West, 1993; Shiba et al., 1991), and stimulates the ATPase activity of RuvB (Mitchell and West, 1994; Shiba et al., 1991). RuvB is the motor that drives branch migration (Adams and West,
multi-stranded DNA
homoduplex
direction of RecA strand exchange Fig. 3. RecA-mediated 4-strand exchange. RecA protein catalyses a 4-strand exchange reaction between two homologous DNA duplexes (i.e. homoduplexes), which become intertwined within the RecA nucleoprotein filament. Pairing results in the formation of multi-stranded DNAs and is followed by strand exchange. RecA protein remains tightly bound to the products of strand exchange (i.e. heteroduplexes) and needs to be recycled at the end of the reaction.
D.E. Adams, SC. West/Mutation Research 337 (1995) 149-159
1995; Miller et al., 1993; Tsaneva et al., 1992; Tsaneva et al., 1993), while RuvA plays an important ancillary role, reducing the requirement for RuvB in branch migration approximately 50-fold. Together RuvA and RuvB form an ATP-dependent DNA helicase that is capable of unwinding a partial duplex up to 558 bp in length with a defined 5’ to 3’ polarity (Tsaneva et al., 1993). The RuvAB DNA helicase activity can be specifically targeted to Holliday junctions, and is stimulated by the presence of the E. cofi SSB protein (Tsaneva and West, 1994). RuvA protein itself stimulates both the rate and extent of RuvB-mediated DNA unwinding (Adams and West, 19951, indicating that RuvA acts as a helicase accessory factor for RuvB. Consistent with this hypothesis is the observation that RuvB protein possesses sequence motifs shared by other E. coli DNA helicases (Lloyd and Sharples, 1993b; Tsaneva et al., 1993). From these observations, it was proposed that RuvAB-catalysed branch migration involves the unwinding and rewinding of duplex DNA near the base of the Holliday junction. At some stage in the reaction, ATP hydrolysis is required. RuvAB form an unusual DNA unwinding machine, however, as ATP hydrolysis is apparently not directly coupled to branch migration (A. Mitchell and S.C.W., unpublished results). Recent experimental studies indicate that RuvAB form a tripartite protein complex on Holliday junctions, as assayed by enzymatic probing and electron microscopy (Hiom and West, 1995; Parsons et al., 1995a). A diagram of this complex is shown in Fig. 4. Consistent with DNase 1 footprinting (Hiom and West, 19951, gel filtration (Mitchell and West, 1994), and electron microscopic studies (Parsons et al., 1995a), tetramers of RuvA protein are depicted as binding to the base of the crossover with the junction arms in a square configuration. Supporting evidence for this unfolded arm geometry has come from band-shift experiments, which showed that RuvA stabilises the unfolded form of a Holliday junction (Parsons et al., 1995a). Also shown in the model is the position of the RuvB protein. Two hexameric rings of RuvB sit on opposite arms of the junction, with duplex DNA passing through the centre of each protein ring. Image analysis and mass measurements have indicated that each RuvB ring contains six protein monomers (Stasiak et al., 1994), leading to
1.53
RuvAB complex
branch migration
Fig. 4. RuvAB-mediated branch migration. RuvA and RuvB form a tripartite protein complex that promotes the branch migration of unfolded Holliday junctions. Two hexameric rings of RuvB protein sit on opposite arms of the junction, with RuvA protein bound at the base. DNA within the cavity of each RuvB ring is thought to be transiently unwound and rewound, leading to twisting and translocation of the DNA in the direction indicated.
