Resolution of single and double INAUGURAL ARTICLE recombination intermediates by GEN1

Rajvee Shah Punatara,1, Maria Jose Martina,1, Haley D. M. Wyatta, Ying Wai Chana, and Stephen C. Westa,2

aDNA Recombination and Repair Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2016.

Contributed by Stephen C. West, December 2, 2016 (sent for review November 10, 2016; reviewed by Lumir Krejci and Lorraine S. Symington) provides an important mechanism for the In both DSBR and single-end invasion, the resulting HJs repair of DNA double-strand breaks. Homologous pairing and need to be processed for efficient segregation at strand exchange lead to the formation of DNA intermediates, in mitosis, thereby promoting genome stability (4). There are which sister chromatids or homologous are co- three pathways for HJ processing (5). In humans, the primary valently linked by four-way Holliday junctions (HJs). Depending pathway involves the BLM -TopoisomeraseIIIα-RMI1- on the type of recombination reaction that takes place, interme- RMI2 (BTR) complex, which promotes the dissolution of diates may have single or double HJs, and their resolution is double Holliday junctions (dHJs) by catalyzing the conver- essential for proper . In mitotic cells, gent migration of each junction followed by topoisomerase- double HJs are primarily dissolved by the BLM helicase- mediated processing of the resulting hemicatenane (6–8). TopoisomeraseIIIα-RMI1-RMI2 (BTR) complex, whereas single HJs This pathway favors the formation of noncross-over products (and double HJs that have escaped the attention of BTR) are re- (NCOs). In yeast, similar reactions are promoted by the - solved by structure-selective endonucleases known as HJ resol- Top3-Rmi1 (STR) complex (9, 10). Persistent dHJs that es- vases. These enzymes are ubiquitous in nature, because they are cape dissolution by BTR/STR are acted on at a later stage of present in , bacteria, archaea, and simple and com- the cell cycle by two distinct and temporally regulated nucle- plex . The human HJ resolvase GEN1 is a member of olytic cleavage pathways that promote HJ resolution. How-

