Holliday Junction-Containing DNA Structures Persist in Cells Lacking Sgs1 Or Top3 Following Exposure to DNA Damage

Holliday junction-containing DNA structures persist in cells lacking Sgs1 or Top3 following exposure to DNA damage

Hocine W. Mankouria,b,1, Thomas M. Ashtona,1, and Ian D. Hicksona,b,2

aWeatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom; and bNordea Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen N, Denmark

Edited* by Stephen C. Kowalczykowski, University of California, Davis, CA, and approved February 14, 2011 (received for review September 22, 2010)

The Sgs1–Rmi1–Top3 “dissolvasome” is required for the mainte- HRR is a conserved cellular process that allows cells to copy nance of genome stability and has been implicated in the process- genetic information from a homologous sequence, and is re- ing of various types of DNA structures arising during DNA required for the efﬁcient repair of DNA breaks and ssDNA gaps plication. Previous investigations have revealed that unprocessed that can arise during S-phase due to DNA damage, discon- (X-shaped) homologous recombination repair (HRR) intermediates tinuities in DNA replication, or the impediment of replication persist when S-phase is perturbed by using methyl methanesulfo- fork (RF) progression. Evidence that BLM functions in HRR is nate (MMS) in Saccharomyces cerevisiae cells with impaired Sgs1 suggested by the fact that BS cells demonstrate elevated levels of or Top3. However, the precise nature of these persistent DNA struc- mitotic recombination, sister chromatid exchanges, and genome instability (1). Furthermore, BLM (either alone or in conjunction tures remains poorly characterized. Here, we report that ectopic α expression of either of two heterologous and structurally unrelated with hTOPOIII , hRMI1, and hRMI2) can resolve different types Escherichia coli of HRR intermediates in vitro, such as D-loops and single or Holliday junction (HJ) resolvases, RusA or human – GEN11-527, promotes the removal of these X-structures in vivo. double Holliday junctions (HJs; refs. 6, 7, and 16 21). Mutation of SGS1 or the Schizosaccharomyces pombe ortholog of BLM/SGS1, Moreover, other types of DNA replication intermediates, including +