the suggestion that hexameric RuvB rings are the active form of the enzyme during branch migration. This suggestion has received indirect experimental support by the finding that RuvAB form stable (RuvA),(RuvB), complexes in solution (Mitchell and West, 1994). To carry out branch migration, RuvAB are thought to catalyse a transient unwinding reaction in the duplex DNA lying within the interior of the RuvB ring, leading to translocation of the DNA through the branch migration complex (Adams and West, 1995; Parsons et al., 1995a; Tsaneva et al., 1993). Repeated cycles of DNA unwinding and rewinding may lead to twisting of the junction arms about their axes, leading to base pair swapping at the crossover. Rapid
154
D.E. Adams, SC. West/Mutation
junction movement is also thought to be facilitated by RuvA holding the junction arms in an unfolded configuration (Parsons et al., 1995a). This model for RuvAB action bears striking resemblance to the DNA unwinding reaction catalysed by the SV40 large T-antigen (Dean and Hurwitz, 1991; Mastrangelo et al., 1989). When this ringshaped DNA helicase binds to its cognate replication origin, two protein rings are seen by electron microscopy to sit at the palindromic origin (Wessel et al., 1992). Each belicase ring contains six protein monomers, similar to RuvB. Unwinding and translocation of DNA through the origin unwinding complex results in the generation of single-stranded DNA, which in turn allows for binding of the DNA polymerase apparatus. RuvB and SV40 large T-antigen thus belong to an emerging group of hexameric DNA helicases, which includes the E. cd DnaB protein (Bujalowski et al., 1994) and T7 helicase/primase (Egelman et al., 1995), that serve important functions during DNA replication, recombination and repair.
5. Role of RecA and RuvAB heterologies
Research 337 (1995) 149-159
but strand exchange is slow and requires large amounts of ATP (Hahn et al., 1988; Morel et al., 1994; Tsaneva et al., 1992). The situation with the RuvAB complex is entirely different. Both genetic (Lloyd and Buckman, 1995; Matic et al., 1995) and biochemical evidence (Iype et al., 1994; Parsons et al., 1995b; Tsaneva et al., 1992) support the idea that RuvAB have a unique role in recombinational repair. For example, when four-strand exchange substrates are heavily irradiated with UV-light in vitro, RecAmediated strand exchange is severely blocked, whereas addition of RuvAB to the strand exchange reaction stimulates recombinational bypass (Tsaneva et al., 1992). In addition, when RuvAB are added to ongoing three-strand RecA reactions between single-stranded circular and linear duplex DNA, where the linear duplex DNA harbours heterologous inserts ranging in size from 198 to 1037 bp, RuvAB was found to greatly stimulate bypass of the large inserts (Iype et al., 1994). This bypass is likely due to RuvAB itself. Recently, RuvAB were shown to unwind DNA heterologies up to 1800 bp in size in the absence of other proteins (Parsons et al., 1995b).
in bypass of DNA
A critical problem in both general genetic recombination and recombinational repair is the promotion of strand exchange through heterologous and damaged DNA sequences. Heterologous insertions, deletions, mismatches and DNA lesions can all potentially disrupt the normal exchange of DNA strands. The mechanism by which branch migration enzymes like RecA and RuvAB promote the movement of Holliday junctions through damaged DNA and DNA heterologies is of great interest, and it is vital for understanding how species barriers are maintained (Matic et al., 1995) and for understanding what limits recombination between closely related gene families (Bianchi and Radding, 1983). Renewed interest in this problem has arisen with the finding that a single base mismatch can significantly impede spontaneous branch migration (Panyutin and Hsieh, 1993). RecA protein is able to promote four-stranded exchanges through short regions of DNA lesions, mismatches, and heterologous insertions ( < 100 bp),
6. RuvAB-mediated filaments
dissociation
of RecA protein
