Title: DNA bending facilitates the error-free DNA damage tolerance pathway and upholds genome integrity
Abstract: Article31 January 2014Open Access DNA bending facilitates the error-free DNA damage tolerance pathway and upholds genome integrity Victor Gonzalez-Huici Victor Gonzalez-Huici Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Barnabas Szakal Barnabas Szakal Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Madhusoodanan Urulangodi Madhusoodanan Urulangodi Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Ivan Psakhye Ivan Psakhye Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Martinsried/Munich, Germany Search for more papers by this author Federica Castellucci Federica Castellucci Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Demis Menolfi Demis Menolfi Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Eerappa Rajakumara Eerappa Rajakumara Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Marco Fumasoni Marco Fumasoni Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Rodrigo Bermejo Rodrigo Bermejo Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Stefan Jentsch Stefan Jentsch Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Martinsried/Munich, Germany Search for more papers by this author Dana Branzei Corresponding Author Dana Branzei Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Victor Gonzalez-Huici Victor Gonzalez-Huici Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Barnabas Szakal Barnabas Szakal Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Madhusoodanan Urulangodi Madhusoodanan Urulangodi Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Ivan Psakhye Ivan Psakhye Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Martinsried/Munich, Germany Search for more papers by this author Federica Castellucci Federica Castellucci Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Demis Menolfi Demis Menolfi Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Eerappa Rajakumara Eerappa Rajakumara Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Marco Fumasoni Marco Fumasoni Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Rodrigo Bermejo Rodrigo Bermejo Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Stefan Jentsch Stefan Jentsch Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Martinsried/Munich, Germany Search for more papers by this author Dana Branzei Corresponding Author Dana Branzei Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy Search for more papers by this author Author Information Victor Gonzalez-Huici1,3,‡, Barnabas Szakal1,‡, Madhusoodanan Urulangodi1, Ivan Psakhye2, Federica Castellucci1, Demis Menolfi1, Eerappa Rajakumara1, Marco Fumasoni1, Rodrigo Bermejo1,4, Stefan Jentsch2 and Dana Branzei 1 1Fondazione Istituto FIRC di Oncologia Molecolare (IFOM), Milan, Italy 2Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Martinsried/Munich, Germany 3Present address: College of Life Sciences, University of Dundee, Dundee, UK 4Present address: Instituto de Biología Funcional y Genómica, CSIC, Universidad de Salamanca, Salamanca, Spain ‡These authors contributed equally to this work. *Corresponding author. Tel: +39-02574303259; Fax: +39-02574303231; E-mail: [email protected] The EMBO Journal (2014)33:327-340https://doi.org/10.1002/embj.201387425 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability. Synopsis The DNA-bending HMG-box protein Hmo1 channels replication-associated lesions into error-free template switching pathways, highlighting the regulatory roles of chromosome architecture upstream of more dedicated repair mechanisms. Replication-associated topological changes foster error-free damage-bypass. The Rad5-mediated error-free damage-bypass is activated by topological changes in S phase. The chromatin architectural protein Hmo1 prevents mutagenic bypass and toxic recombination. Hmo1-mediated DNA bending promotes error-free DNA damage