Cleavage Mechanism of Human Mus81–Eme1 Acting on Holliday-Junction Structures

Cleavage mechanism of human Mus81–Eme1 acting on Holliday-junction structures

Ewan R. Taylor* and Clare H. McGowan*†‡

Departments of *Molecular Biology and †Cell Biology, The Scripps Research Institute, La Jolla, CA 92037

Edited by Stephen J. Elledge, Harvard Medical School, Boston, MA, and approved January 10, 2008 (received for review October 29, 2007) Recombination-mediated repair plays a central role in maintaining Results genomic integrity during DNA replication. The human Mus81–Eme1 Recombinant Human Mus81–Eme1. Truncation analysis of both endonuclease is involved in recombination repair, but the exact Mus81 and Eme1 was used to define the minimal domains structures it acts on in vivo are not known. Using kinetic and enzy- required for endonuclease function [see supplementary infor- matic analysis of highly puriﬁed recombinant enzyme, we ﬁnd mation (SI) Fig. 5]. Versions of each protein containing amino that Mus81–Eme1 catalyzes coordinate bilateral cleavage of acids 260–551 for Mus81 and amino acids 244–571 for Eme1 model Holliday-junction structures. Using a self-limiting, cruciform- were expressed in Escherichia coli. The recombinant truncated containing substrate, we demonstrate that bilateral cleavage occurs complex, which we named EcME, was well expressed, largely sequentially within the lifetime of the enzyme–substrate complex. soluble, and, as detailed below, active. The complex was purified Coordinate bilateral cleavage is promoted by the highly cooperative to apparent homogeneity through affinity chromatography, ion nature of the enzyme and results in symmetrical cleavage of a exchange, and gel filtration steps (Fig. 1a). cruciform structure, thus, Mus81–Eme1 can ensure coordinate, bilateral cleavage of Holliday junction-like structures. Endonuclease Properties of a Minimal Mus81–Eme1 Complex. The activity of the recombinant enzyme was initially tested on three nuclease ͉ recombination repair model substrates, X12, nX12, and 3Ј flap, and each junction was radiolabeled on the 5Ј end of one oligonucleotide (Fig. 1b). The

oligo nucleotide sequences are identical to those used previously (4, BIOCHEMISTRY he maintenance of genomic integrity requires multiple coordi- 16, 23). The X12 substrate has four 25-bp duplex DNA arms, with nated repair processes during DNA replication. Fork-stalling, T a 12-bp homologous core that allows branch migration of the recombination repair, and replication restart create a variety of junction. The nX12 substrate is identical to X12, except that it branched structures that are substrates for endonucleases. The contains a nick at the cross-over point. The 3Ј flap substrate Mus81–Eme1 endonuclease was first identified in budding yeast as contains 50 bp of duplex DNA, a central nick, and 25 bases of a mutant that causes sensitivity to replication-associated genotoxic single-stranded DNA. EcME is able to convert all three substrates stress (1). Fission yeast strains null for either Mus81 or Eme1 are to linear duplex product (Fig. 1b). The kinetic parameters of the exquisitely sensitive to replication stress and are inviable during EcME complex on these substrates were calculated by using meiosis (2, 3). Based on enzyme activity, damage sensitivity, and the nonlinear regression analysis of the reaction velocities obtained rescue of meiotic segregation defects by the prokaryotic resolvase, from experiments in which substrate concentration was varied (SI Ϫ1 RusA, a role in resolving Holliday junctions was proposed for Table 1 and SI Fig. 6). EcME has the highest kcat,0.18s , when Mus81–Eme1 in fission yeast (3). This function is supported by acting on an nX12 substrate. The enzyme functioned catalytically more recent data showing that the accumulation of X structures in in this reaction and converted 95 moles of substrate per mole of Mus81 delete cells in response to replication pausing and at sites of enzymein10min(SI Table 2). Consistent with previous observa- meiotic recombination (4, 5). tions using full-length Mus81–Eme1 (4, 24), EcME processed an Ϫ1 Mus81-null mice are viable, fertile, and have no obvious devel- intact X12 junction less efficiently, with a kcat of 0.0047 s . opmental defects (6, 7). Both mouse and human Mus81- and Nevertheless, at the highest concentration tested, EcME converted Eme1-null cell lines are exquisitely sensitive to interstrand crosslink Ͼ95% of the intact X12 substrate into linear duplex product (Fig. agents including mitomycin C (6–9). Mus81–Eme1 is recruited to 1b). The reduced catalytic efficiency of EcME on an intact X12 was sites of UV irradiation specifically during DNA replication (10). To driven both by a higher Km and a lower catalytic rate. The date, no meiotic defects have been reported in null Mus81 mice or observation that Mus81–Eme1 favors cleavage of nX12 over an X12 Drosophila, however, a role in generating interference-independent suggests that the initial cut on an X12 substrate is rate-limiting, and Ϸ crossovers has been reported for budding yeast and Arabidopsis (6, that it is followed by an 35-fold faster second