′ BIOCHEMISTRY theXPG/Rad2familyof5-flap endonucleases. Biochemical stud- ever, in contrast to the BTR/STR system, these ies of GEN1 revealed that it cleaves synthetic DNA substrates resolve both single HJs and dHJs. Resolution gives rise to both containing a single HJ by a mechanism similar to that shown by cross-over products (COs) and NCOs (11), and the enzymes the prototypic HJ resolvase, RuvC protein, but it is that catalyze these reactions are broadly referred to as HJ unclear whether these substrates fully recapitulate the proper- resolvases (12). ties of recombination intermediates that arise within a physio- The first cellular (prototypic) HJ resolvase identified was the logical context. Here, we show that GEN1 efficiently cleaves both Escherichia coli RuvC protein, with which eukaryotic resolvases single and double HJs contained within large recombination in- are compared (13, 14). RuvC is a homodimeric protein con- termediates. Moreover, we find that GEN1 exhibits a weak se- taining two 19-kDa subunits that interact specifically with HJs. quence preference for incision between two G residues that Binding leads to the formation of an open HJ–RuvC structure, reside in a T-rich region of DNA. These results contrast with those and symmetrically related nicks are introduced into two strands obtained with RuvC, which exhibits a strict requirement for the – consensus sequence 5′-A/ TTG/ -3′. that are diametrically opposed across the junction (15 21). T C Consistent with this mechanism of cleavage, the crystal struc- recombination | repair | resolvase | RuvC | RecA ture of E. coli RuvC shows that the two monomers are related by a dyad axis, in which the two DNA binding clefts are sepa- rated by ∼30 Å (Fig. 2A) (22, 23). The 3D structure of Thermus omologous recombination (HR) plays an important role in thermophilus RuvC bound to an HJ has also been solved and Hthe repair of DNA double-strand breaks resulting from confirms the presence of a twofold symmetric unfolded junction DNA damaging agents, such as ionizing radiation, or the progression of a replication fork through single-strand breaks Significance in DNA (1, 2). Strand break repair by HR generally involves interactions between sister chromatids, which can become covalently linked by four-way junctions known as Holliday Holliday junction resolvases are required for the resolution of junctions (HJs) (3). Although originally proposed to explain recombination intermediates to ensure proper chromosome meiotic in fungal systems, the HJ provides segregation at mitosis. In human cells, the GEN1 protein, a a central tenet that is integral to many mechanisms of re- member of the XPG/Rad2 family of structure-selective endo- nucleases, is specialized for the cleavage of Holliday junction combination in both meiotic and mitotic cells. Two such recombination intermediates. Here, we show that GEN1 cuts pathways of DNA strand break repair that occur in mitotic DNA substrates containing single or double Holliday junctions cells are shown in Fig. 1. In classical double-strand break re- to generate cross-over or noncross-over recombination products. pair (DSBR), a resected DNA end pairs with homologous In contrast to the bacterial Holliday junction resolvase, RuvC, DNA to generate a D-loop structure that provides a 3′ end for A G which prefers to cut junctions at the sequence 5′- /TTT /C -3′, DNA synthesis (gap filling). Capture of the second end fol- GEN1 shows only weak sequence specificity. lowed by more DNA synthesis lead to the formation of DNA intermediates, in which the interacting are joined by Author contributions: R.S.P., M.J.M., and S.C.W. designed research; R.S.P. and M.J.M. two HJs (Fig. 1, Left). In a related but distinct scenario, a performed research; H.D.M.W. and Y.W.C. contributed new reagents/analytic tools; R.S.P., single DNA end, which might arise at a stalled/broken repli- M.J.M., and S.C.W. analyzed data; and R.S.P., M.J.M., and S.C.W. wrote the paper. cation fork, invades homologous DNA to again form a D loop. Reviewers: L.K., Masaryk University; and L.S.S., Columbia University. In this case, however, there is no second end available, and The authors declare no conflict of interest. DNA synthesis at the D loop leads to the formation of a single 1R.S.P. and M.J.M. contributed equally to this work. HJ (Fig. 1, Right). 2To whom correspondence should be addressed. Email: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1619790114 PNAS | January 17, 2017 | vol. 114 | no. 3 | 443–450 Downloaded by guest on September 29, 2021 reported (Fig. 2 C and D). In one study, catalytically inactive – Double Strand Break Repair Single End Invasion human GEN12 505 was bound nonproductively to the duplex arm of an HJ (32), whereas in the other, Chaetomium ther- – mophilum GEN11 487 was captured on the nicked duplex DNA that is produced by HJ resolution (33). Although the well- conserved core is similar to that of other XPG/Rad2 nucleases, GEN1 contains a chromodomain that provides an additional DNA binding site necessary for efficient HJ cleavage (32). In- DSB formation terestingly, the GEN1 nicked duplex structure revealed that the resection & resection XPG/Rad2 fold found in FEN1 and other members of this family are modified for its role in HJ cleavage, with the dimer interface juxtaposing the two nicked