tolerance. Introduction Damaged DNA templates are major obstacles during replication, inducing fork stalling and discontinuities in the replicated chromosomes. DNA damage tolerance (DDT) mechanisms are crucial to promote replication completion by mediating fork restart and filling of DNA gaps (Lopes et al, 2006; Branzei et al, 2008; Daigaku et al, 2010; Karras & Jentsch, 2010; Minca & Kowalski, 2010). Genetic work has delineated two main modes of DDT in all organisms: an error-free mode involving recombination in which one newly synthesized strand is used as a template for replication of the blocked nascent strand, and an error-prone mode involving translesion synthesis (TLS) and which is largely accountable for mutagenesis (reviewed in Friedberg, 2005; Branzei, 2011). Because increased mutations ultimately lead to genome instability and cancer (Nik-Zainal et al, 2012; Alexandrov et al, 2013), the molecular mechanisms underlying DDT pathway choice have implications for understanding cancer etiology and for cancer therapy. At present, the mechanisms underlying the error-free/error-prone DDT pathway switch remain little understood: on one hand, high expression of TLS polymerases in mitosis may represent a passive mechanism that favors error-free damage-bypass early during replication (Waters & Walker, 2006), in line with the observed correlation between replication timing and mutation rates (Lang & Murray, 2011); on the other hand, regulatory mechanisms, such as the ones involving post-translational modification of the polymerase clamp, PCNA, with SUMO and ubiquitin, modulate the recruitment of repair factors and TLS polymerases, thus influencing DDT pathway choice (Bergink & Jentsch, 2009). PCNA modifications with SUMO and ubiquitin are crucial for DDT: mono-ubiquitylation of PCNA promotes translesion polymerase-mediated error-prone DDT (Stelter & Ulrich, 2003), Rad5-Mms2-Ubc13-dependent polyubiquitylation of PCNA acts in conjunction with a subset of homologous recombination factors to mediate error-free DDT by formation of sister chromatid junctions (SCJs) (Branzei et al, 2008; Minca & Kowalski, 2010; Vanoli et al, 2010; Karras et al, 2013), and SUMOylated PCNA recruits Srs2 to chromatin, where it presumably prevents the access of the recombination machinery and inhibits unwanted recombination (Papouli et al, 2005; Pfander et al, 2005; Branzei et al, 2008; Karras et al, 2013). The recombination pathway prevented by SUMOylated PCNA is also known as the salvage pathway of DDT, whereas the Rad5-mediated pathway is commonly referred to as template switching. Notably, both these error-free DDT pathways mediate damage-bypass via the formation of SCJs, but may occupy distinct time windows in relation to DNA replication (Branzei et al, 2008; Karras et al, 2013). Following the formation of damage-bypass SCJs, the Sgs1 helicase, homolog of human BLM that is mutated in cancer-prone Bloom syndrome patients, is thought to process together with the Top3 topoisomerase these intermediates to hemicatenanes, topological structures conjoining two DNA duplexes through a single-strand interlock, (Wu & Hickson, 2003; Liberi et al, 2005; Branzei et al, 2008; Karras & Jentsch, 2010; Cejka et al, 2012). Type IA topoisomerases—Top1 and Top3 in budding yeast—that catalyze strand passage through a reversible, enzyme-bridged, single-strand break can then resolve the resulting hemicatenanes (Wang, 2002). When Sgs1 functionality is impaired, the SCJs arising during error-free DDT are resolved by crossover-prone nucleases (Ashton et al, 2011; Szakal & Branzei, 2013), leading to elevated sister chromatid exchanges and loss of heterozygosity events that may ultimately drive chromosomal instabilities underpinning tumorigenesis (Wechsler et al, 2011; Szakal & Branzei, 2013). High mobility group box (HMGB) proteins are abundant, multifunctional proteins with genome