cut. A nick- 7, 11–13). Also, Mus81 deficiency is lethal when combined with the counternick mechanism, in which a rate-limiting initial cut is disruption of the BLM helicase homologues in budding yeast, followed by a kinetically favored second-strand cleavage reaction, fission yeast, Drosophila, and Arabidopsis, suggesting a conserved has been used to explain the mechanism of action of a number of junction-resolving enzymes (4, 25–28). The kinetic parameters of role for Mus81–Eme1 in recombination repair and possibly Holli- Ј day-junction processing (2, 11, 14, 15). Data from several eukaryotic EcME on a 3 flap suggests that the complex binds this substrate organisms have shown that Mus81–Eme1 has activity on a number relatively poorly, but, once bound, the enzyme has a relatively robust rate of catalysis, with a k of 0.12 sϪ1. of branched DNA structures: Potential in vivo substrates are cat speculated to include forks, flaps, D-loops, and Holliday junctions (3, 4, 16–22). Author contributions: E.R.T. and C.H.M. designed research; E.R.T. performed research; In this study, we use highly purified recombinant Mus81–Eme1 E.R.T. and C.H.M. analyzed data; and E.R.T. and C.H.M. wrote the paper. to test the enzymatic properties and investigate the mechanism of The authors declare no conﬂict of interest. cleavage of model Holliday junctions. We define the catalytic core This article is a PNAS Direct Submission. of the enzyme complex. By using a plasmid-based substrate, we ‡To whom correspondence should be addressed. E-mail: [email protected]. demonstrate that Mus81–Eme1 uses a highly cooperative, coordi- This article contains supporting information online at www.pnas.org/cgi/content/full/ nated mechanism that ensures bilateral, symmetrical cleavage of 0710291105/DC1. Holliday-junction structures. © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710291105 PNAS ͉ March 11, 2008 ͉ vol. 105 ͉ no. 10 ͉ 3757–3762 Downloaded by guest on September 28, 2021 rations is explained by structural predications suggesting that these amino acids do not directly coordinate the catalytic cation and thus play a supporting rather than essential role in catalysis (29). The analogous mutation, D599A, in PfHef results in a similar dimin- ished, but not absent, nuclease activity (29).

Substrate Specificity of Endogenous Mus81–Eme1 Endonuclease Ac- tivity. To test whether the truncations of Mus81 and Eme1 affect substrate preference, we compared the activity of EcME to full- length Mus81–Eme1 purified from baculovirus infected insect cells by anti-FLAG immunoprecipitation (Sf Mus81–Eme1). EcME has the same relative substrate preference as the recombinant full- length version of the protein, nX12 Ͼ 3Ј flap ϾϾ X12 (Fig. 1c), confirming that the truncations do not profoundly alter the substrate preference of the recombinant EcME enzyme. The activity profile of endogenous Mus81–Eme1 from a human cell line (HeLa) was also compared with that of recombinant full-length Mus81– Eme1 from insect cells (Fig. 1c). As with both recombinant versions of the complex, endogenous Mus81–Eme1 converted all three substrates into linear duplex (Fig. 1d). Notably, endogenous Mus81–Eme1 complex has a higher X12/nX12 activity ratio when compared with the recombinant protein. By comparing the amount of product generated at subsaturating levels of Mus81–Eme1 (i.e., 10% of immune precipitate), we find that the endogenous enzyme is only 3-fold less efficient on X12 than nX12 (Fig. 1d), whereas the recombinant insect cell-expressed enzyme is 10-fold less efficient (Fig. 1c). The endogenous enzyme had the same relative rate of Fig. 1. Endonuclease activities of recombinant human Mus81–Eme1. (a) activity when immune-precipitated with either monoclonal mouse Recombinant human Mus81–Eme1 purified from E. coli (EcME). Purified preparations were separated by SDS/PAGE and visualized by Coomassie staining. or polyclonal rabbit anti-Mus81 antibodies (data not shown). (b) Endonuclease activity of EcME on multiple substrates. The X12, nX12, and Gaskell et al. (31) recently concluded that full-length, recombi- 3Ј flap substrates, [200 pM], with 0.02, 0.2, 2, or 20 nM EcME for 20 min. nant, fission yeast Mus81–Eme1 has the same intact X versus nicked Products were separated by native PAGE. Substrates and DNA products are as X activity ratio as the endogenous protein. However, this compar- indicated. Each substrate is illustrated above the gels, and the 5Ј 32P radiolabel ison was based on assays done in two different laboratories (4, 31). is indicated by a circle. (c) Endonuclease activity of Myc.Mus81–Eme1.FLAG In this study, we compared the activity of recombinant full-length purified from Sf9 cells (Sf Mus81–Eme1). The indicated substrates [200 pM] human Mus81–Eme1 to the endogenous complex under identical were incubated with purified SfMus81–Eme1 for 20 min, with either 10%, assay conditions. In these circumstances, increased relative activity ␣ 30%, or 100% of a FLAG immune precipitate being used. Products were on the intact X12 structure is clearly detected. The difference in separated by native PAGE. Substrates and products are as indicated. Percent- age of cleavage (% cleavage) is indicated below the panel, with a dash substrate specificity between the endogenous human and the representing Ͻ1% cut. (d) Endonuclease activity of Mus81–Eme1 immune- recombinant enzyme could be due to the presence of associated precipitated from HeLa cell extracts. The indicated substrates [200 pM] were factors or posttranslational modifications that do not occur in insect incubated with purified Mus81–Eme1, or beads (B), for 20 min, with either cells. 