Title: Rtt105 functions as a chaperone for replication protein A to preserve genome stability
Abstract: Article31 July 2018free access Source DataTransparent process Rtt105 functions as a chaperone for replication protein A to preserve genome stability Shuqi Li Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China Search for more papers by this author Zhiyun Xu State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Jiawei Xu State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Linyu Zuo orcid.org/0000-0003-0477-0304 Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China Search for more papers by this author Chuanhe Yu Department of Pediatrics and Department of Genetics and Development, Institute for Cancer Genetics, Columbia University, College of Physicians and Surgeons, New York, NY, USA Search for more papers by this author Pu Zheng State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Haiyun Gan Department of Pediatrics and Department of Genetics and Development, Institute for Cancer Genetics, Columbia University, College of Physicians and Surgeons, New York, NY, USA Search for more papers by this author Xuezheng Wang Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China Search for more papers by this author Longtu Li State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Sushma Sharma Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden Search for more papers by this author Andrei Chabes orcid.org/0000-0003-1708-8259 Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden Search for more papers by this author Di Li State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Sheng Wang State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Sihao Zheng Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences and the Institute for Advanced Studies, Wuhan University, Wuhan, China Search for more papers by this author Jinbao Li Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences and the Institute for Advanced Studies, Wuhan University, Wuhan, China Search for more papers by this author Xuefeng Chen Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences and the Institute for Advanced Studies, Wuhan University, Wuhan, China Search for more papers by this author Yujie Sun State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Dongyi Xu State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Junhong Han Division of Abdominal Cancer, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, and National Collaborative Center for Biotherapy, Chengdu, China Search for more papers by this author Kuiming Chan Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China Search for more papers by this author Zhi Qi Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China Search for more papers by this author Jianxun Feng Corresponding Author [email protected] orcid.org/0000-0001-8704-5348 Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Qing Li Corresponding Author [email protected] orcid.org/0000-0003-0251-9159 Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Shuqi Li Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China Search for more papers by this author Zhiyun Xu State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Jiawei Xu State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Linyu Zuo orcid.org/0000-0003-0477-0304 Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China Search for more papers by this author Chuanhe Yu Department of Pediatrics and Department of Genetics and Development, Institute for Cancer Genetics, Columbia University, College of Physicians and Surgeons, New York, NY, USA Search for more papers by this author Pu Zheng State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Haiyun Gan Department of Pediatrics and Department of Genetics and Development, Institute for Cancer Genetics, Columbia University, College of Physicians and Surgeons, New York, NY, USA Search for more papers by this author Xuezheng Wang Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China Search for more papers by this author Longtu Li State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Sushma Sharma Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden Search for more papers by this author Andrei Chabes orcid.org/0000-0003-1708-8259 Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden Search for more papers by this author Di Li State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Sheng Wang State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Sihao Zheng Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences and the Institute for Advanced Studies, Wuhan University, Wuhan, China