The E. coli RuvA and RuvB proteins possess another activity that may be important for genetic recombination. Together RuvA and RuvB promote the ATP-dependent dissociation of RecA nucleoprotein filaments from duplex DNA (Adams et al., 1994). This ability is thought to be useful in several regards, both in cleaning up the DNA of the proteins used to create the Holliday junction, and in recycling RecA protein during recombinational repair (Adams et al., 1994; Kuzminov, 1993). The requirement for ATP in the RecA displacement reaction suggests that helicase translocation is required (Adams et al., 1994). Several groups have now obtained evidence for DNA helicases being able to displace tightlybound protein from DNA (Bedinger et al., 1983; Bonne-Andrea et al., 1990; Eggleston et al., 1995; Salinas and Kadadek, 1994; Yancey-Wrona and Matson, 1992). The physical act of threading doublestranded DNA through the cavity of the RuvB ring, coupled with transient DNA unwinding, may provide
D.E. Adams. S.C. West/Mutation
a processive mechanism for RecA displacement. Alternatively, a direct physical interaction between RecA and RuvAB may be required (Adams et al., 1994). At this point in time, the exact mechanism of RuvAB-mediated RecA displacement from duplex DNA is unknown, Nor is it clear whether RuvAB can displace RecA bound to other forms of DNA (Iype et al., 1994). Recently, the T4 dda helicase was shown to dissociate tightly-bound proteins from duplex DNA (Salinas and Kadadek, 1994) and to dissociate synthetic DNA triple helices (Maine and Kodadek, 1994). Perhaps RuvAB can remove RecA from 3- and 4-stranded intermediates and also dissociate the multi-stranded structures made by RecA. The ability of RuvAB to dissociate RecA protein from recombination intermediates may also allow other Holliday junction processing enzymes, like the 19 kDa RuvC protein, to gain access to the Holliday junction, and resolve these junctions by endonucleolytic cleavage (Benson and West, 1994; Dunderdale et al., 1991; Iwasaki et al., 1991). The preference of RuvC for a small 4 bp consensus sequence, S*/$-TI G/C-3’ (B ennett et al., 1993; Shah et al., 1994) may make this enzyme dependent on RuvAB (or another E. c&-encoded branch migration activity) to position the Holliday junction near preferred sites of cleavage (i.e. resolution ‘hotspots’). Recent structural studies of RuvC and RuvC-Holliday junction complexes will lead to rapid progress in our understanding the molecular details of the RuvC junction cleavage reaction (Ariyoshi et al., 1994; Bennett and West, 199Sa; Bennett and West, 1995b). Remarkably, single mutants in ruuA, ruuB, or ruuC are viable, exhibiting only a 2-3-fold deficiency in genetic recombination (Lloyd and Sharples, 1992). These observations imply that there are other pathways for processing of Holliday junctions in E. co/i cells.
7. RecG and UvrD-mediated
branch migration
TWO other E. coli proteins are known to be able to catalyse the branch migration of Holliday junctions in vitro: the 76 kDa RecG protein (Lloyd and Sharples, 1993a) and the 82 kDa UvrD protein (Morel et al., 1993). Both proteins are ATP-dependent DNA
Research 337 (1995) 149-159
155
helicases that unwind duplex DNA in the 3’ to 5’ direction (a polarity opposite to that exhibited by RuvAB). In addition, both proteins can catalyse the branch migration of RecA-made recombination intermediates. The role of RecG and UvrD (in particular) in catalysing the branch migration of Holliday junctions in vivo, however, is less clear than it is for RecA and RuvAB. Mutants in recG exhibit a functional overlap with the ruu genes. Recombination is reduced about 500fold in ruv recG strains, but only Z- to 3-fold in either of the single mutants (Lloyd, 1991). This functional overlap does not extent to DNA repair, however, as recG mutants are much less sensitive to UV irradiation than are single mutants in ruv. Unlike RuvAB, the synthesis of RecG is not induced as part of the SOS response. Sequence analysis of the recG gene shows structural motifs that are conserved in the DExH family of DNA and RNA helicases. This observation suggests that RecG, like RecA and RuvAB, uses a DNA unwinding activity to promote Holliday junction branch migration (Lloyd and Sharples, 1993a). Support for this idea has come from biochemical studies indicating that a single Ala to Val substitution in the helicase III motif of RecG, leads to the production of a mutant RecG protein that is unable to branch migrate