10%, 30%, or 100% of a ␣Mus81 immune precipitate. Bilateral Cleavage of a Cruciform-Containing Plasmid. The cruciform structures formed by extrusion of an inverted repeat sequence in To determine whether the endonuclease activity detected in supercoiled plasmids have been extensively used to study the these assays can exclusively be attributed to Mus81–Eme1, versions enzymatic properties of Holliday junction-resolving enzymes (25– of EcME enzyme lacking potential catalytic residues were made. 27, 32). Cruciforms are formed by intrastrand base pairing of We have previously used Mus81D338/339A, a version of Mus81 that inverted repeat sequences and thus form two hairpin loops ex- has severely compromised, but not completely abolished, endonu- truded from closed circular plasmid DNA. The region at the base clease activity (16, 20). Based on recent structural analysis of related of the stem loops forms a four-way junction (Fig. 2a). A unique nucleases, PfHEF and ApeXPF (29, 30), we predicted that aspartic feature of this model substrate is that the cruciform structure is acid D307 might be critical for catalysis and that changing this stabilized by the free energy of negative supercoiling in the plasmid. residue to alanine would more effectively compromise catalysis. A break in either strand of the DNA leads to relaxation, destabi- Both mutant versions of EcME were expressed in E. coli and lization, and, thus, reabsorption of both hairpins into duplex DNA purified as above. (33). An enzyme that cuts a single strand of DNA will thus generate An X12 substrate labeled on the 5Ј end of oligonucleotide 1 was nicked circular DNA. By contrast, an enzyme that simultaneously incubated with wild-type or mutant versions of EcME, and the cleaves both strands of the plasmid generates linear DNA. Likewise, products were examined after denaturing PAGE (SI Fig. 7). enzymes that make two cleavages sequentially, within the lifetime Denaturing gels were used for this analysis because nicking of a of the protein DNA complex, generate linear product even though single strand is not detected on native gels. A trace of endonuclease cleavage of the two strands is not simultaneous (25–27). activity was detected in EcMED338/339A. Significantly, no cleavage We examined the ability of EcME to process cruciform- was detected when an equivalent amount of EcMED307A was containing plasmid DNA. Supercoiled plasmid pAT25 (25), which incubated with substrate. The low-to-undetectable nuclease func- contains two 25-bp hairpin structures, was incubated with EcME tion of these preparations indicates that the endonuclease activity and samples were taken at different time points. The reaction detected in wild-type preparation of EcME is due to the intrinsic products were analyzed on a native agarose gel. As shown in Fig. 2b, properties of the enzyme complex and that the recombinant the substrate was converted to linear product, indicating that EcME enzyme preparations are free of contaminating nuclease activities. can make two incisions on this substrate. At early time points, when The residual endonuclease activity seen in EcMED338/339A prepa- Ͻ30% of substrate had been processed, similar amounts of nicked

3758 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710291105 Taylor and McGowan Downloaded by guest on September 28, 2021 Fig. 2. Bilateral cleavage mechanism of the Mus81–Eme1 endonuclease. (a) Bilateral cleavage of the pAT25 cruciform-containing plasmid. The extruded cruciform is depicted in its most abundant isomer, i.e., parallel orientation of the junction arms (44). The consequence of cutting the plasmid either at one or two sites is indicated. (b) Time course of EcME [10 nM] cutting pAT25 [20 pM]. Products were visualized by Southern blot. The percentage of each form of pAT25 is plotted; each point represents the average from three experiments with the standard error of the mean shown. The points represent quantifica- tion of supercoiled DNA (black diamonds), linear DNA (blue squares), and nicked DNA (red triangles). BIOCHEMISTRY and linear product were generated. As the reaction proceeded further, the amount of nicked product declined. By the end of the 30-min reaction, when 97% of substrate had been processed, the major product (70%) was linear DNA. The generation of significant quantities of nicked circular DNA early in the reaction suggests that Fig. 3. Cooperative cleavage by Mus81–Eme1. (a) Dose–response of pAT25 the two incisions made by EcME are sequential. This idea is also cleavage versus EcME concentration. pAT25 substrate [20 pM] was incubated supported by the continued appearance of linear product at later with