Human Rad54 protein stimulates human Mus81–Eme1 endonuclease

Olga M. Mazina; Alexander V. Mazin

doi:10.1073/pnas.0807016105

New Research In

Edited by Stephen C. Kowalczykowski, University of California, Davis, CA, and approved September 25, 2008 (received for review July 18, 2008)

Abstract

Rad54, a key protein of homologous recombination, physically interacts with a DNA structure-specific endonuclease, Mus81–Eme1. Genetic data indicate that Mus81–Eme1 and Rad54 might function together in the repair of damaged DNA. In vitro, Rad54 promotes branch migration of Holliday junctions, whereas the Mus81–Eme1 complex resolves DNA junctions by endonucleolytic cleavage. Here, we show that human Rad54 stimulates Mus81–Eme1 endonuclease activity on various Holliday junction-like intermediates. This stimulation is the product of specific interactions between the human Rad54 (hRad54) and Mus81 proteins, considering that Saccharomyces cerevisiae Rad54 protein does not stimulate human Mus81–Eme1 endonuclease activity. Stimulation of Mus81–Eme1 cleavage activity depends on formation of specific Rad54 complexes on DNA substrates occurring in the presence of ATP and, to a smaller extent, of other nucleotide cofactors. Thus, our results demonstrate a functional link between the branch migration activity of hRad54 and the structure-specific endonuclease activity of hMus81–Eme1, suggesting that the Rad54 and Mus81–Eme1 proteins may cooperate in the processing of Holliday junction-like intermediates during homologous recombination or DNA repair.

Homologous recombination (HR) is responsible for the repair of DNA double-stranded breaks (DSB) and faithful chromosome segregation during meiosis (1). The Holliday junction (HJ) is a central intermediate of HR (2). HJs are thought to form during meiotic recombination, DSB repair, and repair of collapsed replication forks (3, 4). At late stages of HR or DNA repair, HJs, which constitute a physical link between DNA molecules, must be resolved to allow chromosome separation. Enzymes that cleave HJs, called HJ resolvases, are structure-specific endonucleases that introduce coordinated single-strand cuts across the junction (5). Several HJ resolvases from bacteriophages, eubacteria, archaea, eukaryotic viruses, and mitochondria have been identified, isolated, and characterized (5). However, the identity of eukaryotic nuclear HJ resolvases is still elusive.

A role for Mus81 in HJ resolution was first proposed in studies on Schizosaccharomyces pombe, where Mus81 was identified as a critical factor for the production of viable spores, survival under conditions that lead to stalling of replication fork progression, and viability in the absence of RecQ helicase, Rqh1 (6). All of the defects in S. pombe mus81 mutants were rescued by expression of RusA, a bacteriophage resolvase that is highly specific for HJs (7). Mus81 protein is widely conserved among eukaryotes, including Saccharomyces cerevisiae (8), S. pombe (6), Arabidopsis thaliana (9), mice (10, 11), and humans (12). Mus81 is related to the XPF family of structure-specific endonucleases, which share a highly conserved motif (V/IERKX₃D) that constitutes an integral part of the endonuclease catalytic site. Mus81 functions as a heterodimer with a noncatalytic partner protein known as Eme1 in fission yeast and humans and Mms4 in budding yeast and Drosophila (13).

The role of Mus81–Eme1 as a component of an authentic nuclear-HJ resolvase remains controversial. There is an important distinction between Mus81–Eme1 and prokaryotic HJ resolvases, like RuvC. The dual incisions made by classical RuvC-like HJ resolvases are symmetrical and therefore yield 2 nicked duplex species, which can be directly rejoined by DNA ligase (5). The dual incisions made by Mus81–Eme1 appear to be asymmetric, yielding linear products with short single-strand gaps or flaps that cannot be joined directly by ligation (12). Thus, Mus81–Eme1 presents a subclass of HJ resolvases that differ from classical RuvC-like resolvases. However, the significance of the differences remains to be further elucidated.

An important HR protein that physically interacts with Mus81 is Rad54 (8, 11, 14). Interestingly, Mus81 protein was first identified in S. cerevisiae through a 2-hybrid screen using Rad54 as the bait (8). Moreover, an S. cerevisiae mus81rad54 double mutant was viable and no more UV-sensitive than the rad54 single mutant, indicating that rad54 is epistatic to mus81 for UV sensitivity and that both proteins act in the same pathway for the tolerance/repair of UV-induced damage. Rad54 is a member of the Swi2/Snf2 family that is evolutionary conserved in eukaryotes (15). In vitro, Rad54 has several important activities, reflecting its multiple functions at different stages of HR (16). Rad54 stimulates DNA strand exchange (17–19) and DNA heteroduplex extension promoted by Rad51 (20), and it can translocate along double-stranded DNA (dsDNA) by using the energy of ATP hydrolysis (19, 21–23), displace Rad51 from dsDNA (24), and remodel chromatin (16). We recently showed that Rad54 is a HJ-processing enzyme that promotes branch migration of HJs (25) and D-loop dissociation (26). Here, we examined the effect of human Rad54 (hRad54) on HJ resolution promoted by human Mus81–Eme1 (hMus81–Eme1) endonuclease in vitro.

Our data demonstrate that hRad54 stimulates hMus81–Eme1 endonuclease activity on a broad range of DNA substrates, including HJ. The stimulation of hMus81–Eme1 resolvase activity is apparently specific for hRad54, because S. cerevisiae Rad54 (yRad54) protein does not stimulate hMus81–Eme1 but inhibits it. Thus, our results demonstrate a functional link between branch migration activity of hRad54 and structure-specific endonuclease activity of hMus81–Eme1.

Results

hRad54 Stimulates hMus81 Endonuclease Activity.

Interaction between Rad54 and Mus81 was shown by using a 2-hybrid method and immunoprecipitation (8). Here, we demonstrated physical interactions between purified hRad54 and hMus81–Eme1 by using an affinity pull-down assay (Fig. S1a). For simplicity, we will refer hereafter to the hMus81–Eme1 heterodimer as hMus81. Then, we asked whether hRad54 has an effect on hMus81 endonuclease activity. In these experiments, we used partially mobile nicked HJs (nHJs) (oligos #369/174/176/181/370) and partial X-junctions (PX-junctions) (oligos #369/174/176/198) (Table S1), in which only a central 12-bp homologous core can branch-migrate. Both DNA substrates are known to be efficiently resolved by Mus81 from S. pombe (see ref. 27 for review) and S. cerevisiae (28). First, we examined the kinetics of nHJ resolution mediated by hMus81 in the presence or absence of hRad54 (Fig. 1 A and B). We found that in the presence of hRad54 (120 nM), the initial rate of nHJ cleavage by Mus81 (2.5 nM) was 6-fold greater than that in the absence of hRad54. The stimulation of hMus81 cleavage activity was noticeable through the entire time course, up to 60 min of the reaction (Fig. 1A). Then, we investigated the effect of hRad54 and hMus81 concentrations on the cleavage efficiency of nHJ and PX-junctions. The stimulation of the nHJ and PX-junction cleavage showed dependence on the hRad54 concentration (Fig. 1C and Fig. S2 a and b). The optimal hRad54 concentration required for Mus81 stimulation on nHJ and PX-junctions was 100–150 nM. This hRad54 concentration corresponds to the stoichiometry of 5–8 protein monomers per DNA molecule, consistent with formation of multimeric hRad54 complexes on nHJ and PX-junctions (29). We also investigated the stimulatory effect of hRad54 at different hMus81 concentrations (Fig. 1D). hRad54 stimulated nHJ cleavage at any tested hMus81 concentration, with the greatest stimulation observed at low hMus81 concentration, 1.25–2.5 nM (Fig. 1D). We further found that the presence of the GST-tag on hRad54 had no noticeable effect on stimulation of hMus81; untagged hRad54 stimulated nHJs resolution by hMus81 to the same extend that GST-tagged hRad54 (Fig. S3). Thus, hRad54 efficiently stimulates the DNA cleavage activity of hMus81.