duplex DNA 3’ 3’ products (33). In contrast to the prokaryotic HJ resolvases, the activities of Yen1 and GEN1 are regulated throughout the cell cycle. In 3’ strand invasion & strand invasion & D-loop formation D-loop formation yeast, Yen1 exhibits low activity in S phase, because cyclin- dependent kinase (Cdk) promotes its phosphorylation, leading to nuclear exclusion and inhibition of catalytic activity by reducing its affinity for DNA (34, 35). Then, later in the cell cycle, Yen1 is dephosphorylated by Cdc14; dephosphorylation promotes nu- clear localization and DNA binding, leading to enzymatic acti- DNA synthesis DNA synthesis vation (36–38). GEN1 is also regulated, but in this case by a & 2nd end capture mechanism that seems to be independent of phosphorylation, because its activity is controlled primarily by nuclear exclusion (39). Thus, GEN1 only gains access to the DNA after breakdown of the nuclear envelope. A second mechanism of HJ resolution involves the 3′-flap endonuclease MUS81-EME1 (Mus81-Mms4 in budding yeast) dHJ formation HJ formation (40–45). HJs are a poor substrate for MUS81-EME1 and a c a Mus81-Mms4, because both prefer to cleave nicked HJs (46– 50). Because cleavage requires structural transitions at the junction point, it is possible that the relaxes the topo- bbdd bb logical constraints that inhibit efficient cleavage of intact HJs. The activity of Mus81-Mms4, which is low in S phase, is acti- a c a vated by Cdk/Cdc5-mediated phosphorylation events that occur in G2/M phase of the cell cycle (34, 35, 51, 52). The Fig. 1. Recombination pathways for the generation of single-HJ and dHJ situation in human cells is similar, except that phosphoryla- intermediates. (Left) DSBR involves end resection, strand invasion, DNA tion stimulates MUS81-EME1 to interact with the conserved synthesis (gap filling), and second-end capture to generate an intermediate GIY-YIG endonuclease SLX1-SLX4 to form an SLX1-SLX4- in which the sister chromatids are covalently joined by a dHJ. Resolution of MUS81-EME1 (SLX-MUS) complex in prometaphase (49, 53, the intermediate can generate COs (cleavage in orientations a and d or b 54). The SLX-MUS complex promotes efficient HJ resolution, and c) or NCOs (cleavage in orientations a and c or b and d). (Right) Single- with SLX1-SLX4 introducing the initial nick at the junction end invasion involves one DNA end, which could be generated by cleavage of a stalled replication fork. End resection and strand invasion lead to the and MUS81-EME1 catalyzing the second incision in the op- formation of a D-loop structure similar to that produced in DSBR. However, posing strand (49). The two cuts occur by a concerted nick and in this instance, there is no second end for capture, and instead, the invading counternick mechanism. 3′ end serves to prime DNA synthesis and form a single HJ. Resolution can Previous biochemical studies of the actions of GEN1 used syn- give rise to a CO (cleavage in orientation a) or NCO (cleavage in orientation thetic HJ substrates formed by annealing four partially comple- b). DSB, double-strand break. mentary or that contain inverted repeat sequences that extrude to form cruciform structures, the base of which mimics a single HJ. Although these model substrates have (Fig. 2B) (24, 25). Although substrate binding occurs in a sequence- provided many important insights into the functions of these en- independent manner, cleavage occurs preferentially at the se- zymes (27, 30, 31), they may not fully represent all aspects of re- ′ A G ′ quence 5 - /TTT /C-3 and generates a pair of nicked duplexes combination intermediates that arise in vivo. We therefore (26). Because of the symmetry of the cleavage reaction, the generated two physiological HJ substrates for additional analysis of resulting nicks can be joined by DNA ligase. the resolution reactions mediated by GEN1: (i) -based The search for eukaryotic equivalents to the RuvC HJ resol- recombination intermediates (α-structures) formed by RecA pro- vase was hindered by the lack of sequence and structural simi- tein-mediated strand exchange (26, 55, 56) and (ii) plasmid-based larities but culminated in the identification of Yen1 and GEN1 substrates containing dHJs (57, 58). from yeast and humans, respectively (27). GEN1/Yen1 belong to the XPG/Rad2 family of 5′-flap endonucleases (28, 29), whereas Resolution of Recombination Intermediates Containing RuvC is related to the retroviral superfamily (25). Single HJs by GEN1 Like RuvC, GEN1 promotes HJ resolution by a coordinated RecA-mediated strand exchange between gapped circular nick and counternick mechanism, but it is currently unknown pDEA-7Z DNA and 3′ 32P-labeled linear duplex pDEA2 DNA whether the eukaryotic HJ resolvases exhibit a defined se- produces recombination intermediates (α-structures) containing a quence specificity (30, 31). GEN1 also promotes symmetrical single HJ (26). The strand exchange reaction proceeds through incisions across the junction, generating nicked duplex products 1,536 bp of homology until it reaches a heterologous block that is that can be ligated. Recently, two crystal structures of active 643 bp in length (Fig. 3A). The resulting products are stable and site-containing truncated forms of GEN1 bound to DNA were were previously used for detailed analysis of HJ resolution by