increasing amounts of Mus81–Eme1 as indicated. Percentage of super- time points, when the substrate has been almost exhausted. The coiled pAT25 substrate cleaved is plotted. Data are from one experiment and relatively slow conversion of nicked to linear product coupled with are representative of four experiments. (b) Activation of EcME by EcMED307A. the observation that 70% of substrate is converted to linear product pAT25 substrate [20 pM] was incubated with a fixed amount of wild-type suggests that EcME restrains the nicked intermediate in a confor- EcME [3 nM] and increasing amounts of EcMED307A. Percentage of product and mation that can be further processed into linear DNA. Data substrate present at each time point is plotted. Data are from one experiment and are representative of three experiments. The points represent supercoiled confirming that EcME cuts at the position of the cruciform is DNA (black diamonds), linear DNA (blue squares), and nicked DNA (red presented in SI Figs. 8 and 9. triangles). (c) Unilateral cleavage by mixed active/inactive EcME. pAT25 substrate [20 pM] was incubated with a fixed amount of wild-type EcME [6 nM] Cooperative Bilateral Cleavage. The dose dependence of cruciform and increasing amounts of EcMED307A. The percentage of product and sub- processing was investigated (Fig. 3a). Increasing concentrations of strate present at each time point is plotted. Data are from one experiment and EcME were incubated with pAT25. Strikingly, the rate of cleavage are representative of three experiments. (d) Gel filtration analysis of EcME. catalyzed by EcME was not directly proportional to the concen- The indicated concentrations of EcME were fractionated on a Superdex S200 tration of protein. In multiple experiments, a marked threshold gel-filtration column. Aliquots of each fraction were separated by SDS/PAGE concentration of enzyme was required to initiate cruciform cleav- gel, and apparent molecular mass was calculated relative to standards. age. The sigmoidal relationship between activity and EcME concentration indicates that the EcME-catalyzed reaction is higher enzyme (Fig. 3b, lanes 5 and 6). In these reactions, Ϸ30% of the than first order. The shape of the curve is most easily explained by substrate was converted to nicked circular DNA. Nicked circular cooperative association of two molecules of EcME with DNA. DNA can only result from unilateral cleavage of the substrate, thus, Similar results were obtained when the substrate concentration was the increase in nicked circular product suggests that dimers in which 100-fold higher (SI Fig. 8c), indicating that the sigmoidal relationship is not due to low substrate concentration. one molecule of Mus81 is active and the other is inactive are If the catalytic activity of EcME depends on the simultaneous formed. The addition of higher concentrations of catalytically presence of two molecules of EcME, then low concentrations of inactive EcME also increased the amount of linear DNA that was wild-type complex may be stimulated by addition of catalytically generated, suggesting that the number or function of active dimers inactive EcME. To test this, an amount of wild-type enzyme below also increases. the threshold required to initiate cleavage was supplemented with The ability of active EcME to function when complexed with increasing concentrations of endonuclease dead, and the ability of inactive EcME was further tested by examining the effects of the mix to cleave cruciform DNA was monitored (Fig. 3b). As titrating endonuclease-dead EcMED307A against a fixed concentra- before, at low concentrations of wild-type enzyme, no processing of tion of wild-type EcME that is sufficient to cleave Ϸ90% of the substrate was detected (Fig. 3b, lane 2), significantly, addition substrate. At a 1:1 ratio of wild-type:inactive EcME (Fig. 3c, lane of endonuclease-dead EcMED307A stimulated cleavage (Fig. 3b 3), the same amount of substrate was processed, indicating that the lanes 4–7). Maximal activity was detected when the concentration presence of the endonuclease-dead enzyme does not significantly of endonuclease-dead enzyme was 2-fold greater than the wild-type inhibit unilateral cleavage catalyzed by the wild-type enzyme. The

Taylor and McGowan PNAS ͉ March 11, 2008 ͉ vol. 105 ͉ no. 10 ͉ 3759 Downloaded by guest on September 28, 2021 decrease in linear product was balanced by an increase in nicked product. As the amount of EcMED307A was increased further, the amount of cleaved substrate decreased. At the highest concentrations of EcMED307A,aϾ90% decrease in linear product and a 30% reduction of nicked product was observed. This result supports earlier evidence suggesting that dimers of active and inactive EcME form under these conditions. The unilateral cleavage seen in these experiments can best be explained if each scission step is catalyzed independently. Taken together, these results suggest that the catalytic function of each unit is independent but cooperative. A coordinate but independent mechanism has been proposed for canonical Holliday junction-resolving enzymes and for restriction endonucleases (25–27, 34, 35).