called rqh1 , also causes genome instability, hyperrecombination, GENETICS stalled replication forks and non-HRR-dependent X-structures, are and sensitivity to DNA-damaging agents (8, 22–25), and Sgs1, like refractory to RusA or GEN11-527, demonstrating specificity of these BLM, is able to unwind HJs in vitro (26). Furthermore, un- HJ resolvases for MMS-induced X-structures in vivo. These data processed HRR intermediates (X-structures) have been directly suggest that the X-structures persisting in cells with impaired observed in methyl methanesulfonate (MMS)-treated sgs1, top3, Sgs1 or Top3 contain HJs. Furthermore, we demonstrate that Sgs1 and rmi1 mutants using 2D gel electrophoresis (27–30). Because directly promotes X-structure removal, because the persistent many of the deleterious phenotypes of sgs1 or rqh1 mutants can be fi structures arising in Sgs1-de cient strains are eliminated when suppressed by the mutation of genes involved in the early steps of Sgs1 is reactivated in vivo. We propose that HJ resolvases and HRR (e.g., RAD51 in S. cerevisiae and rhp51+ in S. pombe; refs. Sgs1–Top3–Rmi1 comprise two independent processes to deal with 31–35), it is likely that these phenotypes are, at least in part, due to HJ-containing DNA intermediates arising during HRR in S-phase. unregulated or incomplete HRR. Although X-shaped HRR structures have been detected by DNA repair | RecQ | helicase | topoisomerase using 2D DNA gel electrophoresis in S. cerevisiae cells with impaired Sgs1, Top3, or Rmi1 (27–30), it is presently unknown he RecQ family of DNA helicases is required for the main- whether different types of DNA structures arise in cells deficient Ttenance of genome stability in all organisms. Mutations in at in these different proteins. Because abolition of Sgs1 activity (or least three (of five) human RecQ helicases is associated with an just its helicase activity) can suppress the poor growth pheno- increased predisposition to the development of cancer and/or types of top3 or rmi1 mutants (8, 13, 14, 36), it has been proposed premature aging (1). Mutations in BLM cause Bloom’s syndrome that Sgs1 may create a DNA intermediate that is toxic in cells (BS), which is associated with increased cancer predisposition, lacking Top3 or Rmi1. One proposal is that the convergent whereas mutations in WRN or RECQ4 cause distinct disorders branch migration of double HJs (dHJs) by Sgs1 creates a hemi- (Werner’s syndrome and Rothmund–Thomson syndrome, re- catenane structure that can only be resolved by Top3 (in con- spectively) characterized by premature aging and some de- junction with Rmi1), in a process known as “dHJ dissolution” velopmental abnormalities. In Saccharomyces cerevisiae, there is (18, 20, 21). However, in vivo evidence for this process is lacking, only one RecQ helicase, called Sgs1. As a consequence, this because the 2D gel methodology cannot definitively distinguish organism has proved particularly useful for genetic analyses, as between different types of joint DNA molecules such as HJs and well as for characterization of the defects arising in cells lacking hemicatenanes. RecQ helicases (2). The X-structures arising in MMS-treated sgs1 mutants have “ ” Sgs1 is thought to be the ortholog of BLM, based on a number been proposed to be pseudo-HJs, consisting of a region of of observations. First, BLM is the only human RecQ helicase hemicatenated nascent DNA and concomitant single-stranded that shares the same structural domain architecture as Sgs1. regions of parental DNA (27). This conclusion was based on the Second, both BLM and Sgs1 associate with conserved interacting in vitro characterization of X-structures, including their ability to partners that are apparently unique for BLM in human cells. More specifically, BLM exists in a complex with a type IA topoisomerase, hTOPOIIIα, and two oligonucleotide/oligosaccharide- Author contributions: H.W.M. and I.D.H. designed research; H.W.M. and T.M.A. per- binding (OB)-fold containing proteins, hRMI1 and hRMI2 (3– formed research; H.W.M. contributed new reagents/analytic tools; H.W.M., T.M.A., and 7); Sgs1 associates with the yeast orthologs of these proteins, I.D.H. and analyzed data; and H.W.M. wrote the paper. Top3 and Rmi1 (8–14). Together, these proteins likely act co- The authors declare no conflict of interest. operatively as a “dissolvasome” that processes multiple kinds of *This Direct Submission article had a prearranged editor. DNA structures arising during DNA replication, repair, recom- 1H.W.M. and T.M.A. contributed equally to this work. bination, and mitosis (15). 