architectural capacity conferred by their ability to bend DNA, in the process creating DNA topologies that can impinge on the assembly of nucleoprotein structures (reviewed in Thomas & Travers, 2001; Stros, 2010). Notably, HMGB1 binds with high affinity to hemicatenanes (Stros et al, 2004; Jaouen et al, 2005). The Saccharomyces cerevisiae HMGB protein, Hmo1 - the closest ortholog of HMGB1 in yeast-, shows synthetic lethal interactions with top3Δ (Gadal et al, 2002), and binds with preference to single stranded (ss) DNA and to DNA with altered conformations, showing reduced DNA sequence specificity (Kamau et al, 2004; Bauerle et al, 2006; Xiao et al, 2010). In addition, in hmo1 mutant cells, spontaneous and damage-induced mutagenesis is increased (Alekseev et al, 2002; Kim & Livingston, 2006, 2009), suggesting a possible role for Hmo1 in DDT or its regulation. It is of note that while mutation rates vary along chromosomes and correlate with replication timing (Lang & Murray, 2011), the underlying mechanisms accounting for the preferred usage of error-free DDT early in S phase remain elusive. Here we show that Hmo1 has an early regulatory role, coincident with DNA replication, in error-free DDT pathway choice by channeling lesions towards the Rad5-Mms2-Ubc13-mediated pathway of template switching, while preventing mutagenic bypass and toxic recombination. We uncover that error-free DDT pathway choice, previously shown to be controlled by SUMOylated PCNA and its interactors Srs2 and Elg1, is uncoupled from the SCJ formation process per se. While Srs2 and Elg1 do not play a discernible role in SCJ formation, Hmo1 affects also this latter process. The time window for Hmo1 action in SCJ formation overlaps with the one of the Rad5-Mms2-Ubc13, being predominant early during replication. Importantly, these Hmo1 functions in error-free DDT are largely mediated via its carboxy (C)-terminal domain, previously shown to promote DNA bending. We additionally find that Hmo1 promotes topological transitions related to catenane/hemicatenane formation/stabilization during unperturbed growth and that this function is also largely dependent on its C-terminal domain. Together, the results indicate that the Hmo1-mediated topological pathway involving DNA bending represents a new replication-associated regulatory mechanism that facilitates error-free DDT and influences the error-free/error-prone DDT switch. Results Hmo1 functionally interacts with the Rad5-Mms2-Ubc13 error-free DDT pathway Hmo1 and its human ortholog, HMGB1, exhibit high affinity for DNA hemicatenanes and other types of DNA with altered conformations such as ssDNA and DNA cruciform structures (Bianchi et al, 1989; Lu et al, 1996; Kamau et al, 2004; Jaouen et al, 2005) forming during replication in unperturbed and genotoxic stress conditions (Lopes et al, 2003, 2006; Liberi et al, 2005; Branzei et al, 2008). Hmo1 is an abundant protein, associated with chromatin throughout the cell-cycle (Bermejo et al, 2009). Following replication in the presence of DNA damage (MMS), we found by ChIP-on-chip a statistically significant co-localization between Hmo1 clusters and the ones of Rfa1, the large subunit of RPA (P-value 1.80E-16), which presumably marks ssDNA regions (Supplementary Fig S1A). Indeed, after treatment with high doses of HU, which blocks replication by depleting dNTP pools, Rfa1 peaks were clustered around early origins of replication and were overlapping with the BrdU peaks marking ongoing DNA replication (Supplementary Fig S1B, P-value 3.10E-17), in line with findings showing that HU treatment induces replication fork stalling and accumulation of ssDNA regions in the proximity of origins of replication (Sogo et al, 2002; Feng et al, 2006). On the other hand, following treatment with sublethal doses of MMS, which does not slow down replication fork progression to the same degree as high