Fig. 1.

hRad54 stimulates hMus81 endonuclease activity. nHJ (20 nM, molecules) were incubated with hRad54 (120 nM, or indicated otherwise) or its dilution buffer. DNA cleavage was initiated by addition of hMus81 (2.5 nM or as indicated otherwise). DNA cleavage was carried out for 20 min or as indicated otherwise. (A) The kinetics of nHJ resolution. The vertical arrow on the nHJ scheme indicates the hMus81 incision site. The arrows at the right of the gel indicate migration of the DNA substrate and the cleavage product. (B) Graphical representation of the data from A. (C) The effect of hRad54 concentration. (D) The effect of hMus81 concentration. The data are the mean of at least 3 measurements, and the error bars represent the SE. *, ³²P-label at the 5′ DNA end.

hRad54 Stimulates hMus81 Cleavage Activity on a Broad Range of DNA Substrates.

We constructed a series of DNA substrates that resemble the structures encountered during HR or stalled replication fork repair, including forked DNA, 3′-flap, replication fork, PX-junction, nHJ, and intact HJ (Table S1). hMus81 showed preference for PX-junctions and nHJ (Fig. 2A, lanes 11 and 14 and Table S1); it cleaves PX-junctions 4-, 6-, and 200-fold more efficiently than 3′-flap, replication fork, and intact HJ, respectively. We found that hRad54 stimulates hMus81 resolution activity on all tested DNA substrates, 2- to 4-fold, except forked DNA, for which we did not observe significant cleavage activity of hMus81, regardless of the hRad54 presence (Fig. 2A, lanes 2 and 3 and Table S1). The observed substrate specificity of hMus81 cleavage is similar to that of Mus81 from yeast (see ref. 27 for review) with the only exception of forked DNA, which is efficiently resolved by yeast Mus81 (28) but not hMus81 (30).

Fig. 2.

hRad54 stimulates hMus81 endonuclease activity on various DNA substrates. (A) hRad54 (120 nM) was incubated with DNA substrates (20 nM, molecules), hMus81 (5 nM, or 10 nM in the case of HJ) was added to initiate DNA cleavage. ³²P-label was in oligonucleotide 369, common for all DNA substrates. Note that cleavage of forked DNA would produce 5′-tailed DNA. (B) Experimental scheme to test hRad54 stimulation of hMus81 on mobile PX-junctions. The PX-junction was designed in such way that branch migration can proceed only in 1 direction of movement, shown by the block arrow. The zig-zag line denotes the poly(dT)₃₀ region. Single base pair heterologies are denoted by AT and GC. (C) PX-junction (20 nM, molecules) cleavage by hMus81 (5 nM) alone (lanes 1–4), in the presence of hRad54 (120 nM) (lanes 5–7), or branch migration by hRad54 (120 nM) (lanes 8–10). In lanes 5–7, hRad54 was added first, followed by immediate addition of hMus81. In lane 11 (denoted 5^spon), hRad54 was replaced with dilution buffer. The arrows at the sides of the gels indicate migration of DNA substrates and products of PX cleavage or branch migration. *, ³²P-label at the 5′ DNA end.

We also tested the effect of hRad54 on hMus81 cleavage activity on fully mobile PX-junctions (oligos #71/169/170/171), which hRad54 can branch-migrate up to the complete separation of DNA molecules (Fig. 2 B and C). The results show that hRad54 stimulates hMus81 cleavage activity on mobile PX-junctions (Fig. 2C, compare lanes 2–4 with lanes 5–7). Thus, hRad54 stimulates hMus81 cleavage activity on a broad range of DNA substrates, which include partially and fully mobile PX-junctions.

hRad54 Protein Stimulates Endonuclease Activity of hMus81 Via Protein-Specific Interaction with hMus81 Protein.

Previously, it was demonstrated that yeast, mouse, and rice Rad54 proteins specifically interact with their cognate Mus81 protein in vivo (8, 11, 14). To determine whether the stimulation of hMus81 observed in our current studies is specific for hRad54, we examined the effect of yRad54 protein on hMus81 cleavage activity. We found that yRad54 does not stimulate hMus81 cleavage activity; in fact, the yield of the cleavage products was inhibited by yRad54 ≈3-fold (Fig. 3, lanes 2 and 4). Importantly, under the tested conditions, yRad54 protein was fully active and efficiently promoted branch migration (data not shown). We also tested whether human RecQ1 helicase, a branch migration protein of the RecQ family (31), can stimulate the cleavage activity of Mus81. However, we found that RecQ1 inhibits Mus81 in a concentration-dependent manner (Fig. S4). Thus, specific protein–protein interactions between hRad54 and hMus81 proteins are required for the stimulation of hMus81 cleavage activity. The cleavage depended entirely on hMus81 endonuclease activity because the endonuclease-deficient hMus81AA mutant did not cleave PX-junctions, regardless of the hRad54 presence (Fig. 3, lanes 5 and 6).

Fig. 3.

Yeast Rad54 inhibits hMus81 resolution activity. hRad54 (120 nM, lanes 3 and 6) or yRad54 (120 nM, lane 4) proteins were incubated with PX-junctions (20 nM, molecules), then hMus81 (5 nM, lanes 2–4) or the hMus81AA mutant (5 nM, lanes 5 and 6) were added to the reaction mixture. In lanes 2 and 5, hRad54 protein was replaced with dilution buffer. *, ³²P-label at the 5′ DNA end.

hRad54 Recruits hMus81 to the Site of DNA Junction Resolution.

To gain more insight into the mechanism of hMus81 stimulation by hRad54, we examined the effect of the order of addition of these proteins to PX-junctions. We used 3 sets of the reactions, in which (i) hRad54 was first mixed with hMus81 and then both enzymes were added to PX-junctions, (ii) hRad54 was preincubated with PX-junctions followed by addition of hMus81, or (iii) hRad54 protein was added to the on-going hMus81 cleavage reaction. The results show that hRad54 stimulates hMus81 resolution activity when hRad54 is added to PX-junctions before hMus81 (Fig. 4A, lanes 4 and 5) or in a complex with hMus81 (Fig. 4A, lane 3). Preincubation time of hRad54 with PX-junctions did not have a significant effect on the stimulation of hMus81 (Fig. 4B, bars 3–5). In contrast, when hRad54 was added to the on-going hMus81 reaction, only weak, if any, stimulation was observed (Fig. 4B, bars 6 and 7).

