Atomic structures and functional implications of the archaeal RecQ-like helicase Hjm

Background  

Pyrococcus furiosus Hjm (PfuHjm) is a structure-specific DNA helicase that was originally identified by in vitro screening for Holliday junction migration activity. It belongs to helicase superfamily 2, and shares homology with the human DNA polymerase Θ (PolΘ), HEL308, and Drosophila Mus308 proteins, which are involved in DNA repair. Previous biochemical and genetic analyses revealed that PfuHjm preferentially binds to fork-related Y-structured DNAs and unwinds their double-stranded regions, suggesting that this helicase is a functional counterpart of the bacterial RecQ helicase, which is essential for genome maintenance. Elucidation of the DNA unwinding and translocation mechanisms by PfuHjm will require its three-dimensional structure at atomic resolution.     

Hjm/Hel308A DNA helicase from Sulfolobus tokodaii promotes replication fork regression and interacts with Hjc endonuclease in vitro

Hjm and Hel308a are novel, RecQ-like DNA helicases recently identified in the euryarchaeotes Pyrococcus furiosus and Methanothermobacter thermautotrophicus, respectively. In this study, an Hjm/Hel308 homologue (designated StoHjm) from Sulfolobus tokodaii, a hyperthermophilic archaeon belonging to the Crenarchaeota subdomain of archaea, was cloned, purified, and characterized. Unlike Hjm and Hel308a, which unwind DNA in a 3'-to-5' direction, StoHjm unwound DNA in both 3'-to-5' and 5'-to-3' directions. Remarkably, StoHjm exhibited structure-specific single-stranded-DNA-annealing and fork regression activities in vitro. In addition, gel filtration, affinity pulldown, and yeast two-hybrid analyses revealed that StoHjm physically interacted with StoHjc, the Holliday junction-specific endonuclease from S. tokodaii. This interaction may have functional significance, because the unwinding activity of StoHjm was inhibited by StoHjc in vitro. These results may suggest that the Hjm/Hel308 family helicases, in association with Hjc endonucleases, are involved in processing of stalled replication forks.     

Branch migration of Holliday junctions: identification of RecG protein as a junction specific DNA helicase.

The product of the recG gene of Escherichia coli is needed for normal recombination and DNA repair in E. coli and has been shown to help process Holliday junction intermediates to mature products by catalysing branch migration. The 76 kDa RecG protein contains sequence motifs conserved in the DExH family of helicases, suggesting that it promotes branch migration by unwinding DNA. We show that RecG does not unwind blunt ended duplex DNA or forked duplexes with short unpaired single-strand ends. It also fails to unwind a partial duplex (52 bp) classical helicase substrate containing a short oligonucleotide annealed to circular single-stranded DNA. However, unwinding activity is detected when the duplex region is reduced to 26 bp or less, although this requires high levels of protein. The unwinding proceeds with a clear 3' to 5' polarity with respect to the single strand bound by RecG. Substantially higher levels of unwinding are observed with substrates containing a three-way duplex branch. This is attributed to RecG's particular affinity for junction DNA which we demonstrate would be heightened by single-stranded DNA binding protein in vivo. Reaction requirements for unwinding are the same as for branch migration of Holliday junctions, with a strict dependence on hydrolysis of ATP. These results define RecG as a new class of helicase that has evolved to catalyse the branch migration of Holliday junctions.     

Helicase-defective RuvB(D113E) promotes RuvAB-mediated branch migration in vitro.

In Escherichia coli, the RuvA and RuvB proteins interact at Holliday junctions to promote branch migration leading to the formation of heteroduplex DNA. RuvA provides junction-binding specificity and RuvB drives ATP-dependent branch migration. Since RuvB contains sequence motifs characteristic of a DNA helicase and RuvAB exhibit helicase activity in vitro, we have analysed the role of DNA unwinding in relation to branch migration. A mutant RuvB protein, RuvB(D113E), mutated in helicase motif II (the DExx box), has been purified to homogeneity. The mutant protein forms hexameric rings on DNA similar to those formed by wild-type protein and promotes branch migration in the presence of RuvA. However, RuvB(D113E) exhibits reduced ATPase activity and is severely compromised in its DNA helicase activity. Models for RuvAB-mediated branch migration that invoke only limited DNA unwinding activity are proposed.     