444 | www.pnas.org/cgi/doi/10.1073/pnas.1619790114 Shah Punatar et al. Downloaded by guest on September 29, 2021 AB INAUGURAL ARTICLE

E. coli RuvC T. thermophilus RuvC bound to HJ

CD BIOCHEMISTRY

H. sapiens GEN1 bound C. thermophilum GEN1 bound non-productively to HJ arm to HJ resolution products

Fig. 2. 3D crystal structures of the RuvC and GEN1 HJ resolvases. Each structure depicts two monomeric subunits: one colored blue, and the other colored magenta. Active site residues are illustrated in ball and stick format and colored in lime and orange. Metal bound in the active site are shown as yellow spheres. The Protein Data Bank ID codes used to generate these structures are as follows: (A) E. coli RuvC, 1HJR; (B) T. thermophilus RuvC–HJ complex, 4LDO; (C) Homo sapiens GEN1 bound to duplex arm, 5T9J; and (D) C. thermophilum GEN1 bound to the nicked duplex products of HJ resolution, 5CO8.

RuvC (13, 56). They, therefore, provide good model substrates for Resolution of dHJs by GEN1 the analysis of GEN1 activity. To determine whether GEN1 acts on dHJs, we prepared a When the α-structures were treated with full-length human GEN1 plasmid substrate that contains two single HJs separated by 32 protein, we observed their efficient resolution into P-labeled 165 bp (Fig. 4A) (57). Previous studies have shown that nucleases, 32 gapped linear and P-labeled nicked circular products consis- such as T7 endonuclease I, resolve this dHJ substrate to pro- 32 tent with resolution in orientation b − b and P-labeled linear duce a large circle of 881 bp, representative of a CO, or two dimer DNA consistent with resolution in orientation a − a (Fig. small circles of 465 and 416 bp that represent the NCOs (57) as 3 B, lanes d–fandC, lanes d–h).Resolutionoccurredineither showninFig.4B. The large circle is often seen as two bands, of two possible orientations at a ratio of ∼1:1 (note that the and these forms are thought to represent distinct topological linear dimer product has two 32P labels, and therefore, this forms because restriction enzyme cleavage converts them to a product band is twice as intense as the nicked circular and single band (57). When the dHJ substrate was treated with GEN1, gapped linear DNA products). The products made by GEN1 we observed the formation of the expected COs and NCOs (Fig. 4 were indistinguishable from those made by E. coli RuvC (Fig. 3 C,lanesc–gandD, lanes c–g). B, lane b and C, lane b) (26). We did not observe cleavage by In contrast to GEN1, RuvC protein did not cleave the dHJ nuclease-dead GEN1E134A,E136A protein as observed previously substrate (Fig. 4E). This lack of cleavage may result from tor- with -based HJs (31). These results confirm that sional constraints that prohibit the conversion of the HJs into GEN1 acts as a prototypic HJ resolvase on single-HJ recombination the open configuration required for HJ cleavage (16, 17). Al- intermediates made by RecA protein. ternatively, it may be because of RuvC’s stringent requirement

Shah Punatar et al. PNAS | January 17, 2017 | vol. 114 | no. 3 | 445 Downloaded by guest on September 29, 2021 * A * linear dimer α-structure

a-a a resolution * * b b gapped linear a b-b + *

*

nicked circle

BC GEN1 (nM) GEN1 (1 nM)

- RuvC 0 0.25 0.5 1 - RuvC 0 1 2.5 5 10 15 Time (min)

α-structure α-structure

linear dimer linear dimer nicked circle nicked circle linear & linear & gapped linear gapped linear abcdef abcdefgh

Fig. 3. Resolution of single-HJ recombination intermediates by GEN1. (A) Schematic showing the α-structure recombination intermediate, in which two DNAs are linked by an HJ, and the products of nucleolytic resolution. The α-structures are made by RecA-mediated homologous pairing and strand exchange between gapped circular and 3′ 32P-labeled linear duplex DNA. Strand exchange proceeds through 1,536 bp of homology up to a heterologous block (green boxes; 643 bp in length). HJ resolution in orientation a − a produces 32P-labeled linear dimer DNA, whereas cleavage in orientation b − b gives rise to 32P-labeled gapped linear DNA and 32P-labeled nicked circular DNA. *3′ 32P-labeled termini. (B and C) Resolution of α-structure recombination intermediates by GEN1. Reactions contained the indicated concentrations of GEN1 or E. coli RuvC HJ resolvase (10 nM) as a control. Products were analyzed by agarose followed by autoradiography.