Fig. 4.

Effect of the order of protein addition on hMus81 stimulation by hRad54. (A) In lane 3, hRad54 (120 nM) and hMus81 (5 nM) were mixed and then added to PX-junctions (20 nM, molecules) followed by a 20-min incubation. In lanes 4 and 5, hRad54 was incubated with PX-junctions for 5 and 10 min, respectively, then hMus81 was added and the cleavage was carried out for 20 min. In lanes 6 and 7, hRad54 was added to an on-going DNA cleavage by hMus81 at 5 and 10 min after initiation, and the reactions were continued for 15 and 10 min, respectively. In lane 2, hRad54 was replaced with dilution buffer, and hMus81 was incubated with PX-junction for 20 min. (B) Graphical representation of the data from A. The data are the mean of at least 3 measurements, and the error bars represent the SE. *, ³²P-label at the 5′ DNA end.

We also tested the ability of hRad54 to stimulate hMus81 cleavage activity in trans. We preincubated hRad54 (120 nM) with a 3-fold excess of supercoiled pUC19 dsDNA (10.8 μM, bp) and then mixed the resultant hRad54-DNA complex with hMus81 followed by addition of PX-junctions (Fig. S5 a and b) or nicked HJs (Fig. S5c). The result shows that the preincubation of hRad54 with pUC19 dsDNA in the presence of ATP or ATPγS, reduces the stimulatory effect of hRad54 on hMus81 resolution activity. In addition, even the residual stimulation could not be attributed to the in trans mechanism, because of possible transfer of hRad54 protein from pUC19 dsDNA onto DNA-junctions, which might cause the observed stimulation by the in cis mechanism. Thus, our results indicate that hRad54 stimulates hMus81 cleavage activity in cis by forming a complex with the DNA-junction, which then interacts with hMus81.

Effect of Nucleotide Cofactors on Stimulation of hMus81 by hRad54.

ATP hydrolysis by hRad54 is essential for branch migration activity and for stimulation of DNA pairing promoted by hRad51 (25, 26). Here, we tested the effect of hRad54 ATPase activity on stimulation of hMus81 resolution activity (Fig. 5A). With nHJ substrate, the greatest cleavage efficiency was observed in the presence of ATP; smaller stimulation was also observed in the presence of ATPγS and ADP. In contrast, in the absence of any nucleotide cofactor, hRad54 inhibited hMus81 cleavage ≈2-fold (Fig. 5A, bar 4). The observed inhibition likely resulted from hRad54 binding to DNA junctions and from shielding the incision site because preincubation of hRad54 with pUC19 dsDNA before addition of DNA junctions, alleviated inhibition of the hMus81 cleavage activity (Fig. S5a, lanes 8 and 9 and Fig. S5 b and c). Then, we tested whether “inhibitory” hRad54 complexes formed on nicked HJ in the absence of any nucleotide cofactor could be converted into complexes that stimulate hMus81 cleavage activity. We found that addition of ATP, ATPγS, or ADP to these hRad54–DNA complexes restored the ability of hRad54 to stimulate hMus81 cleavage (Fig. 5 B and C); ATP was the most efficient in converting of inhibitory hRad54-DNA complexes into stimulatory ones (Fig. 5B, lane 5 and Fig 5C, bar 5).

Fig. 5.

Effect of nucleotide cofactors on the stimulation of hMus81 by hRad54. (A) hRad54 (120 nM) was incubated with nHJ (20 nM, molecules) for 5 min in the presence of ATP (bars 1 and 2), ATPγS (bars 5 and 6), or ADP (bars 7 and 8) or in the absence of a nucleotide cofactor (bars 3 and 4). DNA cleavage was initiated by addition of hMus81 (5 nM) and carried out for 20 min. The ATP regeneration system was included only in the reactions containing ATP. Magnesium acetate concentrations in the presence of ATP, absence of ATP, and presence of ATPγS or ADP were 7.5, 3, and 5 mM, respectively, which were optimal for each reaction. (B) hRad54 protein (120 nM) was incubated with nHJ (20 nM, molecules) for 2 min in the absence of a nucleotide cofactor. Then, the reaction mixture was divided between 5 test tubes. In the first, DNA cleavage was initiated by addition of hMus81 (5 nM, lane 3). In the second, incubation was continued for another 5 min, and then hMus81 was added (lane 4). In the third, fourth, and fifth, ATP, ATPγS, and ADP, respectively, were added and incubation was continued for 5 min, followed by addition of hMus81 (lanes 5–7). After addition of hMus81, all reactions were carried out for 20 min. hMus81 cleavage activity, in the absence of hRad54 and nucleotide cofactors, is shown in lane 2. (C) Graphical representation of the data from A. The data are the mean of 3 measurements, and the error bars represent the SE. *, ³²P-label at the 5′ DNA end.

We concluded that binding of nucleotide cofactors rather than ATP hydrolysis by hRad54 is required for stimulation of hMus81 cleavage activity. Because the hRad54 stoichiometric titration (Fig. 1C) indicates that it stimulates hMus81 cleavage activity likely by forming multimeric complexes on DNA junctions, we suggest that nucleotide cofactors promote formation of a specific type of hRad54–DNA complexes that are proficient in stimulation of hMus81.

hRad54 Does Not Change the Cleavage Specificity of hMus81.

hRad54 binding to DNA junctions may change the conformation of DNA and thereby modify the cleavage specificity of hMus81. Also, protein–protein interactions between hRad54 and hMus81 may affect the cleavage specificity of hMus81. Here, we examined the effect of hRad54 on the hMus81 cleavage pattern.

First, we used mobile PX-junctions (oligos #71/169/170/171) (Fig. 2B) to determine the effect of branch migration on the hMus81 cleavage specificity (Fig. 6). We found that the stimulation of hMus81 endonuclease activity by hRad54 was not accompanied by a change in the cleavage pattern (Fig. 6A, lanes 3 and 4). Three main cleavage sites (R, S, and T) were mapped 2–4 nt 5′ to the junction on strand 71 (Fig. 6B). A plausible explanation of why, during on-going branch migration, the pattern of the cleavage products was not changed (Fig. 6A, lanes 3 and 4) and also (Fig. 2C, lanes 5–7) is the following: When the junction point of the original PX-junction migrates away from the nick, the PX-junction transforms into intact HJ, which resolves by hMus81 with an ≈200-fold lower efficiency than the PX-junction (Fig. 2A, lanes 11, 12, 17, and 18 and Table S1). Thus, sensitivity of the assay allows us to detect only the products of the cleavage of the original PX-junction.