2To whom correspondence should be addressed. E-mail: [email protected]. One cellular process in which BLM and Sgs1 have both been This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. strongly implicated is homologous recombination repair (HRR). 1073/pnas.1014240108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1014240108 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 branch migrate (even in the presence of Mg2+, which generally medium containing MMS or HU at 30 °C for 1 h. Cultures were inhibits HJ migration), their apparent resistance to the RuvC HJ then incubated at 37 °C for a further 6 h to promote robust HJ resolvase, and their partial sensitivity to ssDNA nucleases (27). resolvase activity. Genomic DNA was extracted by using the MMS also causes increased interhomolog X-structures in sgs1 hexadecyltrimethylammonium bromide (CTAB) method to re- diploid cells (37), although the precise nature of these in- strain branch migration of joint (X-shaped) molecules (42). Af- terchromosomal joint DNA molecules remains to be determined. ter 7-h exposure to 0.033% MMS, all DNA replication inter- Interestingly, a recent study demonstrated that interchromosomal mediates had disappeared from ARS305 in WT cells expressing dHJs arise during double-strand break (DSB) repair in mitotic GFP, consistent with the completion of DNA replication at this diploid cells (38). Furthermore, the turnover of these structures region of the genome by this stage. In sgs1 mutants expressing was altered in sgs1 mutants, consistent with a possible role for Sgs1 GFP, bubbles and Y arcs had also disappeared, but, consistent in the prevention and/or direct processing of HJs in vivo (38). In- with previous findings (27, 28, 30), persistent X-structures at ter-sister chromatid X-structures were also identified as inter- ARS305 were observed (Fig. 1B). Previous analyses have iden- mediates of DSB repair in this study, although whether these tified these structures to be Rad51-dependent HRR inter- contained HJs or not could not be determined. Therefore, despite mediates (27). Interestingly, these MMS-induced X-structures several independent studies demonstrating the existence of un- were not detectable in sgs1 cells expressing RusA and GEN11-527 processed X-shaped DNA structures in sgs1 mutants under a (Fig. 1B). All strains examined exhibited similar mid-S-phase number of different experimental conditions, it remains unknown FACS profiles (Fig. S1), suggesting that the observed differences whether these comprise a common type of DNA structure or not. on 2D gels were not attributable to any differences in cell cycle Indeed, the precise nature and abundance of the X-structures progression or MMS-induced checkpoint arrest. We conclude could vary depending on the context (e.g., in DSB repair vs. the that expression of RusA and GEN11-527 leads to a marked re- repair of replication-associated lesions). Consistent with this pro- duction in the level of unprocessed HRR intermediates in MMS- posal, RecQ helicases can process a variety of different DNA treated sgs1 cells. structures in addition to HJs in vitro, and the BLM–hTOPOIIIα– hRMI1–hRMI2 and Sgs1–Top3–Rmi1 complexes probably act as Stalled RFs and Non-HRR-Dependent X-Structures Are Refractory to DNA structure-specific “dissolvasomes” in vivo (15). HJ Resolvases. Biochemical analysis of RusA and GEN11-527 has We sought to characterize the composition of MMS-induced, demonstrated robust, and substrate-specific, HJ resolvase activity replication-associated X-structures by promoting their resolution for these enzymes in vitro (40, 43–47). However, unlike RusA in vivo using heterologous enzymes with well-characterized in (40), GEN11-527 also exhibits some ability to cleave RF-like vitro substrate specificities. Here, we report that ectopic ex- structures in vitro (44, 45). To assess whether the RusA and pression of either of two heterologous HJ resolvases, Escherichia GEN11-527 HJ resolvases demonstrate specificity for MMS- coli RusA or human GEN11-527, enhances the in vivo removal of induced X-structures in vivo, we examined whether they could the MMS-induced X-structures in cells impaired for Sgs1 or cleave other types of replication intermediates detectable using Top3. We propose that the X-structures persisting in both of 2D