A G for the sequence 5′- /TTT /C-3′ for efficient HJ cleavage (26). α-structures were used to determine the sequence preference Consistent with the latter possibility, DNA sequence analyses used by RuvC (26). revealed that there were three sequences present within the Using RuvC as a control, we carried out large-scale resolution 32 153-bp region of homology that are identical to RuvC’spre- reactions with P-labeled α-structures and compared the prod- ferred sequence. However, none of these consensus sequences ucts with those produced by GEN1, as analyzed by denaturing reside at the boundaries of homology/heterology where the gel electrophoresis and visualized by autoradiography. In the absence of heat-induced , 32P-labeled frag- junctions are located the topologically constrained dHJ ∼ substrate. ments of 1.5kbinlengthwereobservedwithbothRuvCand GEN1 (Fig. 5A, lanes b and f), consistent with resolution oc- Sequence Preference for HJ Cleavage by GEN1 curring at sequences located near the heterologous block. α The ability of GEN1 to cleave the dHJ substrate, whereas RuvC However, when the -structures were incubated at 55 °C for 1 h could not, indicated to us that HJ cleavage by GEN1 was un- to induce limited spontaneous branch migration before treat- ment with either RuvC or GEN1, we observed a series of dis- likely to be limited by strict sequence constraints. Instead, we tinct 32P-labeled DNA fragments by denaturing PAGE (Fig. reasoned that GEN1 might act more like the bacteriophage 5A, lanes d and h). The distinct cleavage pattern generated by resolvases T4 endonuclease VII or T7 endonuclease I or the RuvC is caused by HJ resolution at sites that correspond to its archaeal resolvase Hjc, all of which exhibit little or no sequence consensus sequence (26). In contrast, GEN1 generated a sig- specificity (59, 60). nificantly greater number of distinct 32P-labeled DNA prod- The sequence specificity of GEN1 was investigated using the ucts. There are two possibilities to explain this result: (i)GEN1 α -structure DNA substrate. A useful feature of this natural re- has a weak sequence preference, which would lead to cleavage combination intermediate is that, by gentle heating at 55 °C, the at a greater number of sites than observed with RuvC, or HJs can be induced to branch migrate and become distributed (ii) GEN1 has no sequence specificity and simply cleaves HJs when over a large DNA sequence. The migrated junctions, therefore, branch migration has temporarily paused because of sequence/ provide a greater choice of potential cleavage sites than that energy constraints. To test the latter possibility, we included T4 offered by oligonucleotide-based HJs. In previous studies, these endonuclease VII in our analyses, because this resolvase has little

446 | www.pnas.org/cgi/doi/10.1073/pnas.1619790114 Shah Punatar et al. Downloaded by guest on September 29, 2021 ABT7 EndoI INAUGURAL ARTICLE M .01 .02 units bp 881 bp 1517 1200 1000 CO CO 800 dHJ 500 NCO NCO 400

465 bp+ 416 bp

CDGEN1 (nM) GEN1 (20 nM) M -2.55102050 M - 5 10 20 30 60 Time bp (min) 1517 1200 1000 CO CO 800 dHJ dHJ 500 NCO 400 NCO BIOCHEMISTRY

abcdefg abc de f g

E RuvC (nM) M bp - T7 Endo I - 1550 100 250 500 1517

1200 1000 CO 800 dHJ

500 NCO 400

abcde f g hi

Fig. 4. Resolution of dHJs by GEN1. (A) Schematic diagram of the dHJ substrate and the products of resolution. Cleavage of both HJs in the same orientation generates NCOs (circles of 465 and 416 bp), whereas cleavage in the opposite orientation gives rise to a CO (a single 881-bp circle). (B) Resolution of dHJs by T7 endonuclease I. (C and D) Resolution of dHJs by GEN1. (E) Analysis of the actions of RuvC on the dHJ substrate. Products were separated by agarose gel electrophoresis and stained with SYBR gold.