Fig. 6.

Effect of hRad54 on hMus81 cleavage specificity on mobile PX-junctions. The conditions of DNA cleavage by hMus81 were identical to those described in the legend of Fig. 2C. (A) The reactions were carried out either in the absence or presence hRad54 (120 nM) (lanes 3 and 4, respectively) for 5 min and the DNA products were analyzed in a 12% denaturing polyacrylamide gel (19:1). Migration markers were produced by cleavage of oligonucleotides 71 and 169 at A+G positions (lanes 1 and 5, respectively). (B) The cleavage sites (denoted by “q” through “z”) are indicated by arrows. Main cleavage sites are designated by capital letters (R, S, and T). *, ³²P-label at the 5′ DNA end.

Then, we used static PX-junctions (oligos #71/234/271/374) to inspect the effect of hRad54 on hMus81 cleavage specificity. Whereas hRad54 efficiently stimulated hMus81 cleavage activity on static PX-junctions (Fig. S6a), we found no change in the cleavage pattern of hMus81 on static PX-junctions (Fig. S6 b and c). In summary, our results demonstrate that the hRad54-mediated stimulation does not change the cleavage specificity of hMus81.

Discussion

In eukaryotes, HR is generally initiated at DSB (1). First, DSB become processed by exonucleases to generate DNA ends with ssDNA tails. Then, Rad51 protein binds to these tails and performs a search for homologous sequences and subsequent invasion of broken DNA ends into the homologous duplex DNA that then serves as a template for the repair. The invasion produces the HJ. As we recently found, Rad54 catalyzes an ATP-dependent branch migration of HJs (25, 26, 29). It has been previously shown that Rad54 protein interacts physically with Mus81, a structure-specific nuclease, and that this interaction is evolutionarily conserved in eukaryotes (8, 11, 14). Here, we demonstrate that hRad54 also stimulates DNA cleavage activity of hMus81, which may be responsible for resolution of HJ or HJ-like structures. Stimulation of DNA cleavage activity of hMus81 by hRad54, observed in the present paper, may have important biological implications because it shows that branch migration of HJ by hRad54 and HJ resolution by hMus81–Eme1 structure-specific endonuclease are coupled. Remarkably, Zhang et al. (32) recently showed that Bloom's syndrome protein (BLM), another human protein that can promote branch migration of HJ, stimulates hMus81 endonuclease activity as well. These observations position Mus81 at the late stage of recombinational DNA repair, when branch migration of HJ is occurring. Nevertheless, stimulation of Mus81 cleavage activity is specific for hRad54 and BLM, not a generic property of all branch migration proteins, considering that yRad54 and RecQ1 do not stimulate Mus81.

A high affinity of hRad54 protein for branched DNA (25) and dependence of the hMus81 stimulation on species-specific interactions with cognate hRad54, are consistent with a model in which hRad54 targets hMus81 to DNA junctions via protein–protein interactions. This model is also supported by the observation that the stimulation of hMus81 activity occurs only if hRad54 is added to PX-junctions first or, at least, concurrently with hMus81, indicating that stimulation occurs in cis and that formation of hRad54 complexes on DNA junctions is important for hMus81 stimulation.

Our finding that stimulation of hMus81 cleavage activity by hRad54 depends on nucleotide cofactors, especially ATP, is of particular interest. There is an intriguing parallel with Escherichia coli RuvC resolvase. Zerbib et al. (33) showed that stimulation of RuvC by the RuvAB branch migration complex shows dependence on ATP. Because ATP hydrolysis is essential for branch migration activity of RuvAB, they suggested that the process of branch migration is important for RuvC stimulation. In contrast, for hRad54, the process of branch migration appeared to be nonessential for stimulation of hMus81 resolution activity. First, hRad54 stimulates hMus81 on static PX-junctions that are incapable of branch migrating. Second, ATPγS and ADP, which do not support branch migration activity of hRad54, stimulate hMus81 cleavage, albeit to a smaller degree than ATP. Finally, hRad51 that stimulates branch migration activity of hRad54 (34) does not increase the stimulation of the hMus81 cleavage activity by hRad54, in fact Rad51 diminishes this stimulation (Fig. S7).

As we previously showed, hRad54 assembles on DNA a large multimeric complex that promotes DNA branch migration in the presence of ATP (29). Our current stoichiometric titration data indicate that formation of a multimeric hRad54 complex is also required for stimulation of hMus81. It was shown that Rad54 binding to DNA and multimerization occurs in the absence of nucleotide cofactors (35). However, hRad54 forms multimers of different size in the presence and in the absence of ATP (22). We suggest that in the presence of nucleotide cofactors, hRad54 may form nucleoprotein complexes that either efficiently stimulate loading of hMus81 on DNA junctions or facilitate hRad54 dissociation from DNA junctions on hMus81 binding, thereby providing hMus81 with an access for DNA junction. Whereas specific protein–protein interactions are critically important for hMus81 stimulation by hRad54, formation of hRad54 nucleoprotein complexes may also induce conformational changes in DNA junctions, facilitating hMus81 cleavage. HJs are known to form 2 distinct conformations, the open and the stacked, which exist in equilibrium. We previously showed that hRad54 binds preferentially to HJs in the open conformation during branch migration and ATP hydrolysis (29). It is possible that, in the presence of ATP or other nucleotide cofactors, hRad54 may stabilize DNA junctions in the open conformation. This conformation may be preferable for Mus81 because it was reported that several studied junction-specific nucleases show preference for the DNA junctions in the open conformation (36). The proposed mechanism has a precedent: RuvA protein stabilizes HJs in a square-plane (open) conformation, facilitating RuvAB branch migration and, presumably, RuvC cleavage (37).

Stimulation of hMus81 activity by hRad54 in vitro is consistent with genetic data because a functional link between Rad54 and Mus81 is documented. Double-mutant analysis in S. cerevisiae showed that mus81rad54 has a UV-sensitive phenotype that is not more severe than either of the single mutants, indicating that Rad54 and Mus81 act in the same DNA repair pathway, at least in response to UV damage (8). Similarly, Mus81^−/−Rad54^−/− mouse embryonic stem (ES) cells were as hypersensitive to interstrand DNA cross-linking (ICL) agents as Mus81^−/− cells, indicating that Mus81 and Rad54 are involved in the same DNA repair pathway of ICL (11). ICL lesions are total barriers to DNA polymerases. Their repair is thought to depend on the formation of a DSB that is subsequently repaired by HR. However, Mus81 and Rad54 are not obligatory functional partners: Whereas Mus81^−/− ES cells are hypersensitive to hydroxyurea, Rad54^−/− ES cells are not (38). Also, in S. pombe, Mus81 and Rad54 likely act in parallel but distinct pathways of DNA repair because mus81rad54 double mutant has severely reduced viability (39). However, poor growth and hypersensitivity of S. pombe mus81rad54 double mutants to genotoxic agents is suppressed by loss of upstream proteins, such as Rad51 and Rad55 (40), indicating a link between the function of these 2 proteins and HR. Thus, genetic data in mammalian cells and S. cerevisiae, together with our results on hRad54 stimulation of hMus81 endonuclease activity, suggest that Rad54 and Mus81 may cooperate in processing of HJ intermediates that are formed during the repair of DSB or stalled replication forks.