Ternary Structure of Recombinant Mus81–Eme1. The presence of two active sites in a single functional complex is thought to be essential for coordinated bilateral cleavage of duplex DNA in the case of restriction endonucleases (36) and Holliday junctions in the case of resolving enzymes (37–39). Several previously char- acterized resolvases have been shown to exist as highly stable dimeric complexes, whereas others undergo rapid subunit exchange in solution (27, 28). We wished to determine whether EcME can exist as a heterotetramer (containing two molecules of Mus81 and two molecules of Eme1) in solution (Fig. 3d). By using gel filtration, the molecular mass of a 3 ␮M solution EcME was determined to be 82 kDa [elution volume (Ve) ϭ 15.7 ml]. This apparent molecular mass is consistent with the truncated enzyme forming a functional dimer (i.e., one molecule of EcMus81 and one molecule of EcEme1). Curiously, when more concentrated preparations (88 ␮M) were analyzed, EcME was Fig. 4. Symmetrical cleavage of a cruciform structure by Mus81–Eme1. (a) found to elute at a position corresponding to a molecular mass Schematic of ligation assay. The symmetrical cleavage of a cruciform creates nicked hairpin DNA ends that are substrates for DNA ligase. Linear DNA with of 140 kDa (Ve ϭ 14.5 ml), suggesting that the preparation forms a tetrameric complex. sealed hairpins runs as a dimer-length ssDNA circle on a denaturing gel. (b) The observation that EcME can exist in solution as either a pAT25 substrate [200 pM] was incubated with either EcME [10 nM] or BamHI (10 units) and followed by heat inactivation. Where indicated, T4 DNA ligase heterodimer or a heterotetramer is consistent with the cooper- was added to the second incubation. Reaction products were analyzed by ative nature of the enzyme acting on the pAT25 substrate. The either native or denaturing gel electrophoresis and detected by Southern blot. concentration of EcME used in the enzyme assays (Fig. 3 a and (c) pAT25 substrate [2 nM] was incubated with either wild-type or mutant b) is far below the value needed to form a heterotetramer in EcME [30 nM]. The reaction products were used as templates for primer solution, however the presence of cruciform-containing DNA extension reactions by using forward (Fwd) or reverse (Rev) primers as indi- could significantly affect this value by acting as a nucleation site cated. A Maxam–Gilbert sequencing ladder, run on the same gel, is shown at for the enzyme. a lighter exposure. The major EcME cleavage sites on the cruciform in pAT25 are illustrated. Symmetrical Cruciform Cleavage by Mus81–Eme1. Canonical Holliday-junction resolvases cleave junctions such that the ders. As shown in Fig. 4c, the major cleavage sites for EcME linear duplex products contain ligatable nicks that can be mapped to the branch point on the 5Ј side of the hairpin. Several sealed directly by ligation (28). Previous studies examining minor cleavage sites were also detected within the hairpin arms Mus81–Eme1 cleavage of X-shaped oligonucleotide structures found that the enzyme predominantly makes nonsymmetric (Fig. 4c). These results confirm that EcME makes symmetrically cuts that result in nonligatable products (3, 16, 18). To related cuts at the site of the junction. determine the structure of the products generated by EcME Discussion acting on the cruciform, the reaction products were examined both on native and denaturing gels. Symmetrical bilateral The hypothesis that Mus81–Eme1 resolves Holliday junctions in cleavage at the branch point of an extruded cruciform gener- vivo has been challenged on the basis that it preferentially cleaves Ј ates linear DNA containing terminal hairpins that can be 3 flaps and nicked X structures in vitro (8, 17, 22, 40, 41). Our ligated (Fig. 4a) (39). On a native gel, this structure cannot be analysis of truncated recombinant human Mus81–Eme1, full-length distinguished from linear duplex DNA. However, on a dena- recombinant human Mus81–Eme1, and endogenous human turing gel, this structure runs as dimer-length single-stranded Mus81–Eme1 is in agreement with previous studies showing that an DNA (Fig. 4b). As expected, EcME generated product runs at intact X structure is not the preferred in vitro substrate (18, 40). the position of linear DNA both on native and denaturing gels. However, evidence presented here, that Mus81–Eme1 has the Treatment with T4 ligase did not effect the migration on a specific, intrinsic enzymatic properties needed to catalyze coordi- native gel (Fig. 4b Upper). Significantly, denaturing gel analysis nated cleavage of symmetric substrates, containing two indepen- of T4 ligase-treated products revealed the major species (67%) dent target bonds, supports the idea that Mus81–Eme1 acts on to be dimer-length single-stranded circular DNA. The identity intact Holliday junctions in vivo. of the dimer-length ssDNA circle was validated in SI Fig. 10. The use of a cruciform-extruding plasmid as a model Holliday- The generation of a high proportion of ligatable product junction structure has allowed the elucidation of several enzy- suggested that cleavage of pAT25 is predominantly symmetric. matic properties of Mus81–Eme1. Because the cruciform struc- The exact sites of cruciform cleavage were mapped by comparing ture is unstable in a nicked plasmid (SI Fig. 5), we were able to the products of primer-extension reactions to sequencing lad- conclude that two Mus81–Eme1 complexes act sequentially