sequence specificity and would, therefore, be expected to cut primer extension analysis using three 5′ 32P-labeled primers at sites similar to GEN1. We did not observe any significant sim- located at various distances from the initial junction point (Fig. ilarities between the cleavage patterns generated by GEN1 and 6A, primers P1–P3). Cleavage sites close to the heterologous T4 endonuclease VII (Fig. 5B,comparelaneewithlaneg). block were identified using nonbranch-migrated substrates, These results indicate that the cleavage products generated by whereas sites farther away were mapped using branch-migrated GEN1 are not caused by the accumulation of HJs at certain α-structures. In each case, the precise sites of resolution were sequences during branch migration. Instead, it seems that mapped compared with 35S-labeled A, C, G, and T sequencing GEN1 has a sequence preference that is less stringent than that ladders produced from the same primers. Three representative observed with RuvC. primer extension reactions are shown in Fig. 6B.Intotal, To map the sites of HJ cleavage by GEN1, resolution reac- 24 sites of cleavage were mapped, and the sequences in which tions were performed, and the products were denatured for cleavage occurred were aligned and displayed using the WebLogo

Shah Punatar et al. PNAS | January 17, 2017 | vol. 114 | no. 3 | 447 Downloaded by guest on September 29, 2021 results are consistent with a role for GEN1 in resolving re- A RuvC GEN1 combination intermediates in prometaphase in preparation for kb --0 30 60 0 30 60 branch migration (min) the segregation of the sister chromatids by the mitotic spin- 3.0 2.0 dle (39, 61). Although GEN1 and RuvC are structurally unrelated, there 1.5 are a number of biochemical similarities in terms of their mechanism of action: (i) the two proteins are structure-selective endonucleases, (ii) they promote HJ resolution by formation of 1.0 a homodimer–HJ complex, and (iii) resolution occurs by a mechanism that introduces symmetrical incisions across the 0.9 junction, which releases ligatable nicked duplex products. However, unlike RuvC, which preferentially cleaves HJs at the A G preferred DNA sequence 5′- /TTT /C-3′,GEN1exhibitsa weak specificity, with incisions being introduced preferen- 0.8 tially between two G residues, in a region that is T-rich. This general lack of sequence specificity may relate to observations 0.7 indicating that GEN1 acts alone, in contrast to RuvC, which associates with the RuvAB branch migration complex (62, 63) or reflects the essential need to resolve all single HJs and dHJs abcdefgh (and potentially, other DNA secondary structures) that persist until the late stages of the cell cycle. In this regard, GEN1 is T4 functionally similar to the bacteriophage resolvases T4 endo- B RuvC GEN1 Endo VII nuclease VII and T7 endonuclease I, which ensure the resolu- branch kb - - + - + - + tion of all DNA secondary structures before the packaging of migration DNA into the phage heads (64, 65). By analogy, GEN1’sbroad 3.0 substrate specificity and sequence flexibility ensure that per- 2.0 sistent sister chromatid bridges are resolved before chromo- some segregation and cell division (31, 61). 1.5 Materials and Methods Proteins. C-terminally FLAG-His–tagged human GEN1 and nuclease-dead GEN1E134A,E136A proteins were purified from after overexpression from a galactose-inducible promoter (31). E. coli RuvC was 1.0 purified as described (66). T7 endonuclease I, T4 endonuclease VII, E. coli RecA, and terminal transferase were purchased from New England Biolabs. Proteinase K was purchased from Sigma, and the Klenow fragment of DNA − 0.9 polymerase I (exo ) was purchased from Thermo Scientific.