Materials and Methods

Proteins and DNA.

Human and yeast Rad54 were purified as described previously in ref. 19. Untagged hRad54 was produced by proteolytic cleavage of GST-hRad54 with thrombin, as previously described in ref. 15. Recombinant baculoviruses encoding wild-type hMus81 and hMus81AA mutant with 3 haemagglutinin epitopes at the N terminus and hEme1 with a Flag epitope at the C terminus, were expressed in Sf21 insect cells (12). The hMus81–Eme1 heterodimer and hMus81AA–Eme1 mutant were purified to near homogeneity on the Anti-FLAG agarose as described in SI Text (Fig. S1b). DNA substrates (Table S2) were prepared as described in ref. 41 and stored at −20 °C. Oligonucleotides were labeled with [γ-³²P]ATP and T4 polynucleotide kinase, as described previously in ref. 42.

Endonuclease Assays.

The hRad54 protein (120 nM) was preincubated with ³²P-labeled synthetic nHJ or PX-junction (20 nM, molecules) in a branch migration buffer containing 25 mM Tris-acetate (pH 7.5), 2 mM ATP, 2 mM DTT, 7.5 mM magnesium acetate (or indicated otherwise), 100 μg/mL BSA, the ATP regenerating system (10 units/mL creatine phosphokinase and 10 mM creatine phosphate) for 5 min at 30 °C. Then, Mus81–Eme1 complex (5 nM or indicated otherwise) was added to initiate DNA cleavage and the incubation was continued for another 20 min (or indicated otherwise). The reaction products were analyzed by electrophoresis in either native 8% polyacrylamide gels (29:1) or denaturing 12% polyacrylamide gels (19:1) containing 8 M urea, as described in SI Text.

Endonuclease and Branch Migration Assays.

For the experiment in Fig. 2C, hMus81–Eme1 complex (5 nM) alone, hRad54 protein (120 nM) alone, or both hMus81–Eme1 complex and hRad54 proteins were mixed with ³²P-labeled synthetic PX-junctions (oligos #71/169/170/171) (20 nM, molecules) in branch migration buffer described above. The reaction mixtures were incubated at 30 °C. Aliquots (10 μl) were withdrawn from the reaction mixtures at the indicated points of time and analyzed by electrophoresis in native 10% polyacrylamide gel (17:1) as described in SI Text.

Acknowledgments

We thank S. Kowalczykowski (University of California, Davis, CA) for yeast Rad54 protein, N. Thomä (Friedrich Miescher Institute, Basel, Switzerland) for untagged hRad54, and C. McGowan (Scripps, CA) for recombinant viruses expressing hMus81, hMus81AA mutant, and hEme1; M. Rossi and D. Bugreev for the comments and discussion. This work was supported by National Institutes of Health Grants CA100839 and MH084119 and Leukemia and Lymphoma Society Scholar Award 1054-09 (to A.V.M.).

Footnotes

¹To whom correspondence should be addressed at:
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, 245 North 15th Street, New College Building, Room 10103, Philadelphia, PA 19102-1192.
E-mail: amazin{at}drexelmed.edu
Author contributions: O.M.M. and A.V.M. designed research; O.M.M. performed research; O.M.M. and A.V.M. analyzed data; and O.M.M. and A.V.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0807016105/DCSupplemental.
© 2008 by The National Academy of Sciences of the USA