Dissection of the functional domains of an archaeal Holliday junction helicase

Helicases and nucleases form complexes that play very important roles in DNA repair pathways some of which interact with each other at Holliday junctions. In this study, we present in vitro and in vivo analysis of Hjm and its interaction with Hjc in Sulfolobus. In vitro studies employed Hjm from the hyperthermophilic archaeon Sulfolobus tokodaii (StoHjm) and its truncated derivatives, and characterization of the StoHjm proteins revealed that the N-terminal module (residues 1-431) alone was capable of ATP hydrolysis and DNA binding, while the C-terminal one (residues 415-704) was responsible for regulating the helicase activity. The region involved in StoHjm-StoHjc (Hjc from S. tokodaii) interaction was identified as part of domain II, domain III (Winged Helix motif), and domain IV (residues 366-645) for StoHjm. We present evidence supporting that StoHjc regulates the helicase activity of StoHjm by inducing conformation change of the enzyme. Furthermore, StoHjm is able to prevent the formation of Hjc/HJ high complex, suggesting a regulation mechanism of Hjm to the activity of Hjc. We show that Hjm is essential for cell viability using recently developed genetic system and mutant propagation assay, suggesting that Hjm/Hjc mediated resolution of stalled replication forks is of crucial importance in archaea. A tentative pathway with which Hjm/Hjc interaction could have occurred at stalled replication forks is discussed.     

Functional interactions between the holliday junction resolvase and the branch migration motor of Escherichia coli.

Homologous recombination generates genetic diversity and provides an important cellular pathway for the repair of double-stranded DNA breaks. Two key steps in this process are the branch migration of Holliday junctions followed by their resolution into mature recombination products. In E.coli, branch migration is catalysed by the RuvB protein, a hexameric DNA helicase that is loaded onto the junction by RuvA, whereas resolution is promoted by the RuvC endonuclease. Here we provide direct evidence for functional interactions between RuvB and RuvC that link these biochemically distinct processes. Using synthetic Holliday junctions, RuvB was found to stabilize the binding of RuvC to a junction and to stimulate its resolvase activity. Conversely, RuvC facilitated interactions between RuvB and the junction such that RuvBC complexes catalysed branch migration. The observed synergy between RuvB and RuvC provides new insight into the structure and function of a RuvABC complex that is capable of facilitating branch migration and resolution of Holliday junctions via a concerted enzymatic mechanism.     

Characterisation of RuvAB-Holliday junction complexes by glycerol gradient sedimentation.

The Escherichia coli RuvA and RuvB proteins interact specifically with Holliday junctions to promote ATP-dependent branch migration during genetic recombination and DNA repair. In the work described here, glycerol gradient centrifugation was used to investigate the requirements for the formation of pre-branch migration complexes. Since gradient centrifugation provides a simple and gentle method to analyse relatively unstable protein-DNA complexes, we were able to detect RuvA- and RuvAB-Holliday junction complexes without the need for chemical fixation. Using 35S-labelled RuvA protein and 3H-labelled Holliday junctions, we show that RuvA acts as a helicase accessory factor that loads the RuvB helicase onto the Holliday junction by structure-specific interactions. The resulting complex contained both RuvA and RuvB, as detected by Western blotting using serum raised against RuvA and RuvB. The stoichiometry of binding was estimated to be approximately four RuvA tetramers per junction. Formation of the RuvAB-Holliday junction complex required the presence of divalent metal ions and occurred without the need for ATP. However, the stability of the complex was enhanced by the presence of ATP gamma S, a non-hydrolysable ATP analogue. The data support a model for branch migration in which structure-specific binding of Holliday junctions by RuvA targets the assembly of hexameric RuvB rings on DNA. Specific loading of the RuvB ring helicase by RuvA is likely to be the initial step towards ATP-dependent branch migration.     

RuvAB-mediated branch migration does not involve extensive DNA opening within the RuvB hexamer

The Escherichia coli RuvA and RuvB proteins promote the branch migration of Holliday junctions during the late stages of homologous recombination and DNA repair (reviewed in [1]). Biochemical and structural studies of the RuvAB-Holliday junction complex have shown that RuvA binds directly to the Holliday junction [2] [3] [4] [5] [6] and acts as a specificity factor that promotes the targeting of RuvB [7] [8], a hexameric ring protein that drives branch migration [9] [10] [11]. Electron microscopic visualisation of the RuvAB complex revealed that RuvA is flanked by two RuvB hexamers, which bind DNA arms that lie diametrically opposed across the junction [8]. ATP-dependent branch migration occurs as duplex DNA is pumped out through the centre of each ring. Because RuvB possesses well-conserved helicase motifs and RuvAB exhibits a 5'-3' DNA helicase activity in vitro [12], the mechanism of branch migration is thought to involve DNA opening within the RuvB ring, which provides a single strand for the unidirectional translocation of the protein along DNA. We have investigated whether the RuvB ring can translocate along duplex DNA containing a site-directed interstrand psoralen crosslink. Surprisingly, we found that the crosslink failed to inhibit branch migration. We interpret these data as evidence against a base-by-base tracking model and suggest that extensive DNA opening within the RuvB ring is not required for DNA translocation by RuvB.     