DNA. Recombination intermediates containing a single HJ (α-structures) were generated by RecA-mediated strand exchange between gapped circular 0.8 pDEA-7Z (15 μM) and 3′ 32P end-labeled linearized pDEA2 plasmid DNA (15 μM) (26, 67). After 30 min at 37 °C, the reactions were stopped and abcdefg deproteinized, and the DNA was purified through a 3.5-mL Sepharose CL-2B column equilibrated with 20 mM Tris·HCl, pH 8.0, 5 mM MgCl , 1 mM DTT, Fig. 5. Sequence preference for HJ cleavage by GEN1. (A) Large-scale res- 2 and 100 μg/mL BSA. olution reactions (200 μL) contained 32P-labeled α-structures with either RuvC (10 nM; lanes b–d) or GEN1 (1 nM; lanes f–h). Before the addition of For some experiments, the HJ was migrated away from the heterologous protein, spontaneous branch migration of the HJ was induced by incubation block by incubation of the column-purified intermediates at 55 °C. The at 55 °C for the times indicated. 32P-labeled resolution products were sep- branch migration reaction was stopped by chilling on ice, and the re- arated by denaturing PAGE and autoradiography. (B) Comparison of the combination intermediates were used immediately. cleavage profiles by GEN1 (1 nM), RuvC (10 nM), and T4 endonuclease VII dHJ-containing substrates were prepared as described (57). (0.5 U/μL). Reactions were carried out as in A. The recombination interme- 32 diates were branch-migrated where indicated (+). 32P-labeled products were Endonuclease Assays. Reactions (10 μL) containing 3′ P-labeled α-structures analyzed by denaturing PAGE and autoradiography. (1.5 nM) and purified enzymes were incubated at 37 °C for 10 min unless otherwise stated. The cleavage buffer was 50 mM Tris·HCl, pH 8.0, 1 mM

MgCl2, 1 mM DTT, and 100 μg/mL BSA. For reactions containing RuvC or T4 endonuclease VII, the cleavage buffer was adjusted to 10 mM MgCl2.Be- tool (weblogo.berkeley.edu). This analysis revealed that cleavage fore use, GEN1 was diluted in buffer containing 50 mM Tris·HCl, pH 7.5, occurred preferentially but not exclusively between two G resi- 250 mM NaCl, 10% (vol/vol) glycerol, 1 mM DTT, and 0.05% Nonidet-P40. dues and that T residues were common at positions −3 and −4 All other proteins were diluted in 50 mM Tris·HCl, pH 8.0, 100 mM NaCl, (Fig. 6C, Upper). However, a comparison with 17 cleavage sites 10% (vol/vol) glycerol, 1 mM DTT, and 100 μg/mL BSA. Reactions were produced by RuvC (26) (Fig. 6C, Lower) emphasizes that GEN1 stopped by addition of 2 μL6× gel loading dye (New England Biolabs), and fails to exhibit a strong preference for a defined consensus the products were analyzed by 1.1% agarose gel electrophoresis in the sequence. presence of 0.5 μg/mL ethidium bromide. DNA was visualized by either In conclusion, we have extended our biochemical analysis ethidium bromide staining or autoradiography. μ of the GEN1 HJ resolvase using DNA substrates that better ForanalysisbydenaturingPAGE, reactions were scaled up to 200 L. Reactions were stopped and deproteinized by the addition of 0.8% SDS, represent recombination intermediates that are formed in · α 20 mM Tris HCl, pH 7.4, and 2 mg/mL proteinase K followed by incubation vivo. Using RecA-generated -structures that contained single at 37 °C for 10 min. The DNA was then phenol-chloroform–extracted, HJs and a plasmid-based substrate that contains two HJs ethanol-precipitated, and resuspended in 5 μL loading dye [80% (vol/vol) separated by 165 bp, we found that GEN1 promoted the effi- formamide, 0.1% bromophenol blue, 0.1% xylene cyanol]. Samples were cient resolution of both single-HJ and dHJ structures. The denatured by heating at 95 °C for 3 min and analyzed by electrophoresis

448 | www.pnas.org/cgi/doi/10.1073/pnas.1619790114 Shah Punatar et al. Downloaded by guest on September 29, 2021 A B INAUGURAL ARTICLE - GA TC RuvC GEN1