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(2000) Damage tolerance protein Mus81 associates with the FHA1 domain of checkpoint kinase Cds1. Mol Cell Biol 20:8758–8766.
OpenUrl Abstract/FREE Full Text
↵
1. Bolt EL,
2. Lloyd RG
(2002) Substrate specificity of RusA resolvase reveals the DNA structures targeted by RuvAB and RecG in vivo. Mol Cell 10:187–198.
OpenUrl CrossRef PubMed
↵
1. Interthal H,
2. Heyer WD
(2000) MUS81 encodes a novel helix-hairpin-helix protein involved in the response to UV- and methylation-induced DNA damage in Saccharomyces cerevisiae. Mol Gen Genet 263:812–827.
OpenUrl CrossRef PubMed
↵
1. Hartung F,
2. Suer S,
3. Bergmann T,
4. Puchta H
(2006) The role of AtMUS81 in DNA repair and its genetic interaction with the helicase AtRecQ4A. Nucleic Acids Res 34:4438–4448.
OpenUrl Abstract/FREE Full Text
↵
1. McPherson JP,
2. et al.
(2004) Involvement of mammalian Mus81 in genome integrity and tumor suppression. Science 304:1822–1826.
OpenUrl Abstract/FREE Full Text
↵
1. Hanada K,
2. et al.
(2006) The structure-specific endonuclease Mus81-Eme1 promotes conversion of interstrand DNA crosslinks into double-strands breaks. EMBO J 25:4921–4932.
OpenUrl CrossRef PubMed
↵
1. Chen XB,
2. et al.
(2001) Human Mus81-associated endonuclease cleaves Holliday junctions in vitro. Mol Cell 8:1117–1127.
OpenUrl CrossRef PubMed
↵
1. Ciccia A,
2. McDonald N,
3. West SC
(2008) Structural and functional relationships of the XPF/MUS81 family of proteins. Annu Rev Biochem 77:259–287.
OpenUrl CrossRef PubMed
↵
1. Mimida N,
2. et al.
(2007) Two alternatively spliced transcripts generated from OsMUS81, a rice homolog of yeast MUS81, are up-regulated by DNA-damaging treatments. Plant Cell Physiol 48:648–654.
OpenUrl Abstract/FREE Full Text
↵
1. Thoma NH,
2. et al.
(2005) Structure of the SWI2/SNF2 chromatin-remodeling domain of eukaryotic Rad54. Nat Struct Mol Biol 12:350–356.
OpenUrl CrossRef PubMed
↵
1. Heyer WD,
2. Li X,
3. Rolfsmeier M,
4. Zhang XP
(2006) Rad54: The Swiss Army knife of homologous recombination? Nucleic Acids Res 34:4115–4125.
OpenUrl Abstract/FREE Full Text
↵
1. Petukhova G,
2. Stratton S,
3. Sung P
(1998) Catalysis of homologous DNA pairing by yeast Rad51 and Rad54 proteins. Nature 393:91–94.
OpenUrl CrossRef PubMed
↵
1. Sigurdsson S,
2. Van Komen S,
3. Petukhova G,
4. Sung P
(2002) Homologous DNA pairing by human recombination factors Rad51 and Rad54. J Biol Chem 277:42790–42794.
OpenUrl Abstract/FREE Full Text
↵
1. Mazina OM,
2. Mazin AV
(2004) Human Rad54 protein stimulates DNA strand exchange activity of hRad51 protein in the presence of Ca2+. J Biol Chem 279:52042–52051.
OpenUrl Abstract/FREE Full Text
↵
1. Solinger JA,
2. Lutz G,
3. Sugiyama T,
4. Kowalczykowski SC,
5. Heyer WD
(2001) Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA strand exchange via specific interactions with the presynaptic Rad51 nucleoprotein filament. J Mol Biol 307:1207–1221.
OpenUrl CrossRef PubMed
↵
1. Van Komen S,
2. Petukhova G,
3. Sigurdsson S,
4. Stratton S,
5. Sung P
(2000) Superhelicity-driven homologous DNA pairing by yeast recombination factors Rad51 and Rad54. Mol Cell 6:563–572.
OpenUrl CrossRef PubMed
↵
1. Ristic D,
2. Wyman C,
3. Paulusma C,
4. Kanaar R
(2001) The architecture of the human Rad54-DNA complex provides evidence for protein translocation along DNA. Proc Natl Acad Sci USA 98:8454–8460.
OpenUrl Abstract/FREE Full Text
↵
1. Amitani I,
2. Baskin RJ,
3. Kowalczykowski SC
(2006) Visualization of Rad54, a chromatin remodeling protein, translocating on single DNA molecules. Mol Cell 23:143–148.
OpenUrl CrossRef PubMed
↵
1. Solinger JA,
2. Kiianitsa K,
3. Heyer WD
(2002) Rad54, a Swi2/Snf2-like recombinational repair protein, disassembles Rad51:dsDNA filaments. Mol Cell 10:1175–1188.
OpenUrl CrossRef PubMed
↵
1. Bugreev DV,
2. Mazina OM,
3. Mazin AV
(2006) Rad54 protein promotes branch migration of Holliday junctions. Nature 442:590–593.
OpenUrl CrossRef PubMed
↵
1. Bugreev DV,
2. Hanaoka F,
3. Mazin AV
(2007) Rad54 dissociates homologous recombination intermediates by branch migration. Nat Struct Mol Biol 14:746–753.
OpenUrl CrossRef PubMed
↵
1. Whitby MC
(2004) Junctions on the road to cancer. Nat Struct Mol Biol 11:693–695.
OpenUrl CrossRef PubMed
↵
1. Ehmsen KT,
2. Heyer WD
(2008) Saccharomyces cerevisiae Mus81-Mms4 is a catalytic, DNA structure-selective endonuclease. Nucleic Acids Res 36:2182–2195.
OpenUrl Abstract/FREE Full Text
↵
1. Mazina OM,
2. Rossi MJ,
3. Thomaa NH,
4. Mazin AV
(2007) Interactions of human rad54 protein with branched DNA molecules. J Biol Chem 282:21068–21080.
OpenUrl Abstract/FREE Full Text
↵
1. Ciccia A,
2. Constantinou A,
3. West SC
(2003) Identification and characterization of the human mus81-eme1 endonuclease. J Biol Chem 278:25172–25178.
OpenUrl Abstract/FREE Full Text
↵
1. Bugreev DV,
2. Brosh RM, Jr,
3. Mazin AV
(2008) RECQ1 possesses DNA branch migration activity. J Biol Chem 283:20231–20242.
OpenUrl Abstract/FREE Full Text
↵
1. Zhang R,
2. et al.
(2005) BLM helicase facilitates Mus81 endonuclease activity in human cells. Cancer Res 65:2526–2531.
OpenUrl Abstract/FREE Full Text
↵
1. Zerbib D,
2. Mezard C,
3. George H,
4. West SC
(1998) Coordinated actions of RuvABC in Holliday junction processing. J Mol Biol 281:621–630.
OpenUrl CrossRef PubMed
↵
1. Rossi MJ,
2. Mazin AV
(2008) Rad51 protein stimulates the branch migration activity of rad54 protein. J Biol Chem 283:24698–24706.
OpenUrl Abstract/FREE Full Text
↵
1. Petukhova G,
2. Van Komen S,
3. Vergano S,
4. Klein H,
5. Sung P
(1999) Yeast Rad54 promotes Rad51-dependent homologous DNA pairing via ATP hydrolysis-driven change in DNA double helix conformation. J Biol Chem 274:29453–29462.
OpenUrl Abstract/FREE Full Text
↵
1. Biertumpfel C,
2. Yang W,
3. Suck D
(2007) Crystal structure of T4 endonuclease VII resolving a Holliday junction. Nature 449:616–620.
OpenUrl CrossRef PubMed
↵
1. Ariyoshi M,
2. Nishino T,
3. Iwasaki H,
4. Shinagawa H,
5. Morikawa K
(2000) Crystal structure of the holliday junction DNA in complex with a single RuvA tetramer. Proc Natl Acad Sci USA 97:8257–8262.
OpenUrl Abstract/FREE Full Text
↵
1. Hanada K,
2. et al.
(2007) The structure-specific endonuclease Mus81 contributes to replication restart by generating double-strand DNA breaks. Nat Struct Mol Biol 14:1096–1104.
OpenUrl CrossRef PubMed
↵
1. Doe CL,
2. Whitby MC
(2004) The involvement of Srs2 in post-replication repair and homologous recombination in fission yeast. Nucleic Acids Res 32:1480–1491.
OpenUrl Abstract/FREE Full Text
↵
1. Osman F,
2. Whitby MC
(2007) Exploring the roles of Mus81-Eme1/Mms4 at perturbed replication forks. DNA Repair 6:1004–1017.
OpenUrl CrossRef PubMed
↵
1. Bugreev DV,
2. Mazina OM,
3. Mazin AV
(2006) Analysis of branch migration activities of proteins using synthetic DNA substrates. Nat Protoc, 2010.1038/nprot.2006.217.
↵
1. Mazin AV,
2. Alexeev AA,
3. Kowalczykowski SC
(2003) A novel function of Rad54 protein. Stabilization of the Rad51 nucleoprotein filament. J Biol Chem 278:14029–14036.
OpenUrl Abstract/FREE Full Text

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Proceedings of the National Academy of Sciences: 105 (47)

Table of Contents

Submit

[1] ↵
Sung P,
Klein H
(2006) Mechanism of homologous recombination: Mediators and helicases take on regulatory functions. Nat Rev Mol Cell Biol 7:739–750.
OpenUrl CrossRef PubMed

[2] Sung P,

[3] Klein H

[4] ↵
Liu Y,
West SC
(2004) Happy Hollidays: 40th anniversary of the Holliday junction. Nat Rev Mol Cell Biol 5:937–944.
OpenUrl CrossRef PubMed

[5] Liu Y,

[6] West SC

[7] ↵
Cox MM,
et al.
(2000) The importance of repairing stalled replication forks. Nature 404:37–41.
OpenUrl CrossRef PubMed

[8] Cox MM,

[9] et al.