3760 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710291105 Taylor and McGowan Downloaded by guest on September 28, 2021 within the lifetime of the enzyme–substrate complex. The sig- crossovers are greatly reduced in fission yeast mutants of Mus81– moidal relationship between enzyme concentration and activity, Eme1 (3, 5, 19). However, the role of recombination repair in coupled with stimulation of wild-type enzyme by the addition of mitotic cells, maintenance of genomic integrity, and facilitating catalytically inactive EcME, demonstrates that the presence of replication restart is somewhat different from its role in meiosis. In two catalytic units is either required for or greatly stimulates mitosis, crossovers occurring between sister chromatids are genet- cleavage of the first strand of DNA. These data suggest that ically silent and are therefore assayed by monitoring sister- allosteric interaction between one Mus81–Eme1 complex and chromatid exchange. Disruption of Eme1 in mouse embryonic stem another promotes catalysis. It is also possible that binding of a cells leads to a slight increase, not a decrease, in sister-chromatid heterotetrameric enzyme complex to DNA manipulates the exchange (8). Thus, if Mus81–Eme1 acts on D-loops/nicked Hol- junction into a more favorable conformer. These properties are liday junctions during mitosis in mammalian cells, it is not apparent consistent with those expected of an endonuclease that acts on in assays of sister-chromatid exchange. symmetric substrates containing two equivalent cleavage sites. Given evidence that Mus81–Eme1 can exist in different multi- The data also suggest that dimerization is an inherent property meric states (Fig. 3d) (20, 31) and the possibility that cofactors may of the Mus81–Eme1 complex. Dimerization of endogenous modulate function, it would be interesting to see whether the human enzyme has previously been described (20) and was enzyme functions in different modes in different biological circum- recently confirmed for recombinant yeast proteins (31). We stances. The Mus81/XPF/Hef-related endonuclease from Drosoph- found that high concentrations of EcME are needed to form a ila, Mei9, functions in nucleotide excision repair and interstrand heterotetramer in solution. However, in vivo complex formation crosslink repair in somatic cells (45, 46). Within meiosis, Mei9 is may be promoted by interaction with DNA, by amino terminal required to generate crossovers (47). Intriguingly, the crossover sequences that are not essential for catalysis, and perhaps by function, interstrand crosslink repair function, but not the nucle- other protein factors. otide-excision repair function of Mei9 depend on an associated A nick-counternick mechanism has previously been suggested as protein, Mus312 (48). Mus312 homologues have not been described the mechanism by which resolvases could use independently func- in higher eukaryotes, and, indeed, the region that Mus312 binds in tioning catalytic units to coordinate bilateral cleavage of Holliday Mei9 is not conserved in Mus81 (49). Nevertheless, based on the junctions (4, 25–27, 37). Another mechanism used by endonucle- enhanced X12 activity seen for endogenous Mus81–Eme1, it is ases to ensure cleavage of duplex DNA is cooperativity. In the case tempting to speculate that either changes in the dimerization state, of type IIS restriction endonucleases, cooperativity and biological or association with other proteins factors, might direct Mus81–

function are driven by induced dimerization of the enzyme subunits Eme1 activity to different structures. BIOCHEMISTRY (35, 36). The highly cooperative kinetic mechanism proposed for Mus81–Eme1 avoids first-strand cleavage unless second-strand Methods cleavage is ensured. Endonuclease Assays on Radiolabeled Substrate. The radiolabeled junctions Previous studies found that Mus81–Eme1 cuts oligonucleotide were produced and used as previously detailed (4, 16). Reaction products were X-shaped structures by cleaving strands of opposite polarity (3, analyzed by native or denaturing PAGE as outlined in ref. 16. 16, 18). However, the majority of these products were cut nonsymmetrically and contained short flaps or gaps and could Endonuclease Assays on pAT25. Reactions containing pAT25 were initiated by addition of MgCl2 to 2.5 mM, incubated at 37°C for 20 min, and terminated by not be directly ligated in vitro (3, 16, 18). In contrast, by using a using stop buffer (10 mM EDTA, 1 mg/ml proteinase K, 0.4% SDS). Products cruciform-containing plasmid as a substrate, we find that cleav- were separated by electrophoresis through 1% agarose at 45 V for 16 h. DNA age by Mus81–Eme1 is largely symmetric and a significant was transferred by Southern blotting. pAT25 DNA was detected by using a 32P fraction of the products can be ligated directly. Both oligonu- random-labeled probe. Signals were quantified by using a Storm 860 Phos- cleotide X structures and cruciforms mimic Holliday junctions, phorImager (Molecular Dynamics). however, structural differences may account for differences in the way they are cleaved. For example, the arms of oligonucle- Ligation Assay on pAT25. 0.2 nM pM pAT25 was incubated in endonuclease otide-based substrates stack in an antiparallel conformation (42, buffer [50 mM Tris (pH 8.0), 20 mM NaCl, 2.5 mM MgCl2, 1 mM DTT and 100 43), whereas the arms of plasmid-extruded cruciforms predom- ␮g/ml BSA] with either 10 nM EcME or 10 units of BamHI (NEB) for 2 min at inantly stack in a parallel orientation (44). Equally, it is possible 37°C. The reactions were terminated by heating at 70°C for 20 min. T4 DNA ligase (5U) and 1X T4 ligase buffer (Invitrogen) were added, and the reaction that the cooperativity seen when Mus81–Eme1 acts on the was incubated at room temperature for 30 min. Reaction products were cruciform structure promotes symmetric cleavage. Further stud- separated by either native or denaturing agarose gel electrophoresis (39) and ies are required to test the contribution of each of these detected by Southern blotting. SI Methods contains detailed descriptions of properties to the symmetrical cleavage of the cruciform junction. enzyme purification, pAT25 purification, and the mapping of pAT25 cut sites. The efficient cleavage of nicked X structures by Mus81–Eme1 has been interpreted as a mechanism, which, if Mus81–Eme1 acts ACKNOWLEDGMENTS. We thank D. M. J. Lilley (University of Dundee, on D-loops before they mature into fully formed X-structures, Dundee, Scotland) for the kind donation of the pAT25 substrate and his encouragement, V. Blais for valuable technical support, and members of the would favor crossovers in meiosis (4, 19). This is an exciting cell cycle group for their input and reading of the manuscript. This work is possibility because it provides a de facto mechanism by which supported by a Leukemia Lymphomia Society Career Development Award (to Holliday-junction resolution can ensure crossover. Indeed, meiotic E.R.T.) and National Cancer Institute grant support (to C.H.M.).