gels. To test this, we directly analyzed the fate of DNA these mutants contain one or more HJs. Furthermore, because replication intermediates arising at ARS305 in response to HU. X-structure processing in Sgs1-deficient strains can also be pro- In the presence of 0.2 M HU, we observed robust ARS305 or- moted by the reactivation of Sgs1 in vivo, we propose that the igin firing (as revealed by the presence of bubbles, Y arcs, and Sgs1 complex directly processes HJ-containing DNA structures origin-associated X-structures) in WT cells and sgs1 mutants arising during HRR, facilitating their elimination. expressing GFP (Fig. 1C). Previous analyses have demonstrated that the origin-associated X-structures are normal DNA replica- Results tion intermediates that are not dependent on DNA damage or Heterologous HJ Resolvases Diminish the MMS-Induced X-Structures HRR proteins for their formation (42). Furthermore, the for- in sgs1 Mutants. To determine the specific nature of the MMS- mation and disappearance of these structures is unaffected in sgs1 induced X-structures arising in haploid sgs1 mutants, we in- mutants (27). ARS305 origin-firing was also unaffected in sgs1 vestigated whether any proteins with well-characterized in vitro mutants expressing RusA or GEN11-527 (Fig. 1C). Therefore, substrate specificities could diminish the level of the X-structures ectopic expression of these HJ resolvases does not noticeably when ectopically expressed in vivo. Interestingly, expression of affect any early DNA replication intermediates arising at ARS305. two heterologous HJ resolvases, E. coli RusA and a fragment of Cultures were then incubated at 37 °C for a further 6 h in the human GEN1, GEN11-527, have been demonstrated to suppress presence of 0.2 M HU, as described in Fig. 1B for MMS-treated the MMS- and UV-sensitivity of S. pombe rqh1 mutants (39–41), cells. We observed that the levels of the different DNA replication suggesting that unprocessed HJs could be directly responsible intermediates detectable after 1 h in 0.2 M HU were substantially for various rqh1 phenotypes. We therefore examined whether reduced in all strains examined by 7 h in the NcoI–NcoI 5-kb expression of these HJ resolvases could reduce the level of ARS305 fragment under analysis. The main species of DNA the X structures in MMS-treated sgs1 mutants using 2D gel replication intermediates present in the NcoI–NcoI fragment now electrophoresis. consisted of large Ys and origin-associated X-structures (Fig. 1C). To permit inducible expression of heterologous proteins and Enhancing the exposure of these data revealed that low levels of their efficient targeting to the nucleus, NLS–GFP (control), intact bubble structures were also detectable in these extracts NLS–RusA–GFP, and NLS–GEN11-527-GFP proteins were (Fig. S2). Furthermore, because these DNA replication inter- subcloned into pYES2, allowing galactose-inducible protein ex- mediates were unaffected by the expression of HJ resolvases, we pression. These proteins will henceforth be denoted as GFP, conclude that RusA and GEN11-527 do not cleave RFs or origin- RusA, or GEN11-527, respectively. Proteins were induced (by the associated (Rad51-independent) X-structures in vivo. addition of 2% galactose) during α-factor arrest and throughout To test whether the reduction in replication intermediates the subsequent analyses. Samples of wild-type (WT)–GFP, sgs1– in the NcoI–NcoI fragment at the 7-h time point was due to GFP, sgs1–RusA, and sgs1–GEN11-527 cells were taken at specific RFs migrating outside of the 5-kb fragment under analysis, or intervals following the release from G1 arrest to observe DNA whether HU-induced RF collapse had occurred, we processed replication intermediates on 2D gels originating from the early- the DNA samples using different restriction enzymes that permit firing replication origin, ARS305. Parallel analyses, using the analysis of replication intermediates in the region just distal to – same G1-arrested/galactose-induced culture, were performed to the NcoI NcoI fragment (see Fig. 1D for a diagrammatic rep- directly compare the cellular responses to MMS and to the DNA resentation). Analysis of a BamHI–SpeI ARS305 fragment replication inhibitor, hydroxyurea (HU; Fig. 1A). To permit ef- revealed that RFs and origin-associated X-structures were deficient origin firing, cells were released from G1 arrest into tectable after 7 h of 0.2 M HU treatment, suggesting that RF