Search for more papers by this author Jinbao Li Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences and the Institute for Advanced Studies, Wuhan University, Wuhan, China Search for more papers by this author Xuefeng Chen Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences and the Institute for Advanced Studies, Wuhan University, Wuhan, China Search for more papers by this author Yujie Sun State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Dongyi Xu State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Junhong Han Division of Abdominal Cancer, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, and National Collaborative Center for Biotherapy, Chengdu, China Search for more papers by this author Kuiming Chan Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China Search for more papers by this author Zhi Qi Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China Search for more papers by this author Jianxun Feng Corresponding Author [email protected] orcid.org/0000-0001-8704-5348 Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Qing Li Corresponding Author [email protected] orcid.org/0000-0003-0251-9159 Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Author Information Shuqi Li1,‡, Zhiyun Xu2,‡, Jiawei Xu2,‡, Linyu Zuo1,3, Chuanhe Yu4, Pu Zheng2, Haiyun Gan4, Xuezheng Wang1, Longtu Li2, Sushma Sharma5, Andrei Chabes5, Di Li2, Sheng Wang6, Sihao Zheng7, Jinbao Li7, Xuefeng Chen7, Yujie Sun6, Dongyi Xu2, Junhong Han8, Kuiming Chan9, Zhi Qi1,3, Jianxun Feng *,1,2 and Qing Li *,1,2 1Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China 2State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China 3Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China 4Department of Pediatrics and Department of Genetics and Development, Institute for Cancer Genetics, Columbia University, College of Physicians and Surgeons, New York, NY, USA 5Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden 6State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing, China 7Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences and the Institute for Advanced Studies, Wuhan University, Wuhan, China 8Division of Abdominal Cancer, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, and National Collaborative Center for Biotherapy, Chengdu, China 9Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 10 62752516; Fax: +86 10 62754427; E-mail: [email protected] *Corresponding author. Tel: +86 10 62752516; Fax: +86 10 62754427; E-mail: [email protected] EMBO J (2018)37:e99154https://doi.org/10.15252/embj.201899154 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 Generation of single-stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA-binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA–ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the Saccharomyces cerevisiae protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA–importin interaction and also promotes RPA binding to ssDNA directly in vitro, but is not present in the final RPA–ssDNA complex. Single-molecule studies reveal that Rtt105 affects the binding mode of RPA to ssDNA. These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates its loading onto ssDNA at replication forks. Synopsis Rtt105 constitutes a new chaperone for the single-stranded DNA (ssDNA) binding protein RPA in yeast, facilitating its nuclear import as well as modulating its ssDNA binding mode. Rtt105 is a novel RPA-binding protein. Rtt105 mediates RPA–importin interactions. Rtt105 promotes RPA binding to ssDNA substrates in vitro but does not exist in the final RPA-ssDNA complex. Rtt105 is important for RPA loading at replication forks. Rtt105 is critical for genome stability. Introduction DNA replication is tightly regulated at multiple levels to ensure that the genome is both accurately and completely duplicated during each cell cycle (Bell & Dutta, 2002; Burgers & Kunkel, 2017). Faults in this regulation can lead to replication errors, and consequently, genome instability. Understanding carcinogenesis and a variety of other diseases (Aguilera & García-Muse, 2013; Jackson et al, 2014) therefore requires a detailed understanding of how replication is mediated. The generation of single-stranded DNA (ssDNA) templates is necessary for DNA replication, but ssDNA is susceptible to secondary structure formation and digestion by nucleases. Therefore, ssDNA exposed during DNA replication must be protected and stabilized, a function performed by replication protein A (RPA; Chase & Williams, 1986). RPA is an evolutionarily conserved protein complex present in all eukaryotes and regulates both DNA