The phage T4 protein UvsW drives Holliday junction branch migration

The phage T4 UvsW protein has been shown to play a crucial role in the switch from origin-dependent to recombination-dependent replication in T4 infections through the unwinding of origin R-loop initiation intermediates. UvsW also functions with UvsX and UvsY to repair damaged DNA through homologous recombination, and, based on genetic evidence, has been proposed to act as a Holliday junction branch migration enzyme. Here we report the purification and characterization of UvsW. Using oligonucleotide-based substrates, we confirm that UvsW unwinds branched DNA substrates, including X and Y structures, but shows little activity in unwinding linear duplex substrates with blunt or single-strand ends. Using a novel Holliday junction-containing substrate, we also demonstrate that UvsW promotes the branch migration of Holliday junctions efficiently through more than 1000 bp of DNA. The ATP hydrolysis-deficient mutant protein, UvsW-K141R, is unable to promote Holliday junction branch migration. However, both UvsW and UvsW-K141R are capable of stabilizing Holliday junctions against spontaneous branch migration when ATP is not present. Using two-dimensional agarose gel electrophoresis we also show that UvsW acts on T4-generated replication intermediates, including Holliday junction-containing X-shaped intermediates and replication fork-shaped intermediates. Taken together, these results strongly support a role for UvsW in the branch migration of Holliday junctions that form during T4 recombination, replication, and repair.     

A mutation in helicase motif III of E. coli RecG protein abolishes branch migration of Holliday junctions.

The RecG protein of Escherichia coli catalyses branch migration of Holliday junctions made by RecA and dissociates synthetic X junctions into duplex products in reactions that require hydrolysis of ATP. To investigate the mode of action of this enzyme a chromosomal mutation that inactivates recG (recG162) was cloned and sequenced. The recG162 mutation is a G:C to A:T transition, which produces an Ala428 to Val substitution in the protein. This change affects a motif (motif III) in the protein that is highly conserved in DNA and RNA helicases. RecG162 protein was purified and shown to retain the ability to bind synthetic X and Y junctions. However, it does not dissociate these junctions and fails to catalyse branch migration of Holliday junction intermediates purified from a RecA strand exchange reaction. RecG162 retains a DNA-dependent ATPase activity, but this is much reduced relative to the wild-type protein, especially with single-stranded DNA as a co-factor. These results suggest that branch migration by RecG is related to a junction-targeted DNA helicase activity.     

RuvA is a sliding collar that protects Holliday junctions from unwinding while promoting branch migration

The RuvAB proteins catalyze branch migration of Holliday junctions during DNA recombination in Escherichia coli. RuvA binds tightly to the Holliday junction, and then recruits two RuvB pumps to power branch migration. Previous investigations have studied RuvA in conjunction with its cellular partner RuvB. The replication fork helicase DnaB catalyzes branch migration like RuvB but, unlike RuvB, is not dependent on RuvA for activity. In this study, we specifically analyze the function of RuvA by studying RuvA in conjunction with DnaB, a DNA pump that does not work with RuvA in the cell. Thus, we use DnaB as a tool to dissect RuvA function from RuvB. We find that RuvA does not inhibit DnaB-catalyzed branch migration of a homologous junction, even at high concentrations of RuvA. Hence, specific protein-protein interaction is not required for RuvA mobilization during branch migration, in contrast to previous proposals. However, low concentrations of RuvA block DnaB unwinding at a Holliday junction. RuvA even blocks DnaB-catalyzed unwinding when two DnaB rings are acting in concert on opposite sides of the junction. These findings indicate that RuvA is intrinsically mobile at a Holliday junction when the DNA is undergoing branch migration, but RuvA is immobile at the same junction during DNA unwinding. We present evidence that suggests that RuvA can slide along a Holliday junction structure during DnaB-catalyzed branch migration, but not during unwinding. Thus, RuvA may act as a sliding collar at Holliday junctions, promoting DNA branch migration activity while blocking other DNA remodeling activities. Finally, we show that RuvA is less mobile at a heterologous junction compared to a homologous junction, as two opposing DnaB pumps are required to mobilize RuvA over heterologous DNA.     