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1014240108 Mankouri et al. Downloaded by guest on October 2, 2021 GENETICS

Fig. 1. Expression of HJ resolvases reduces the levels of MMS-induced X-structures in sgs1 mutants. (A) Outline of the experimental protocol. Cultures

were released from G1 arrest at 30 °C and incubated at 37 °C after 1 h to promote robust HJ resolvase activity. (B) Analysis of DNA structures in WT–GFP, sgs1–GFP, sgs1–RusA, and sgs1–GEN11-527 strains after exposure to MMS. DNA replication intermediates were analyzed in a 5-kb NcoI–NcoI ARS305 fragment by 2D gel electrophoresis after 7-h exposure to 0.033% MMS. The key on the right denotes DNA structures that can be identiﬁed by the 2D gel technique. The arrow denotes X-shaped structures present in sgs1 mutants. (C) Analysis of DNA structures after exposure to HU. DNA replication intermediates were analyzed as in B, except after 1 h or 7 h of exposure to 0.2 M HU. (D) A schematic diagram of ARS305 highlighting the relevant restriction sites. The DNA extracts from cells exposed to 0.2 M HU for 7 h (C) were digested with BamHI and SpeI and then analyzed by 2D gel electrophoresis.

integrity (and ongoing RF progression) remained largely intact HJ Resolvases Do Not Prevent X-Structure Accumulation in sgs1 in the presence of HU (Fig. 1D). Flow cytometric data also con- Mutants. Next, we analyzed the mechanism of X-structure re- firmed that DNA replication proceeded, albeit very slowly, in all moval by HJ resolvases in sgs1 mutants. We analyzed DNA strains in the presence of 0.2 M HU (Fig. S1). Furthermore, the structures at ARS305 after 3 h of 0.033% MMS treatment be- replication intermediates detectable at this time were again un- cause the ARS305 fragment under analysis is generally fully affected by expression of HJ resolvases in sgs1 mutants (Fig. 1D). replicated in the presence of MMS by 2–3 h, which coincides These data indicate that the HJ resolvases analyzed here do not with the peak levels of Rad51-dependent X-structures in sgs1 cleave, or prevent the maturation of, other types of DNA rep- mutants (27, 28, 30). After 3-h exposure to 0.033% MMS, we lication intermediates detectable at ARS305. This result suggests observed that X-structures were detectable in sgs1 mutants that RusA and GEN11-527 retain their biochemically character- expressing GFP, RusA, or GEN11-527 (Fig. 2). However, quan- ized substrate specificities (40, 43–47) when expressed in S. cer- tification of relative X-structure intensity revealed a small re- evisiae and therefore implies that the MMS-induced X-structures duction in the levels of X-structures in sgs1 cells expressing RusA contain one or more HJs, or that they arise from a HJ-containing or GEN11-527 (28% and 35%, respectively). We propose that the precursor. Furthermore, we conclude that the RAD51-independent expression of HJ resolvases is not able to strongly counteract the origin-associated X-structures do not contain HJs, in agreement rapid surge in X-structure formation that occurs in the first 2–3h with previous findings (42). of 0.033% MMS treatment in sgs1 mutants (27, 28, 30). After

Mankouri et al. PNAS Early Edition | 3of6 Downloaded by guest on October 2, 2021 Fig. 2. HJ resolvases do not prevent the accumulation of MMS-induced X- structures in sgs1 mutants. sgs1–GFP, sgs1–RusA, and sgs1–GEN11-527 strains

were released from G1 arrest into medium containing 0.033% MMS at 30 °C. Protein expression was induced during G1 arrest and throughout the subsequent incubation. After 3 h, cells were incubated at 37 °C (to promote robust HJ resolvase activity) for a further 5 h. DNA replication intermediates were analyzed around ARS305 by 2D gel electrophoresis at the indicated times, as per Fig. 1B. X-shaped DNA structures are denoted by arrows.