replication initiation and elongation (Wobbe et al, 1987; Fairman & Stillman, 1988; Wold & Kelly, 1988; Brill & Stillman, 1989; Wold, 1997). RPA is also important during DNA damage repair and recombination, and RPA-coated ssDNA plays a role in the activation of the DNA replication checkpoint pathway and the nucleosome assembly pathway (Zou & Elledge, 2003; Maréchal & Zou, 2015; Liu et al, 2017; Zhang et al, 2017). RPA consists of the three related subunits Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae, with apparent masses of approximately 70, 30, and 14 kDa, respectively (Brill & Stillman, 1991). Each RFA gene is essential in budding yeast (Brill & Stillman, 1991), and all three subunits are required for the formation of the functional RPA complex. In vitro, RPA binds to ssDNA via six oligonucleotide binding (OB)-fold domains with affinities of up to ~ 10−7–10−10 M and a defined 5′→3′ polarity (Bochkarev et al, 1997; Bochkareva et al, 2001, 2002; Fanning et al, 2006; Fan & Pavletich, 2012; Brosey et al, 2013). This high binding affinity has led to the assumption that RPA functions passively and directly binds to exposed ssDNA, including that at the replication fork. However, in vitro binding affinity measurements are not expected to represent all aspects of regulation in living cells. Moreover, previous studies have shown that the binding affinity of RPA for short oligonucleotides is dependent on ssDNA length, and RPA has multiple modes of binding ssDNA determined by the length of ssDNA that it contacts and the number of OB-fold domains involved (Kim et al, 1994; Bastin-Shanower & Brill, 2001; Bochkareva et al, 2001; Kolpashchikov et al, 2001; Fanning et al, 2006). These results raise the possibility that other proteins can alter the binding mode of RPA and thereby regulate its function in replication and other DNA metabolic pathways. The regulator of Ty1 transposition (RTT) genes were first identified in S. cerevisiae in a screen for deficiency in Ty1 transposon transposition (Scholes et al, 2001). 13 of the 21 RTT genes identified in this screen were found to be previously characterized, with confirmed functions in DNA damage response (Game & Mortimer, 1974; Ajimura et al, 1993; Watt et al, 1996; Fortin & Symington, 2002). The other 8 RTT genes were initially uncharacterized (Scholes et al, 2001), but several studies have since shown that some of these 8 genes function in DNA-processing pathways. For example, Rtt101 is a member of the cullin family of ubiquitin ligases and is required for promoting replication fork progression (Michel et al, 2003; Luke et al, 2006; Zaidi et al, 2008), and functions in the regulation of DNA replication-coupled nucleosome assembly via the ubiquitination of H3 (Han et al, 2013). Rtt108, also named MMS1, interacts with Rtt101, and the Rtt101MMS1 complex is the budding yeast counterpart of the mammalian CUL4DDB1 ubiquitin ligase (Zaidi et al, 2008). Rtt109 is the acetyltransferase for histone H3 lysine 56 and is critical for genome integrity in part through its role in DNA replication-coupled nucleosome assembly (Driscoll et al, 2007; Pursell et al, 2007; Fillingham et al, 2008; Li et al, 2008; Burgess et al, 2010). Rtt106 is a histone chaperone that recognizes acetylation on lysine 56 of H3 and plays an important role in transcriptional silencing (Huang et al, 2005, 2007; Li et al, 2008; Su et al, 2012; Zunder et al, 2012). Rtt110, also named Elg1, forms an alternative RFC-like complex (Ben-Aroya et al, 2003; Kanellis et al, 2003) that unloads the PCNA clamp during DNA replication (Kubota et al, 2013; Gazy et al, 2015). Therefore, the functions of most of the RTT genes have been elucidated with the exception of RTT105 (YER104W). A functional dissection of protein complexes based on genetic interactions clustered RTT105 among DNA replication factors, as it shares a similar genetic interaction profile with RFA1 and RFA2 (Collins et al, 2007). However, the biochemical function of Rtt105 remains largely unknown. Here, we show that the Rtt105 protein interacts directly with RPA. We find that cells lacking Rtt105 exhibit defects in the association of RPA with ssDNA at DNA replication forks, and reduced DNA synthesis under replication stress. Mechanistically, we show that Rtt105 promotes both nuclear import of RPA and RPA binding to ssDNA. In vitro single-molecule and EMSA studies reveal that Rtt105 alters the interaction mode of RPA with ssDNA. We propose a model whereby Rtt105 is analogous to histone chaperones, which mediate histone import and promote nucleosome formation using histones and double-stranded DNA (dsDNA): Rtt105 