Mutational analysis of the functional motifs of RuvB, an AAA+ class helicase and motor protein for holliday junction branch migration

Escherichia coli RuvB protein, together with RuvA, promotes branch migration of Holliday junctions during homologous recombination and recombination repair. The RuvB molecular motor is an intrinsic ATP-dependent DNA helicase with a hexameric ring structure and its architecture has been suggested to be related to those of the members of the AAA+ protein class. In this study, we isolated a large number of plasmids carrying ruvB mutant genes and identified amino acid residues important for the RuvB functions by examining the in vivo DNA repair activities of the mutant proteins. Based on these mutational studies and amino acid conservation among various RuvBs, we identified 10 RuvB motifs that agreed well with the features of the AAA+ protein class and that distinguished the primary structure of RuvB from that of typical DNA/RNA helicases with seven conserved helicase motifs.     

Branch migration of three-strand recombination intermediates by RecG, a possible pathway for securing exchanges initiated by 3''-tailed duplex DNA.

RecG protein is required for normal levels of recombination and DNA repair in Escherichia coli. This 76 kDa polypeptide is a junction-specific DNA helicase that acts post-synaptically to drive branch migration of Holliday junction intermediates made by RecA during the strand exchange stage of recombination. To gain further insight into the role of RecG, we studied its activity on three-strand intermediates formed by RecA between circular single-stranded and linear duplex DNAs. Once RecA is removed, RecG drives branch migration of these intermediates by a junction-targeted activity that depends on hydrolysis of ATP. RuvAB has a similar activity. However, when RecG is added to a RecA strand exchange reaction it severely reduces the accumulation of joint molecule intermediates by driving branch migration of junctions in the reverse direction to that catalysed by RecA strand exchange. In comparison, RuvAB has little effect on the reaction. We discuss how reverse branch migration by RecG, which acts counter of the 5'-->3' polarity of RecA binding and strand exchange, could serve to promote or abort the early stages of recombination, depending on the orientation of the single DNA strand initiating the exchange relative to the adjacent duplex region.     

Processing of DNA structures via DNA unwinding and branch migration by the S. cerevisiae Mph1 protein

The budding yeast Mph1 protein, the putative ortholog of human FANCM, possesses a 3' to 5' DNA helicase activity and is capable of disrupting the D-loop structure to suppress chromosome arm crossovers in mitotic homologous recombination. Similar to FANCM, genetic studies have implicated Mph1 in DNA replication fork repair. Consistent with this genetic finding, we show here that Mph1 is able to mediate replication fork reversal, and to process the Holliday junction via DNA branch migration. Moreover, Mph1 unwinds 3' and 5' DNA Flap structures that bear key features of the D-loop. These biochemical results not only provide validation for a role of Mph1 in the repair of damaged replication forks, but they also offer mechanistic insights as to its ability to efficiently disrupt the D-loop intermediate.     

Polarity and bypass of DNA heterology during branch migration of Holliday junctions by human RAD54, BLM, and RECQ1 proteins

Several proteins have been shown to catalyze branch migration (BM) of the Holliday junction, a key intermediate in DNA repair and recombination. Here, using joint molecules made by human RAD51 or Escherichia coli RecA, we find that the polarity of the displaced ssDNA strand of the joint molecules defines the polarity of BM of RAD54, BLM, RECQ1, and RuvAB. Our results demonstrate that RAD54, BLM, and RECQ1 promote BM preferentially in the 3'→5' direction, whereas RuvAB drives it in the 5'→3' direction relative to the displaced ssDNA strand. Our data indicate that the helicase activity of BM proteins does not play a role in the heterology bypass. Thus, RAD54 that lacks helicase activity is more efficient in DNA heterology bypass than BLM or REQ1 helicases. Furthermore, we demonstrate that the BLM helicase and BM activities require different protein stoichiometries, indicating that different complexes, monomers and multimers, respectively, are responsible for these two activities. These results define BM as a mechanistically distinct activity of DNA translocating proteins, which may serve an important function in DNA repair and recombination.     

Processing of recombination intermediates by the RecG and RuvAB proteins of Escherichia coli.