1. Interthal H, Heyer WD (2000) MUS81 encodes a novel helix-hairpin-helix protein 7. Dendouga N, et al. (2005) Disruption of murine Mus81 increases genomic instability involved in the response to UV- and methylation-induced DNA damage in Saccharo- and DNA damage sensitivity but does not promote tumorigenesis. Mol Cell Biol myces cerevisiae. Mol Gen Genet 263:812–827. 25:7569–7579. 2. Boddy MN, et al. (2000) Damage tolerance protein Mus81 associates with the FHA1 8. Abraham J, et al. (2003) Eme1 is involved in DNA damage processing and maintenance domain of checkpoint kinase Cds1. Mol Cell Biol 20:8758–8766. of genomic stability in mammalian cells. EMBO J 22:6137–6147. 3. Boddy MN, et al. (2001) Mus81-Eme1 are essential components of a Holliday junction 9. Hiyama T, et al. (2006) Haploinsufﬁciency of the Mus81-Eme1 endonuclease activates resolvase. Cell 107:537–548. the intra-S-phase and G2/M checkpoints and promotes rereplication in human cells. 4. Gaillard PH, Noguchi E, Shanahan P, Russell P (2003) The endogenous Mus81-Eme1 Nucleic Acids Res 34:880–892. complex resolves Holliday junctions by a nick and counternick mechanism. Mol Cell 10. Gao H, Chen XB, McGowan CH (2003) Mus81 endonuclease localizes to nucleoli 12:747–759. and to regions of DNA damage in human S-phase cells. Mol Biology Cell 5. Cromie GA, et al. (2006) Single Holliday junctions are intermediates of meiotic recom- 14:4826–4834. bination. Cell 127:1167–1178. 11. Johnson-Schlitz D, Engels WR (2006) Template disruptions and failure of double 6. McPherson JP, et al. (2004) Involvement of mammalian Mus81 in genome integrity and Holliday junction dissolution during double-strand break repair in Drosophila BLM tumor suppression. Science 304:1822–1826. mutants. Proc Natl Acad Sci USA 103:16840–16845.

Taylor and McGowan PNAS ͉ March 11, 2008 ͉ vol. 105 ͉ no. 10 ͉ 3761 Downloaded by guest on September 28, 2021 12. de los Santos T, et al. (2003) The Mus81/Mms4 endonuclease acts independently of 31. Gaskell LJ, Osman F, Gilbert RJ, Whitby MC (2007) Mus81 cleavage of Holliday junctions: double-Holliday junction resolution to promote a distinct subset of crossovers during A failsafe for processing meiotic recombination intermediates? EMBO J 26:1891–1901. meiosis in budding yeast. Genetics 164:81–94. 32. Lilley DM, Hallam LR (1984) Thermodynamics of the ColE1 cruciform. Comparisons 13. Berchowitz LE, Francis KE, Bey AL, Copenhaver GP (2007) The role of AtMUS81 in between probing and topological experiments using single topoisomers. J Mol Biol interference-insensitive crossovers in A. thaliana. PLoS Genet 3:e132. 180:179–200. 14. Mullen JR, Kaliraman V, Ibrahim SS, Brill SJ (2001) Requirement for three novel protein 33. Murchie AI, Lilley DM (1992) Supercoiled DNA, cruciform structures. Methods Enzymol complexes in the absence of the Sgs1 DNA helicase in Saccharomyces cerevisiae. 211:158–180. Genetics 157:103–118. 34. Vanamee ES, Santagata S, Aggarwal AK (2001) FokI requires two specific DNA sites for 15. Hartung F, Suer S, Bergmann T, Puchta H (2006) The role of AtMUS81 in DNA repair and cleavage. J Mol Biol 309:69–78. its genetic interaction with the helicase AtRecQ4A. Nucleic Acids Res 34:4438–4448. 35. Bitinaite J, Wah DA, Aggarwal AK, Schildkraut I (1998) FokI dimerization is required for 16. Chen XB, et al. (2001) Human Mus81-associated endonuclease cleaves Holliday junc- DNA cleavage. Proc Natl Acad Sci USA 95:10570–10575. tions in vitro. Mol Cell 8:1117–1127. 36. Gemmen GJ, Millin R, Smith DE (2006) Tension-dependent DNA cleavage by restriction 17. Kaliraman V, Mullen JR, Fricke WM, Bastin-Shanower SA, Brill SJ (2001) Functional endonucleases: Two-site enzymes are ‘‘switched off’’ at low force. Proc Natl Acad Sci overlap between Sgs1-Top3 and the Mms4-Mus81 endonuclease. Genes Dev 15:2730– USA 103:11555–11560. 2740. 