HU concentrations, Rfa1 peaks were spread over much larger regions (Supplementary Fig S1A), supporting the notion that during replication in the presence of genotoxic stress, DNA gaps persist behind replication forks (Lopes et al, 2006). Coating of ssDNA gaps with RPA facilitates the recruitment of the Rad18 ubiquitin ligase (Davies et al, 2008), which together with the Rad6 ubiquitin conjugating enzyme and the Rad5-Mms2-Ubc13 ubiquitylation complex, induces PCNA mono- and polyubiquitylation (Hoege et al, 2002) and mediates postreplicative DDT (Daigaku et al, 2010; Karras & Jentsch, 2010). The overlap between Hmo1 and Rfa1 clusters in MMS-treated cells (Supplementary Fig S1A), together with previous reports indicating a role for Hmo1 in the control of mutagenesis (Alekseev et al, 2002; Kim & Livingston, 2006), prompted us to investigate a possible involvement of Hmo1 in DDT and the metabolism of DNA structures arising during recombination-mediated damage-bypass. Two genetic pathways, the Rad51 and the Rad5-Mms2-Ubc13 pathways were identified to contribute to error-free DDT (Branzei et al, 2008; Karras et al, 2013). While hmo1Δ cells had wild-type (WT) levels of MMS resistance and the hmo1Δ mutation did not increase or rescue the MMS sensitivity of rad51Δ cells (data not shown and see below), it partially but discernibly suppressed the damage sensitivity of rad5Δ cells in two different yeast backgrounds, DF5 (Fig 1A) and W303 (see below), suggesting a functional interaction between Hmo1 and Rad5. We further examined if this genetic relationship extended to other factors involved in PCNA polyubiquitylation. We found that the hmo1Δ mutation also partly suppressed the MMS sensitivity associated with null mutations in MMS2 and UBC13 (Fig 1B), indicating that Hmo1 affects the usage of the Rad5-Mms2-Ubc13 error-free DDT pathway. Figure 1. Hmo1 interacts functionally with the Rad5-Mms2-Ubc13 error-free DDT pathway HMO1 deletion rescues the MMS sensitivity of rad5Δ. wt (FY0113), hmo1Δ (HY3956), rad5Δ (HY0516), rad5Δ hmo1Δ (HY1518) cells were spotted. HMO1 deletion rescues the MMS sensitivity of mms2Δ and ubc13Δ. wt (FY0113), hmo1Δ (HY1508), mms2Δ (HY0518), ubc13Δ (FY1490), mms2Δ hmo1Δ (HY1519), and ubc13Δ hmo1Δ (HY3959) were spotted. HMO1 deletion rescues the cold sensitivity of pol32Δ. wt (FY0090), hmo1Δ (HY2714), pol32Δ (HY2719) and hmo1Δ pol32Δ (HY2706) were spotted. Hmo1 does not affect PCNA modifications with ubiquitin and SUMO. Western blot of Pol30 (PCNA) in an hmo1-AID conditional mutant (HY2174) following or not Hmo1 depletion by addition of auxin (Ax) before G1 arrest and release into MMS-containing media. Ubiquitylated and SUMOylated species are indicated. Hmo1 depletion control and Pgk1, used as loading control, are shown below. To the right, controls for lack of PCNA polyubiquitylation (ubc13Δ, Y2620), or SUMOylation (siz1Δ, Y1630), or both (pol30-RR, FY1487). Asterisks denote cross-reactive proteins. Download figure Download PowerPoint To further test Hmo1 implication in error-free DDT, we used a recently elucidated genetic readout (Karras & Jentsch, 2010). Deletion of POL32, encoding a nonessential subunit of the replicative DNA polymerase δ (Polδ) that is required for DNA synthesis during template switching (Vanoli et al, 2010), generates replication stress accompanied by cold sensitivity and induction of error-free DDT – and therefore of PCNA polyubiquitylation (Karras & Jentsch, 2010; Karras et al, 2013). Because mutations affecting PCNA polyubiquitylation (mms2Δ, ubc13Δ, rad5Δ, and pol30-K164R) suppress the cold sensitivity of pol32Δ cells (Karras et al, 2013), suppressors of the pol32Δ cold sensitivity phenotype are potentially new components or regulators of the error-free DDT pathway. We found that hmo1Δ also partly suppressed the slow growth phenotype at low temperatures of pol32Δ cells (Fig 1C), similarly to mutations