The RuvAB, RuvC and RecG proteins of Escherichia coli process intermediates in recombination and DNA repair into mature products. RuvAB and RecG catalyse branch migration of Holliday junctions, while RuvC resolves these structures by nuclease cleavage around the point of strand exchange. The overlap between RuvAB and RecG was investigated using synthetic X- and Y-junctions. RuvAB is a complex of RuvA and RuvB, with RuvA providing the DNA binding subunit and RuvB the ATPase activity that drives branch migration. Both RuvA and RecG form defined complexes with each of the junctions. The gel mobilities of these complexes suggests that the X-junction attracts two tetramers of RuvA, but mainly monomers of RecG. Dissociation of the junction in the presence of ATP requires high levels of RuvAB. RecG is shown to have a much higher specific activity to the extent that very little of this protein would be required to match RuvAB in vivo. Both proteins also dissociate a Y-junction, which is consistent with helicase activity. However, RecG shows no ability to unwind more conventional substrates and the suggestion is made that its helicase activity is directed towards specific DNA structures such as junctions.     

Biological roles of the Escherichia coli RuvA, RuvB and RuvC proteins revealed

In Escherichia coli, the ruvA, ruvB and ruvC gene products are required for genetic recombination and the recombinational repair of DNA damage. New studies suggest that these three proteins function late in recombination and process Holliday junctions made by RecA protein-mediated strand exchange. In vitro, RuvA protein binds a Holliday junction with high affinity and, together with RuvB (an ATPase), promotes ATP-dependent branch migration of the junction leading to the formation of heteroduplex DNA. The third protein, RuvC, which acts independently of RuvA and RuvB, resolves recombination intermediates by specific endonucleolytic cleavage of the Holliday junction.     

Escherichia coli RuvBL268S: a mutant RuvB protein that exhibits wild-type activities in vitro but confers a UV-sensitive ruv phenotype in vivo.

The RuvABC proteins of Escherichia coli process recombination intermediates during genetic recombination and DNA repair. RuvA and RuvB promote branch migration of Holliday junctions, a process that extends heteroduplex DNA. Together with RuvC, they form a RuvABC complex capable of Holliday junction resolution. Branch migration by RuvAB is mediated by RuvB, a hexameric ring protein that acts as an ATP-driven molecular pump. To gain insight into the mechanism of branch migration, random mutations were introduced into the ruvB gene by PCR and a collection of mutant alleles were obtained. Mutation of leucine 268 to serine resulted in a severe UV-sensitive phenotype, characteristic of a ruv defect. Here, we report a biochemical analysis of the mutant protein RuvBL268S. Unexpectedly, the purified protein is fully active in vitro with regard to its ATPase, DNA binding and DNA unwinding activities. It also promotes efficient branch migration in combination with RuvA, and forms functional RuvABC-Holliday junction resolvase complexes. These results indicate that RuvB may perform some additional, and as yet undefined, function that is necessary for cell survival after UV-irradiation.     

The Annealing Helicase and Branch Migration Activities of Drosophila HARP

HARP (SMARCAL1, MARCAL1) is an annealing helicase that functions in the repair and restart of damaged DNA replication forks through its DNA branch migration and replication fork regression activities. HARP is conserved among metazoans. HARP from invertebrates differs by the absence of one of the two HARP-specific domain repeats found in vertebrates. The annealing helicase and branch migration activity of invertebrate HARP has not been documented. We found that HARP from Drosophila melanogaster retains the annealing helicase activity of human HARP, the ability to disrupt D-loops and to branch migrate Holliday junctions, but fails to regress model DNA replication fork structures. A comparison of human and Drosophila HARP on additional substrates revealed that both HARPs are competent in branch migrating a bidirectional replication bubble composed of either DNA:DNA or RNA:DNA hybrid. Human, but not Drosophila, HARP is also capable of regressing a replication fork structure containing a highly stable poly rG:dC hybrid. Persistent RNA:DNA hybrids in vivo can lead to replication fork arrest and genome instability. The ability of HARP to strand transfer hybrids may signify a hybrid removal function for this enzyme, in vivo.     

Novel endonuclease in Archaea cleaving DNA with various branched structure

We identified a novel structure-specific endonuclease in Pyrococcus furiosus. This nuclease contains two distinct domains, which are similar to the DEAH helicase family at the N-terminal two-third and the XPF endonuclease superfamily at the C-terminal one-third of the protein, respectively. The C-terminal domain has an endonuclease activity cleaving the DNA strand at the 5'-side of nicked or flapped positions in the duplex DNA. The nuclease also incises in the proximity of the 5'-side of a branch point in the template strand for leading synthesis in the fork-structured DNA. The N-terminal helicase may work cooperatively to change the fork structure suitable for cleavage by the C-terminal endonuclease. This protein, designated as Hef (helicase-associated endonuclease for fork-structured DNA), may be a prototypical enzyme for resolving stalled forks during DNA replication, as well as working at nucleotide excision repair.