an additional 5-h incubation at 37 °C, we observed that X- structures were still evident in sgs1 mutants expressing GFP, but they were no longer detectable in sgs1 mutants expressing RusA or GEN11-527 (Fig. 2). We therefore propose that the majority (>65%) of sgs1 X-structures are removed by HJ resolvases only after they form. We acknowledge, however, that the 28–35% Fig. 3. HJ resolvases remove X-structures arising in sgs1 and TOP3Y356F decrease in X-structure accumulation in sgs1 strains expressing mutants. (A) Outline of the experimental protocol. (B) Protein extracts were D70N HJ resolvases could also be due to the additional cleavage of an prepared from G1-arrested sgs1–GFP, sgs1–RusA, sgs1–RusA , sgs1– X-structure precursor by these enzymes. GEN11-527, and sgs1–GEN11-527 D157A strains induced with 2% galactose, and equivalent levels of protein were resolved by SDS/PAGE. Levels of the in- Nuclease Activity of HJ Resolvases Is Required to Diminish X- dicated protein were determined by Western blotting. (C) DNA replication Structures in sgs1 Mutants. To test whether X-structure removal intermediates around ARS305 were analyzed in the indicated sgs1 strains by using HJ resolvases in sgs1 cells was dependent on there being either 2D gel electrophoresis after 5-h recovery from MMS treatment. X-shaped an extended S-phase arrest or a prolonged exposure to MMS (Figs. DNA structures are denoted by arrows. (D) HJ resolvases process X-structures arising in cells impaired for Top3. WT–GFP, WT–RusA, and WT–GEN11-527 1B and 2), we analyzed strains for their recovery from 0.033% MMS strains were transformed with pWJ1347 (GAL1–TOP3Y356F). Coexpression of treatment (Fig. 3A). Protein expression was induced during α-factor proteins was induced during G1 arrest and throughout the subsequent in- arrest (Fig. 3B) and was maintained throughout the subsequent cubation. DNA replication intermediates around ARS305 were analyzed in analyses. After 3 h of 0.033% MMS exposure, sgs1 cells were har- the indicated strains by 2D gel electrophoresis after 5 h of recovery from vested, washed, and released into drug-free medium at 37 °C. Also, to MMS treatment. investigate whether the nuclease activity of RusA and GEN11-527 is required for sgs1 X-structure removal, we compared the effects of expressing biochemically validated, nuclease-defective alleles of poor growth rate and the rapid appearance of suppressor mutations RusA (RusAD70N)andGEN11-527 (GEN11-527 D157A; refs. 44, 48, (8, 13, 14, 36). To permit the acute impairment of Top3 function in and 49; Fig. 3B). All strains examined were proficient in recovery vivo, we therefore used a previously validated, dominant-negative from MMS-induced S-phase arrest and successfully completed allele of TOP3, TOP3Y356F, that, when overexpressed, causes the DNA replication by 5 h after MMS removal, as revealed by FACS accumulation of Rad51-dependent X-structures after 2-h exposure analysis (Fig. S3).Wenote,however,thatGEN11-527 expression did to 0.033% MMS (29, 50). Consistent with our previous findings (29), cause a very small, but reproducible, delay in sgs1 S-phase progression overexpression of TOP3Y356F caused persistent MMS-induced X- inthepresenceof0.033%MMS.WeconcludethatexpressionofHJ structures that were still detectable at 5 h after recovery from resolvases does not cause, or exacerbate, any significant DNA rep- 0.033% MMS (Fig. 3D), similar to what was observed in sgs1 lication defects in sgs1 mutants, consistent with our observations that mutants (Fig. 3C). Interestingly, we observed that expression of HJ resolvases do not cleave RFs, or hinder their progression, in vivo RusA and GEN11-527 also diminished the level of TOP3Y356F X- (Fig. 1 C and D). At 5 h after the removal of MMS, we observed that structures (Fig. 3D). We conclude that heterologous HJ resolvases unprocessed X-structures were still evident at ARS305 in sgs1–GFP can remove X-structures arising in cells with impaired Sgs1 or Top3, strains (Fig. 3C). Interestingly, we again observed that sgs1 X-struc- suggesting that a common type of HJ-containing DNA structure tures were not detectable in cells expressing RusA or GEN11-527 at 5 likely persists in both of these mutants. h after the removal of MMS (Fig. 3C). Importantly, this effect was dependent on the nuclease activity of both proteins, because the Reactivation of Sgs1 Removes X-Structures in sgs1-36 Mutants. nuclease-defective alleles were indistinguishable from the GFP Having demonstrated that unprocessed HJ-containing struc- control in demonstrating prominent X-structures (Fig. 3C). tures persist in sgs1 mutants, we examined whether the normal function of Sgs1 is to prevent these structures from accumulat- HJ Resolvases also Diminish Persistent X-Structures Arising in Cells ing, or whether Sgs1 primarily acts to directly eliminate them Lacking Functional Top3. To determine whether the reduction in in vivo. We note, however, that these roles are not necessarily X-structures caused by RusA and GEN11-527 in vivo is specific for mutually exclusive. To test these possibilities, we used a validated X-structures arising in sgs1 mutants, we assessed whether these HJ temperature-sensitive mutant of sgs1, sgs1-36, that permits the resolvases could also diminish MMS-induced X-structures arising in temporary, and reversible, inactivation of Sgs1 (51). Strains were cells impaired for Top3. Although MMS-induced X-structures have grown at 25 °C, arrested in G1 at the restrictive temperature been detected in top3 deletion mutants (27), it is not feasible to grow (35 °C), and then released into drug-free medium containing large cultures of plasmid-transformed top3 mutants because of their 0.033% MMS. At the restrictive temperature (35 °C), sgs1-36