in other components of the PCNA polyubiquitylation pathway, although to a smaller degree than those mutations (Supplementary Fig S1C). We note that hmo1Δ was reported to suppress the temperature sensitivity of other DNA Polδ mutants (Kim & Livingston, 2009), thus resembling also in this respect deletions of RAD18, RAD5 and MMS2-UBC13 (Giot et al, 1997; Branzei et al, 2002, 2004). We then analyzed if Hmo1 affects PCNA post-translational modifications. Because hmo1Δ strains are slow growing, showing slower progression throughout the cell-cycle (Lu et al, 1996), and PCNA modifications with SUMO and ubiquitin are expected to be sensitive to cell-cycle changes and replication delays (Hoege et al, 2002), we established a conditional degron system (hmo1-AID), in which Hmo1 depletion is induced by addition of auxin (Nishimura et al, 2009). Reduced levels of Hmo1 did not discernibly affect PCNA modifications with ubiquitin and SUMO (Fig 1D), suggesting that the effects manifested by Hmo1 on the Rad5-mediated error-free DDT pathway (Fig 1A and B) are not caused by alterations in PCNA modifications. Hmo1 roles in DDT regulation and SCJ formation are manifested during DNA replication While the ability of cells to deal with exogenous DNA damage is not affected by restricting the expression of key DDT genes to the G2/M phase of the cell-cycle (Daigaku et al, 2010; Karras & Jentsch, 2010), other results suggest an early role for the Rad5 pathway during replication and SCJ formation (Branzei et al, 2008; Minca & Kowalski, 2010; Karras et al, 2013). To address if the role(s) of Hmo1 in regulating the Rad5 pathway (see Fig 1) are normally manifested in S- or G2/M phases of the cell-cycle, or independently of the cell-cycle phase, we applied the S and G2 tags to HMO1. These tags restrict the expression of tagged proteins to specific phases of the cell-cycle, due to control elements of cyclin Clb6 or Clb2, respectively (Karras & Jentsch, 2010; Hombauer et al, 2011). When the S-tag- and G2-tag-containing DNA cassettes were integrated directly upstream of the HMO1 open reading frame at its endogenous locus, the resulting fusion proteins were indeed largely restricted during the cell-cycle as assessed by comparing the expression of these proteins with the ones of Clb2 (Fig 2A). When we further combined these hmo1 alleles with a rad5Δ mutation, we found that specifically the G2-HMO1 allele resembled hmo1Δ in its ability to suppress rad5Δ MMS sensitivity. Thus, Hmo1 role in regulating the Rad5 pathway is manifested during replication. Figure 2. The roles of Hmo1 in Rad5 pathway regulation and SCJ formation are manifested during DNA replication S-tag HMO1 (S-HMO1, HY4324) and G2-tag HMO1 (G2-HMO1, HY4325) cells were arrested in G1 phase and released into YPD at 28°C. Samples were collected at the indicated time points for Western blot analysis. The cell cycle progression was monitored using anti-Clb2 antibody; Pgk1 was used for loading control. Specifically the G2-HMO1 allele partially rescues the MMS sensitivity of rad5Δ cells. wt (FY1296), hmo1Δ (HY1507), S-HMO1 (HY4324), G2-HMO1 (HY4325), rad5Δ (HY2682), rad5Δ hmo1Δ (HY3633), S-HMO1 rad5Δ (HY4355) and G2-HMO1 rad5Δ (HY4359) were spotted. Hmo1 promotes SCJ formation during template switching in S phase. HMO1-AID sgs1Δ (HY2176) cells were synchronized with alpha-factor (aF) and divided into two identical parts. One half of the culture was treated with auxin and released into YPD media containing 0.033% MMS in the presence of auxin (+), the other half was released into MMS-containing media without auxin treatment. At the indicated time points samples were taken for 2D gel, FACS and Western blot analysis. During quantification the highest value obtained for the X-molecules was considered as 100%. The efficiency of Hmo1 depletion was analyzed with anti-Hmo1 antibody via immunoblotting. Pgk1 was used for loading