[10] ↵
Seigneur M,
Bidnenko V,
Ehrlich SD,
Michel B
(1998) RuvAB acts at arrested replication forks. Cell 95:419–430.
OpenUrl CrossRef PubMed

[11] Seigneur M,

[12] Bidnenko V,

[13] Ehrlich SD,

[14] Michel B

[15] ↵
Lilley DM,
White MF
(2001) The junction-resolving enzymes. Nat Rev Mol Cell Biol 2:433–443.
OpenUrl CrossRef PubMed

[16] Lilley DM,

[17] White MF

[18] ↵
Boddy MN,
et al.
(2000) Damage tolerance protein Mus81 associates with the FHA1 domain of checkpoint kinase Cds1. Mol Cell Biol 20:8758–8766.
OpenUrl Abstract/FREE Full Text

[19] Boddy MN,

[20] et al.

[21] ↵
Bolt EL,
Lloyd RG
(2002) Substrate specificity of RusA resolvase reveals the DNA structures targeted by RuvAB and RecG in vivo. Mol Cell 10:187–198.
OpenUrl CrossRef PubMed

[22] Bolt EL,

[23] Lloyd RG

[24] ↵
Interthal H,
Heyer WD
(2000) MUS81 encodes a novel helix-hairpin-helix protein involved in the response to UV- and methylation-induced DNA damage in Saccharomyces cerevisiae. Mol Gen Genet 263:812–827.
OpenUrl CrossRef PubMed

[25] Interthal H,

[26] Heyer WD

[27] ↵
Hartung F,
Suer S,
Bergmann T,
Puchta H
(2006) The role of AtMUS81 in DNA repair and its genetic interaction with the helicase AtRecQ4A. Nucleic Acids Res 34:4438–4448.
OpenUrl Abstract/FREE Full Text

[28] Hartung F,

[29] Suer S,

[30] Bergmann T,

[31] Puchta H

[32] ↵
McPherson JP,
et al.
(2004) Involvement of mammalian Mus81 in genome integrity and tumor suppression. Science 304:1822–1826.
OpenUrl Abstract/FREE Full Text

[33] McPherson JP,

[34] et al.

[35] ↵
Hanada K,
et al.
(2006) The structure-specific endonuclease Mus81-Eme1 promotes conversion of interstrand DNA crosslinks into double-strands breaks. EMBO J 25:4921–4932.
OpenUrl CrossRef PubMed

[36] Hanada K,

[37] et al.

[38] ↵
Chen XB,
et al.
(2001) Human Mus81-associated endonuclease cleaves Holliday junctions in vitro. Mol Cell 8:1117–1127.
OpenUrl CrossRef PubMed

[39] Chen XB,

[40] et al.

[41] ↵
Ciccia A,
McDonald N,
West SC
(2008) Structural and functional relationships of the XPF/MUS81 family of proteins. Annu Rev Biochem 77:259–287.
OpenUrl CrossRef PubMed

[42] Ciccia A,

[43] McDonald N,

[44] West SC

[45] ↵
Mimida N,
et al.
(2007) Two alternatively spliced transcripts generated from OsMUS81, a rice homolog of yeast MUS81, are up-regulated by DNA-damaging treatments. Plant Cell Physiol 48:648–654.
OpenUrl Abstract/FREE Full Text

[46] Mimida N,

[47] et al.

[48] ↵
Thoma NH,
et al.
(2005) Structure of the SWI2/SNF2 chromatin-remodeling domain of eukaryotic Rad54. Nat Struct Mol Biol 12:350–356.
OpenUrl CrossRef PubMed

[49] Thoma NH,

[50] et al.

[51] ↵
Heyer WD,
Li X,
Rolfsmeier M,
Zhang XP
(2006) Rad54: The Swiss Army knife of homologous recombination? Nucleic Acids Res 34:4115–4125.
OpenUrl Abstract/FREE Full Text

[52] Heyer WD,

[53] Li X,

[54] Rolfsmeier M,

[55] Zhang XP

[56] ↵
Petukhova G,
Stratton S,
Sung P
(1998) Catalysis of homologous DNA pairing by yeast Rad51 and Rad54 proteins. Nature 393:91–94.
OpenUrl CrossRef PubMed

[57] Petukhova G,

[58] Stratton S,

[59] Sung P

[60] ↵
Sigurdsson S,
Van Komen S,
Petukhova G,
Sung P
(2002) Homologous DNA pairing by human recombination factors Rad51 and Rad54. J Biol Chem 277:42790–42794.
OpenUrl Abstract/FREE Full Text

[61] Sigurdsson S,

[62] Van Komen S,

[63] Petukhova G,

[64] Sung P

[65] ↵
Mazina OM,
Mazin AV
(2004) Human Rad54 protein stimulates DNA strand exchange activity of hRad51 protein in the presence of Ca2+. J Biol Chem 279:52042–52051.
OpenUrl Abstract/FREE Full Text

[66] Mazina OM,

[67] Mazin AV

[68] ↵
Solinger JA,
Lutz G,
Sugiyama T,
Kowalczykowski SC,
Heyer WD
(2001) Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA strand exchange via specific interactions with the presynaptic Rad51 nucleoprotein filament. J Mol Biol 307:1207–1221.
OpenUrl CrossRef PubMed

[69] Solinger JA,

[70] Lutz G,

[71] Sugiyama T,

[72] Kowalczykowski SC,

[73] Heyer WD

[74] ↵
Van Komen S,
Petukhova G,
Sigurdsson S,
Stratton S,
Sung P
(2000) Superhelicity-driven homologous DNA pairing by yeast recombination factors Rad51 and Rad54. Mol Cell 6:563–572.
OpenUrl CrossRef PubMed

[75] Van Komen S,

[76] Petukhova G,

[77] Sigurdsson S,

[78] Stratton S,

[79] Sung P

[80] ↵
Ristic D,
Wyman C,
Paulusma C,
Kanaar R
(2001) The architecture of the human Rad54-DNA complex provides evidence for protein translocation along DNA. Proc Natl Acad Sci USA 98:8454–8460.
OpenUrl Abstract/FREE Full Text

[81] Ristic D,

[82] Wyman C,

[83] Paulusma C,

[84] Kanaar R

[85] ↵
Amitani I,
Baskin RJ,
Kowalczykowski SC
(2006) Visualization of Rad54, a chromatin remodeling protein, translocating on single DNA molecules. Mol Cell 23:143–148.
OpenUrl CrossRef PubMed

[86] Amitani I,

[87] Baskin RJ,

[88] Kowalczykowski SC

[89] ↵
Solinger JA,
Kiianitsa K,
Heyer WD
(2002) Rad54, a Swi2/Snf2-like recombinational repair protein, disassembles Rad51:dsDNA filaments. Mol Cell 10:1175–1188.
OpenUrl CrossRef PubMed

[90] Solinger JA,

[91] Kiianitsa K,

[92] Heyer WD

[93] ↵
Bugreev DV,
Mazina OM,
Mazin AV
(2006) Rad54 protein promotes branch migration of Holliday junctions. Nature 442:590–593.
OpenUrl CrossRef PubMed