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1014240108 Mankouri et al. Downloaded by guest on October 2, 2021 that normally persist in S. cerevisiae sgs1 and TOP3Y356F strains. Given that both RusA and GEN11-527 exhibit robust HJ processing activity in vitro (40, 43–47) and do not appear to non- specifically cleave other types of (Rad51-independent) DNA replication intermediates in vivo, we propose that the Rad51- dependent X-structures we detect in sgs1 and TOP3Y356F strains probably contain HJs between sister chromatids. However, we note that an ideal specificity control would be a Rad51-dependent X-structure that definitively does not contain HJs. However, to our knowledge, a mutant exhibiting such a structure has not been identified. Previous studies have suggested that the sgs1 MMS-induced X-structures are “pseudo-Holliday junctions,” consisting of a region of hemicatenated nascent DNA and concomitant parental ssDNA (27). However, this type of DNA structure would not be efficiently cleaved by RusA or GEN11-527, which both demonstrate a preference for specific nucleotide sequences being present at the four-way junction center for efficient cutting (44, 46). Furthermore, because the X-structures arising in TOP3Y356F cells are also cleaved by RusA and GEN11- 527, we suggest that the poor growth of top3 strains is unlikely to be caused by unresolved hemicatenane structures created by Sgs1 during HRR. We note, however, that we cannot rule out the possibility that different types of X-structures could exhibit some degree of interconversion and that HJ resolvase cleavage occurs once X-structures temporarily contain one or more canonical HJs. Our data are in agreement with a recent study revealing the first demonstration of HJs arising as intermediates of DSB repair