control. Download figure Download PowerPoint The culmination of error-free DDT is the formation of SCJs, later resolved by Sgs1-Top3 (Branzei et al, 2008). To address if Hmo1 also affects the formation or the stability of SCJs generated during error-free DDT, we studied by 2D gel electrophoresis the profile of replication intermediates arising at an early, efficient origin of replication, ARS305, when yeast cells replicate in media containing MMS (Fig 2B). Because in sgs1Δ cells the processing of the resulting recombination intermediates is impaired and SCJs forming during error-free DDT accumulate (Liberi et al, 2005; Branzei et al, 2008), we used this genetic background as a tool to address a possible role for Hmo1 in this process. Furthermore, since hmo1Δ strains are slow-growing (Lu et al, 1996) and the profile of replication intermediates can be severely impacted by the cell-cycle/replication status, we used again the hmo1-AID degron system described above (see Fig 1D) to induce Hmo1 depletion. sgs1Δ hmo1-AID cells grow normally, but Hmo1 depletion at the beginning of replication correlated with a decrease in the amount of SCJs (Fig 2B, 60–120 min panels), which gradually increased following prolonged MMS treatment (Fig 2B, 180–240 min panels). Thus, Hmo1 facilitates SCJ formation/stability in the same time window with the one reported for Rad5-Mms2-Ubc13 (Karras et al, 2013), being predominant early during replication. Furthermore, these results indicate that Hmo1 depletion does not significantly impair the functionality of the salvage recombination pathway that normally promotes SCJ formation later in the cell-cycle (Branzei et al, 2008; Karras et al, 2013). To examine if the above 2D gel results might reflect a role for Hmo1 in promoting SCJ stability rather than their formation, we used again an sgs1Δ hmo1-AID strain but induced Hmo1-AID depletion after the initiation of SCJ formation (1 h after the cells were released from G1 arrest into S phase, Supplementary Fig S2). Although under these conditions Hmo1 depletion also occurred efficiently, it did not anymore correlate with reduced SCJ levels (Supplementary Fig S2), in contrast to its effect at the beginning of replication (Fig 2B, 60–120 min panels). Thus, following genotoxic stress, Hmo1 facilitates the usage of the Rad5 pathway, promoting template switching accompanied by SCJ formation early in S phase. Hmo1 is a novel regulator of the DDT pathway choice that acts in parallel with Elg1 and Srs2 To understand the molecular mechanism by which Hmo1 facilitates the execution of the Rad5 pathway, we attempted to identify Hmo1 interacting proteins, using a candidate approach as well as yeast two-hybrid screens. We found initially by two-hybrid that Elg1, a regulator of the Rad5 pathway and a binding partner of PCNA (Parnas et al, 2010; Kubota et al, 2013), interacts physically with Hmo1. We then examined this interaction by in vivo pull-down assays. To this end, we purified recombinant GST and GST-Hmo1, immobilized these proteins on glutathione-sepharose beads, and incubated the beads with total cell lysates prepared from Elg1-FLAG yeast strains. In this way, we found that Elg1 is efficiently pulled-down to Hmo1 beads, even when the extract was treated with ethidium bromide, thus suggesting that the interaction between Hmo1 and Elg1 is not bridged by DNA (Fig 3A). Figure 3. Hmo1 acts in parallel with Elg1 and Srs2 to promote Rad5-mediated error-free DDT Hmo1 interacts physically with Elg1. In vivo pull-down assay. Recombinant GST-Hmo1 protein was tested for its ability to bind endogenous Elg1. The amount of GST and GST-Hmo1 protein used is shown by Ponceau staining. Total cell lysates prepared from yeast cells expressing Elg1-FLAG tagged strain (HY1976) were incubated with GST or GST-Hmo1 in the presence or absence of ethidium bromide. The protein complex formed on the