DNA, despite retaining its ATPase and junction-binding activity (Sharples et al., 1994). Mutations in this motif also inhibit the eIF-4A RNA helicase, which acts during protein translation (Pause and Sonenberg, 1992). When RecG protein is added to ongoing RecAmediated 4-strand exchange reactions in vitro, reverse branch migration of the junction is observed (Whitby et al., 1993). This reverse branch migration activity may serve to resolve junctions in vivo by running the junction points off a free DNA end. Alternatively, it may serve an anti-recombinogenic function. In support of this hypothesis, recent genetic data indicate an important role for RecG in limiting recombination-dependent stable DNA synthesis (Hong et al., 1995). With four E. cofi proteins (i.e. RecA, RuvAB and RecG) already known to catalyse branch migration in vivo, is there any need (or evidence) for more such activities? The E. coli UvrD protein (helicase II) stimulates RecA-mediated strand exchange in vitro
156
D.E. Adams, S.C. West/Mutation Research 337 (1995) 149-159
and it also catalyses branch migration in the absence of RecA (Morel et al., 1993). In agreement with these biochemical observations, genetic data indicate that certain MM-Dmutants are deficient in recombination (Horii and Clark, 1973; Morel et al., 1993). But some are normal or even exhibit elevated frequencies of recombination (Horii and Clark, 1973; Morel et al.. 1993). Furthermore, purified UvrD can reverse RecA-mediated synapsis in vitro and even inhibit RecA-mediated homologous DNA pairing (Morel et al., 1993). Hence the precise role for UvrD in recombination remains unclear. In principal, it is possible that any DNA helicase may be able to catalyse branch migration, since DNA unwinding is involved. The question is one of specificity. The bacteriophage T4 dda helicase, for example, stimulates UvsX-mediated strand exchange in vitro, but does not catalyse branch migration on its own (Kodadek and Albert& 1987). In addition, the T4 gene 41 DNA helicase can also stimulate UvsXmediated strand exchange, provided its helicase accessory factor, gene 59 protein, is present (Salinas and Kodadek, 1995). Together, gene 41 helicase and gene 59 protein promote the branch migration of three-way junctions in the absence of UvsX. The adapter protein gene 59 does not act like RuvA in RuvAB reactions, however, by targeting its cognate helicase directly to Holliday junctions. Instead, it acts hy targeting Ihe gene 41 helicase directly to DNA bound by the T4 single-stranded DNA binding protein, gene 32 protein, through specific proteinprotein interactions. Thus nature has evolved at least two independent means of targeting DNA helicases to recombination intermediates: either through specific protein-DNA interactions (e-g. RuvAB and RecG to Holliday junctions) or through specific protein-protein interactions (e.g. T4 gene 41/59/32 proteins).
8. Concluding
remarks
DNA helicases play diverse roles in genetic recombination and DNA repair, including (i) the generation of recombinogenic single-stranded DNA ends; (ii) the unwinding of DNA damages; (iii) anti-recombinogenic functions; and (iv) the branch migration of Holliday junctions. Holliday junctions are
more stable than was originally thought and it is now known that they do not spontaneously branch migrate under physiological reaction conditions. Current models of helicase-mediated branch migration postulate that helicase activity is used to unwind base pairs at the strand exchange fork, resulting in the swapping of base pairs and heteroduplex DNA formation. DNA helicase activity is likely also to be used to unwind large blocks of DNA heterology and DNA lesions during recombination and DNA repair. Currently, little or nothing is known about the mechanics of Holliday junction branch migration in higher cells. The recent excitement generated by the study of prokaryotic branch migration proteins is likely to spill over soon and lead to the discovery of eukaryotic homologues of these remarkable enzymes.
Acknowledgements We are grateful to Alison Mitchell, Carol Parsons and Andrzej Stasiak for access to recent and unpublished data. Our research is supported entirety by the Imperial Cancer Research Fund.
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