in mitotic cells and a defect in the prevention and/or processing GENETICS of these HJs in sgs1 mutants (38). Although X-structure inter- fi mediates of DSB repair were detectable in both haploid and Fig. 4. Reactivation of Sgs1 removes X molecules in SGS1-de cient cells. (A) fi Conditional inactivation of SGS1 causes unprocessed X-structures after ex- diploid WT cells, con rmation that these contained HJs was only + fi “ ” posure to MMS. SGS1 ,sgs1Δ, and sgs1-36 strains were released from G1 possible in diploid cells (due to strand-speci c probing of Mom arrest at 35 °C into medium containing 0.033% MMS at the indicated tem- and “Dad” recombinant homologous chromosomes). Our data peratures. DNA replication was monitored after 2 h by 2D gel electropho- independently verify that unprocessed HJ-containing structures resis and flow cytometry. X-shaped DNA structures are denoted by arrows. arise in haploid sgs1 cells and suggest that the processing of HJs (B) After 2 h of MMS-treatment at 35 °C, sgs1Δ and sgs1-36 cells were har- is altered in sgs1 mutants during both DSB repair and the repair vested, washed, and resuspended in drug-free medium at 35 °C or 25 °C, as of replication-induced lesions. Furthermore, we also propose indicated. The phenotype of the sgs1-36 strain (denoted as sgs1 or SGS1+)at that Sgs1 functions in the direct removal of HJ-containing each step of the protocol is indicated in the schematic diagram. (C) DNA intermediates, because the reactivation of Sgs1 in sgs1-36 strains replication intermediates around ARS305 were analyzed in the indicated promotes the processing of otherwise persistent X-structures. strains by 2D gel electrophoresis. Because unprocessed HJ-containing DNA structures also persist in TOP3Y356F cells, our data are consistent with Sgs1 and Δ Top3 acting together, rather than sequentially, to directly pro- mutants resemble sgs1 mutants and accumulate MMS-induced cess interchromatid HJs arising during HRR of MMS-induced X-structures at ARS305 (Fig. 4A). As expected, MMS-induced X- + DNA lesions. One possibility is that X-structures comprise dHJs structures were not detectable in SGS1 cells or in sgs1-36 that can be removed by Sgs1–Top3–Rmi1-mediated dHJ disso- mutants returned to the permissive temperature (25 °C) imme- lution (18, 20, 21, 52). Top3 is likely to be required for relieving diately after the release from G1 arrest (Fig. 4A). the inevitable buildup of torsional stress associated with con- After 2 h of treatment with 0.033% MMS at 35 °C, we followed vergent branch migration of HJs, because Sgs1 or (Drosophila) sgs1Δ the fate of MMS-induced X-structures in an strain and in BLM alone are incapable of extensive convergent branch mi- sgs1-36 the mutant at both the permissive and the restrictive gration of dHJs in the absence of Top3 or TopoIIIα, respectively B sgs1Δ temperature (Fig. 4 ). In the mutant, X-structures were (52, 53). Another possibility is that X-structures comprise single detectable at 4 h after MMS removal at both 25 °C and 35 °C, HJs and that Sgs1 and Top3 act cooperatively to process these. sgs1 demonstrating that X-structure persistence is independent For example, partially extended D-loops would contain a four- of temperature under these conditions (Fig. 4C). Similarly, sgs1- way junction that could theoretically either be cleaved by HJ 36 sgs1 strains held at 35 °C after the MMS treatment resembled resolvases or disrupted by the helicase activity of Sgs1. Future deletion strains and exhibited unprocessed X-structures. In- studies could be aimed at testing whether the controlled ex- sgs1-36 terestingly, strains returned to the permissive temperature pression of a HJ resolvase can ameliorate certain BS phenotypes, immediately after the removal of MMS did not exhibit unpro- as has been observed for human WS cells and S. pombe rqh1 C cessed X-structures after 4 h in drug-free medium (Fig. 4 ). mutants expressing heterologous HJ resolvases (39–41, 54). Therefore, reactivation of Sgs1 in the sgs1-36 strain background promotes the processing of MMS-induced X-structures, in Materials and Methods a manner similar to that observed for sgs1 mutants expressing HJ – S. cerevisiae Strains and Plasmids. The genotypes of strains used in this study resolvases (Figs. 1 3). Together, these data are consistent with can be found in Table S1. Details of the plasmids used can be found in a role for Sgs1 in the direct removal of HJ-containing structures SI Materials and Methods. arising during MMS-induced HRR. Growth Conditions, Cell Synchronization, and Flow Cytometry Analysis. Strains Discussion were grown at 30 °C, unless indicated otherwise. Strains were synchronized

In this study, we have demonstrated that expression of two evo- in G1 with 5–20 μg/mL α-factor mating pheromone as described (29). For lutionarily divergent HJ resolvases, E. coli RusA and human protein induction, galactose was added during G1 arrest and maintained at 1-527 GEN1 , can diminish the level of MMS-induced X-structures 2% throughout the subsequent analyses. Release of cells from α-factor ar-

Mankouri et al. PNAS Early Edition | 5of6 Downloaded by guest on October 2, 2021 rest, or after MMS treatment, was achieved by centrifugation, washing, and Western Blot Analysis Protein extraction, SDS/PAGE, and Western blot analysis resuspension in drug-free medium. Cell cycle progression was monitored by were performed as described (29). The anti-GFP antibody (Roche Diagnostics) ﬂ using ow cytometry as described (29). was used at a dilution of 1:1,000.

2D Gel Electrophoresis. The CTAB method of DNA extraction and 2D gel ACKNOWLEDGMENTS. We thank Drs. S. Brill, L. Cox, M. A. Resnick, procedures were performed as described (42). Unless stated otherwise, R. Rothstein, M. Seki, S. West, and M. Whitby for strains and plasmids; and DNA extracts were digested with NciI and NcoI before running the ﬁrst- Drs. P. McHugh and W. Niedzwiedz and various members of the I.D.H. dimension gels. Quantiﬁcation of X molecules on 2D gels was performed as laboratory for helpful discussions. This work was supported by Cancer described (30). Research UK and the Nordea Foundation.

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