37. White MF, Lilley DM (1997) The resolving enzyme CCE1 of yeast opens the structure of 18. Constantinou A, Chen XB, McGowan CH, West SC (2002) Holliday junction resolution the four-way DNA junction. J Mol Biol 266:122–134. in human cells: Two junction endonucleases with distinct substrate specificities. EMBO 38. Shah R, Cosstick R, West SC (1997) The RuvC protein dimer resolves Holliday junctions J 21:5577–5585. by a dual incision mechanism that involves base-specific contacts. EMBO J 16:1464– 19. Osman F, Dixon J, Doe CL, Whitby MC (2003) Generating crossovers by resolution of 1472. nicked Holliday junctions: A role for Mus81-Eme1 in meiosis. Mol Cell 12:761–774. 39. Iwasaki H, Takahagi M, Shiba T, Nakata A, Shinagawa H (1991) Escherichia coli RuvC 20. Blais V, et al. (2004) RNA interference inhibition of Mus81 reduces mitotic recombi- protein is an endonuclease that resolves the Holliday structure. EMBO J 10:4381–4389. nation in human cells. Mol Biol Cell 15:552–562. 40. Ciccia A, Constantinou A, West SC (2003) Identification and characterization of the 21. Doe CL, Ahn JS, Dixon J, Whitby MC (2002) Mus81-Eme1 and Rqh1 involvement in human mus81-eme1 endonuclease. J Biol Chem 278:25172–25178. processing stalled and collapsed replication forks. J Biol Chem 277:32753–32759. 41. Whitby MC (2005) Making crossovers during meiosis. Biochem Soc Trans 33:1451–1455. 22. Hollingsworth NM, Brill SJ (2004) The Mus81 solution to resolution: Generating meiotic 42. Duckett DR, et al. (1988) The structure of the Holliday junction, and its resolution. Cell crossovers without Holliday junctions. Genes Dev 18:117–125. 55:79–89. 23. Parsons CA, Kemper B, West SC (1990) Interaction of a four-way junction in DNA with 43. McKinney SA, Declais AC, Lilley DM, Ha T (2003) Structural dynamics of individual T4 endonuclease VII. J Biol Chem 265:9285–9289. 24. Fricke WM, Bastin-Shanower SA, Brill SJ (2005) Substrate specificity of the Saccharo- Holliday junctions. Nat Struct Biol 10:93–97. myces cerevisiae Mus81-Mms4 endonuclease. DNA Rep 4:243–251. 44. Shlyakhtenko LS, Potaman VN, Sinden RR, Lyubchenko YL (1998) Structure and dy- 25. Giraud-Panis MJ, Lilley DM (1997) Near-simultaneous DNA cleavage by the subunits of namics of supercoil-stabilized DNA cruciforms. J Mol Biol 280:61–72. the junction-resolving enzyme T4 endonuclease VII. EMBO J 16:2528–2534. 45. Boyd JB, Golino MD, Setlow RB (1976) The mei-9 alpha mutant of Drosophila mela- 26. Fogg JM, Schofield MJ, Declais AC, Lilley DM (2000) Yeast resolving enzyme CCE1 makes nogaster increases mutagen sensitivity and decreases excision repair. Genetics 84:527– sequential cleavages in DNA junctions within the lifetime of the complex. Biochemistry 544. 39:4082–4089. 46. Sekelsky JJ, McKim KS, Chin GM, Hawley RS (1995) The Drosophila meiotic recombi- 27. Fogg JM, Lilley DM (2000) Ensuring productive resolution by the junction-resolving nation gene mei-9 encodes a homologue of the yeast excision repair protein Rad1. enzyme RuvC: Large enhancement of the second-strand cleavage rate. Biochemistry Genetics 141:619–627. 39:16125–16134. 47. Baker BS, Carpenter AT (1972) Genetic analysis of sex chromosomal meiotic mutants in 28. Lilley DM, White MF (2001) The junction-resolving enzymes. Nat Rev 2:433–443. Drosophilia melanogaster. Genetics 71:255–286. 29. Nishino T, Komori K, Ishino Y, Morikawa K (2003) X-ray and biochemical anatomy of an 48. Yildiz O, Majumder S, Kramer B, Sekelsky JJ (2002) Drosophila MUS312 interacts with archaeal XPF/Rad1/Mus81 family nuclease: Similarity between its endonuclease do- the nucleotide excision repair endonuclease MEI-9 to generate meiotic crossovers. Mol main and restriction enzymes. Structure 11:445–457. Cell 10:1503–1509. 30. Newman M, et al. (2005) Structure of an XPF endonuclease with and without DNA 49. Yildiz O, Kearney H, Kramer BC, Sekelsky JJ (2004) Mutational analysis of the Drosoph- suggests a model for substrate recognition. EMBO J 24:895–905. ila DNA repair and recombination gene mei-9. Genetics 167:263–273.

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