P1P2 5’ 3’ 5’ P3

3’ α-structure

C abcdef g incision site GEN1 2 bits 1 BIOCHEMISTRY

-1-1-2-3-4-5-6 +1 +2 +3 +4 +5 +6 2 RuvC incision site bits 1

-1-1-2-3-4-5-6 +1 +2 +3 +4 +5 +6 Nucleotide position relative to incision site

Fig. 6. Identification of the preferred sites of HJ cleavage by GEN1. (A) Schematic diagram indicating the α-structure and primers (P1–P3) used to map the sites of cleavage. (B) Reactions were carried out as described for Fig. 5, except that the α-structures were generated from unlabeled DNAs, and the resolution products were then subjected to primer extension using 5′ 32P-labeled primers. The products of primer extension (lanes a, b, and g) were analyzed by de- naturing PAGE alongside 35S-labeled A, C, G, and T sequencing ladders. (Top) Cleavage sites close to the heterologous block were identified using nonbranch- migrated substrates, whereas (Middle and Bottom) sites farther away were mapped using branch-migrated α-structures. Three different oligonucleotides were used to map the cleavage sites: (Top) primer P1, (Middle) primer P2, and (Bottom) primer P3. (C, Upper) Twenty-four cleavage sites produced by GEN1 were aligned, and the occurrence of nucleotides A, C, G, and T at each position was determined and displayed using WebLogo. (C, Lower) For comparison, the RuvC cleavage sequence is also indicated.

through 4% (wt/vol) polyacrylamide gels containing 7 M urea. Gels were oligonucleotides and the Klenow fragment of DNA polymerase I as described dried, and the DNA was visualized by autoradiography. (26). The sequences of the primers are Reactions (20 μL) containing dHJ DNA (2.15 nM) were incubated for Primer P1: 5′-GGACCGCTGATCGTCACGGCGATTTATG-3′, 30 min at 37 °C, using essentially the same conditions as described above and stopped by addition of 1% SDS, 20 mM EDTA, and 100 μg/mL pro- Primer P2: 5′-CTTTTGCTCACATGTTCTTTCCTGCGTTATC-3′, and teinase K. The DNA was deproteinized by phenol-chloroform extraction, Primer P3: 5′-GTATTCTATAGTGTCACTAAATAGCTTGGC-3. ethanol-precipitated, and resuspended in 10 mM Tris·HCl, pH 8.0, and 0.1 mM EDTA. The reaction products were then separated by electro- Primers P1–P3 were also used to generate 35S-labeled DNA sequencing phoresis through 2% (wt/vol) agarose and visualized by staining with ladders from plasmid pDEA2 using a Sequenase Kit, version 2.0 (Affymetrix). SYBR Gold (Life Technologies). DNA products were analyzed on 6% (wt/vol) polyacrylamide gels containing 7 M urea followed by autoradiography of the dried gels. Sites of cleavage Primer Extension Analysis. Large-scale (200 μL) resolution reactions con- were aligned and analyzed using WebLogo (weblogo.berkeley.edu). taining α-structures were performed as above, except that unlabeled recombination intermediates (1.5 nM) were used. The products were ACKNOWLEDGMENTS. This work was supported by the Francis Crick In- deproteinized by addition of 0.8% SDS, 20 mM Tris·HCl, pH 7.4, and stitute (which receives core funding from Cancer Research United Kingdom, 2 mg/mL proteinase K followed by incubation at 37 °C for 10 min. DNA the Medical Research Council, and Wellcome Trust Grant FC10212). Addi- was phenol-chloroform–extracted, ethanol-precipitated, and resus- tional funding was provided by European Research Council Grant ERC-ADG- pended in 10 mM Tris·HCl, pH 8.0, and 1 mM EDTA. Primer extension 666400 and the Louis-Jeantet Foundation. M.J.M. and Y.W.C. were recipients analysis of the resolution products was then performed using 5′ 32P-labeled of Marie Curie and European Organization fellowships.

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