[94] Bugreev DV,

[95] Mazina OM,

[96] Mazin AV

[97] ↵
Bugreev DV,
Hanaoka F,
Mazin AV
(2007) Rad54 dissociates homologous recombination intermediates by branch migration. Nat Struct Mol Biol 14:746–753.
OpenUrl CrossRef PubMed

[98] Bugreev DV,

[99] Hanaoka F,

[100] Mazin AV

[101] ↵
Whitby MC
(2004) Junctions on the road to cancer. Nat Struct Mol Biol 11:693–695.
OpenUrl CrossRef PubMed

[102] Whitby MC

[103] ↵
Ehmsen KT,
Heyer WD
(2008) Saccharomyces cerevisiae Mus81-Mms4 is a catalytic, DNA structure-selective endonuclease. Nucleic Acids Res 36:2182–2195.
OpenUrl Abstract/FREE Full Text

[104] Ehmsen KT,

[105] Heyer WD

[106] ↵
Mazina OM,
Rossi MJ,
Thomaa NH,
Mazin AV
(2007) Interactions of human rad54 protein with branched DNA molecules. J Biol Chem 282:21068–21080.
OpenUrl Abstract/FREE Full Text

[107] Mazina OM,

[108] Rossi MJ,

[109] Thomaa NH,

[110] Mazin AV

[111] ↵
Ciccia A,
Constantinou A,
West SC
(2003) Identification and characterization of the human mus81-eme1 endonuclease. J Biol Chem 278:25172–25178.
OpenUrl Abstract/FREE Full Text

[112] Ciccia A,

[113] Constantinou A,

[114] West SC

[115] ↵
Bugreev DV,
Brosh RM, Jr,
Mazin AV
(2008) RECQ1 possesses DNA branch migration activity. J Biol Chem 283:20231–20242.
OpenUrl Abstract/FREE Full Text

[116] Bugreev DV,

[117] Brosh RM, Jr,

[118] Mazin AV

[119] ↵
Zhang R,
et al.
(2005) BLM helicase facilitates Mus81 endonuclease activity in human cells. Cancer Res 65:2526–2531.
OpenUrl Abstract/FREE Full Text

[120] Zhang R,

[121] et al.

[122] ↵
Zerbib D,
Mezard C,
George H,
West SC
(1998) Coordinated actions of RuvABC in Holliday junction processing. J Mol Biol 281:621–630.
OpenUrl CrossRef PubMed

[123] Zerbib D,

[124] Mezard C,

[125] George H,

[126] West SC

[127] ↵
Rossi MJ,
Mazin AV
(2008) Rad51 protein stimulates the branch migration activity of rad54 protein. J Biol Chem 283:24698–24706.
OpenUrl Abstract/FREE Full Text

[128] Rossi MJ,

[129] Mazin AV

[130] ↵
Petukhova G,
Van Komen S,
Vergano S,
Klein H,
Sung P
(1999) Yeast Rad54 promotes Rad51-dependent homologous DNA pairing via ATP hydrolysis-driven change in DNA double helix conformation. J Biol Chem 274:29453–29462.
OpenUrl Abstract/FREE Full Text

[131] Petukhova G,

[132] Van Komen S,

[133] Vergano S,

[134] Klein H,

[135] Sung P

[136] ↵
Biertumpfel C,
Yang W,
Suck D
(2007) Crystal structure of T4 endonuclease VII resolving a Holliday junction. Nature 449:616–620.
OpenUrl CrossRef PubMed

[137] Biertumpfel C,

[138] Yang W,

[139] Suck D

[140] ↵
Ariyoshi M,
Nishino T,
Iwasaki H,
Shinagawa H,
Morikawa K
(2000) Crystal structure of the holliday junction DNA in complex with a single RuvA tetramer. Proc Natl Acad Sci USA 97:8257–8262.
OpenUrl Abstract/FREE Full Text

[141] Ariyoshi M,

[142] Nishino T,

[143] Iwasaki H,

[144] Shinagawa H,

[145] Morikawa K

[146] ↵
Hanada K,
et al.
(2007) The structure-specific endonuclease Mus81 contributes to replication restart by generating double-strand DNA breaks. Nat Struct Mol Biol 14:1096–1104.
OpenUrl CrossRef PubMed

[147] Hanada K,

[148] et al.

[149] ↵
Doe CL,
Whitby MC
(2004) The involvement of Srs2 in post-replication repair and homologous recombination in fission yeast. Nucleic Acids Res 32:1480–1491.
OpenUrl Abstract/FREE Full Text

[150] Doe CL,

[151] Whitby MC

[152] ↵
Osman F,
Whitby MC
(2007) Exploring the roles of Mus81-Eme1/Mms4 at perturbed replication forks. DNA Repair 6:1004–1017.
OpenUrl CrossRef PubMed

[153] Osman F,

[154] Whitby MC

[155] ↵
Bugreev DV,
Mazina OM,
Mazin AV
(2006) Analysis of branch migration activities of proteins using synthetic DNA substrates. Nat Protoc, 2010.1038/nprot.2006.217.

[156] Bugreev DV,

[157] Mazina OM,

[158] Mazin AV

[159] ↵
Mazin AV,
Alexeev AA,
Kowalczykowski SC
(2003) A novel function of Rad54 protein. Stabilization of the Rad51 nucleoprotein filament. J Biol Chem 278:14029–14036.
OpenUrl Abstract/FREE Full Text

[160] Mazin AV,

[161] Alexeev AA,

[162] Kowalczykowski SC

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New Research In

Human Rad54 protein stimulates human Mus81–Eme1 endonuclease

Abstract

Results

hRad54 Stimulates hMus81 Endonuclease Activity.

hRad54 Stimulates hMus81 Cleavage Activity on a Broad Range of DNA Substrates.

hRad54 Protein Stimulates Endonuclease Activity of hMus81 Via Protein-Specific Interaction with hMus81 Protein.

hRad54 Recruits hMus81 to the Site of DNA Junction Resolution.

Effect of Nucleotide Cofactors on Stimulation of hMus81 by hRad54.

hRad54 Does Not Change the Cleavage Specificity of hMus81.

Discussion

Materials and Methods

Proteins and DNA.

Endonuclease Assays.

Endonuclease and Branch Migration Assays.

Acknowledgments

Footnotes

References

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Human Rad54 protein stimulates human Mus81–Eme1 endonuclease

Abstract

Results

hRad54 Stimulates hMus81 Endonuclease Activity.

hRad54 Stimulates hMus81 Cleavage Activity on a Broad Range of DNA Substrates.

hRad54 Protein Stimulates Endonuclease Activity of hMus81 Via Protein-Specific Interaction with hMus81 Protein.

hRad54 Recruits hMus81 to the Site of DNA Junction Resolution.

Effect of Nucleotide Cofactors on Stimulation of hMus81 by hRad54.

hRad54 Does Not Change the Cleavage Specificity of hMus81.

Discussion

Materials and Methods

Proteins and DNA.

Endonuclease Assays.

Endonuclease and Branch Migration Assays.

Acknowledgments

Footnotes

References

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