beads was analyzed by immunoblotting using anti-FLAG antibody. HMO1 and ELG1 deletions additively rescue the MMS sensitivity of rad5Δ. wt (HY4104), rad5Δ (HY4098), rad5Δ hmo1Δ (HY4127), rad5Δ elg1Δ (HY4056) and rad5Δ hmo1Δ elg1Δ (HY4073) cells were spotted. HMO1 deletion rescues the MMS sensitivity of rad5Δ cells by suppressing the recombination pathway. wt (FY0113), hmo1Δ (HY3957), rad5Δ (HY0516), rad5Δ hmo1Δ (HY1518), rad5Δ hmo1Δ rad51Δ (HY3943), rad5Δ rad51Δ (HY3948), hmo1Δ rad51Δ (HY3946) and rad51Δ (HY2651) strains were spotted. The survival of hmo1Δ ubc13Δ cells in MMS depends on the mutagenic pathway involving the translesion synthesis polymerase Rev3. wt (FY0090), hmo1Δ (HY1508), ubc13Δ (FY1490), rev3Δ (HY4416), hmo1Δ ubc13Δ (HY3960), hmo1Δ rev3Δ (HY4439), ubc13Δ rev3Δ (HY4417) and ubc13Δ rev3Δ hmo1Δ (HY4440) strains were spotted. Download figure Download PowerPoint The elg1Δ mutation suppresses the sensitivity of rad5Δ, ubc13Δ, and mms2Δ cells to MMS by a similar degree as the one conferred by hmo1Δ (Fig 3B, note the growth defect associated with hmo1Δ). However, the combination of hmo1Δ and elg1Δ mutations leads to a much better suppression of the rad5Δ sensitivity than the one conferred by single mutations (Fig 3B), attesting to the individual roles of Elg1 and Hmo1 in error-free DDT regulation and indicating that the distinct modulatory actions of Elg1 and Hmo1 on the Rad5 pathway are potentially coordinated via their physical interaction. While the mechanism by which Elg1 regulates the Rad5 pathway remains elusive, it possibly involves a joint action of Elg1 with Srs2, the other known regulator of the Rad5-mediated DDT branch that acts by affecting the choice of the recombinational repair pathway (Rong et al, 1991; Papouli et al, 2005; Pfander et al, 2005). The interplay between Srs2 and Elg1 in error-free DDT regulation was suggested by their preferential binding to SUMOylated PCNA (Papouli et al, 2005; Pfander et al, 2005; Parnas et al, 2010) and the observation that simultaneous deletion of SRS2 and ELG1 leads to a growth impairment that is partly improved by a SUMOylation-defective allele of PCNA (Parnas et al, 2010). The proposed mechanism envisages that while Srs2 disrupts toxic recombination events and makes space for the action of the Rad5 pathway (Aboussekhra et al, 1992; Krejci et al, 2003; Veaute et al, 2003; Papouli et al, 2005; Pfander et al, 2005), Elg1 may help unload (SUMOylated) PCNA from chromatin to facilitate DNA repair (Parnas et al, 2010; Kubota et al, 2013). To further investigate the mechanism by which Hmo1 modulates Rad5-mediated DDT, we aimed at identifying the DDT pathways required for viability in rad5Δ hmo1Δ and ubc13Δ hmo1Δ cells. Similarly to the case previously elucidated for Srs2 (Rong et al, 1991; Aboussekhra et al, 1992; Papouli et al, 2005; Pfander et al, 2005), we found that the viability of rad5Δ hmo1Δ depended on the salvage recombination pathway involving Rad51 (Fig 3C) and the recently identified 9-1-1 activities (Karras et al, 2013) (Supplementary Fig S3A), but not on Ubc13 (Supplementary Fig S3B). In addition, Hmo1 was not required for the viability of rad5Δ srs2Δ cells exposed to MMS (Supplementary Fig S3C, note the growth defect associated with hmo1Δ). This latter result, together with the 2D gel analysis data showing that Hmo1 is dispensable for the formation of late SCJs (Fig 2B), likely arising via the action of the salvage pathway of recombination (Branzei et al, 2008; Karras et al, 2013), indicates that Hmo1 is not required for the execution of the salvage recombination pathway. Furthermore, we found that the viability conferred by HMO1 deletion in mutants defective in the PCNA polyubiquitylation pathway of template switching depends on the TLS polymerase, Rev3 (Fig 3D). Thus, defects in the PCNA polyubiquitylation pathway in WT cells causes MMS hypersensitivity, whereas