is an RPA chaperone that both escorts RPA during nuclear import and promotes the formation of RPA–ssDNA at replication forks. Results Rtt105 interacts directly with RPA We purified Rtt105 from yeast cells using a TAP tag and identified co-purified proteins using mass spectrometry. There were two groups of top hits among the co-purified proteins: the three subunits of RPA, and Kap95 (YLR347C), a karyopherin-beta protein essential for the nuclear import of many proteins, including RPA (Belanger et al, 2011; Fig 1A and Table EV1). To characterize the Rtt105–RPA interaction, we performed reciprocal immunoprecipitation (IP) of TAP-tagged Rfa1 and detected Rtt105 in the Rfa1-associated complex (Fig EV1A). Moreover, the RPA–Rtt105 interaction peaked during S phase and diminished at G2/M phase (Fig 1B). In vitro pull-down assays revealed that purified recombinant maltose-binding protein-tagged Rtt105 (MBP-Rtt105) pulled down recombinant RPA in a concentration-dependent manner, whereas MBP alone did not bind RPA (Fig 1C). These results establish that Rtt105 binds directly to RPA. Figure 1. Rtt105 binds RPA in vivo and in vitro Identification of Rtt105-TAP-associated proteins. Purified proteins were resolved on a 4–12% gel, revealed by silver staining, and identified by mass spectrometry (Table EV1). Asterisk (*) indicates non-specific band also in control. Cells containing Rfa1-TAP were synchronized at G1, S, and G2/M phases and used for the TAP purification. Rfa1-TAP-associated protein complexes were analyzed by Western blot using CBP, Rfa2, and Rtt105 antibodies (left panel). DNA content was monitored by flow cytometry (right panel). MBP-Rtt105 pulled down recombinant RPA, resolved on SDS–PAGE gels and visualized by Coomassie Brilliant Blue (CBB) staining. MBP was used as a negative control. GST-tagged full-length (FL) and Rtt105 deletion mutants (schematic of the Rtt105 truncations is shown in the upper panel) were purified and used to pull down Rfa1 protein. GST proteins were used as a negative control. Isolated protein complexes were resolved on 15% SDS–PAGE gels and visualized by CBB staining. Rfa1-TAP purification was performed using Rfa1-TAP rtt105Δ strains expressing WT and indicated Rtt105 mutant forms. pRS313 serves as vector control (Vc). Source data are available online for this figure. Source Data for Figure 1 [embj201899154-sup-0006-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Mapping key amino acids for the Rtt105–RPA interaction Identification of RPA–Rtt105 interaction by tandem affinity purification (TAP). TAP-tagged Rfa1 was purified from yeast cells with or without Flag-tagged Rtt105 (Rtt105-Flag), and the co-purified proteins were resolved on SDS–PAGE and analyzed by Western blotting using antibodies against calmodulin binding peptide (CBP) and Flag. A no-TAP tag strain was used as a negative control. The Rtt105 E160A and E171AL172A (EL) mutations attenuate the RPA–Rtt105 interaction. Left panel: purified recombinant GST-tagged Rtt105 WT and mutant proteins analyzed by SDS–PAGE with CBB staining. Asterisk (*) indicates non-specific band; right panel: GST pull-down assay was performed to determine the interaction of RPA with Rtt105 mutants. The bead-bound proteins were resolved by SDS–PAGE gels and subjected to silver staining. Download figure Download PowerPoint To identify the Rfa1 binding region on Rtt105, we deleted sections of Rtt105 in 50-amino acid intervals, yielding four truncated forms of Rtt105: Δ2–52, Δ53–103, Δ104–154, and Δ155–208 (Fig 1D). We found that deletion of the C-terminal 50 amino acids (Δ155–208 = Rtt105Δ4) results in a loss of binding to Rfa1 in vitro (Fig 1D). Mutations of several conserved residues at the Rtt105 C-terminus, including E160A, D169A, and E171L172AA, reduce the interaction of Rtt105 with RPA both in vivo and in vitro, with the E171L172AA (EL) mutant exhibiting a more pronounced effect (Figs 1E and EV1B). These results indicate that the C-terminus of Rtt105 mediates the RPA–Rtt105 interaction. Rtt105 is important for RPA binding at replication forks We noticed that in the yeast genome database, RTT105 deletion cells are listed as inviable. However, using standard methods we successfully generated haploid rtt105Δ mutant cells in a W303 background (Appendix Fig S1A and B). To confirm the viability of rtt105Δ cells, we also constructed the null mutant cells in several additional genetic backgrounds, namely S288C, DBY747, and BY4742. rtt105Δ mutant cells in these backgrounds are also viable (Appendix Fig S1A and B). Moreover, rtt105Δ cells in all these strain backgrounds are sensitive to various DNA-damaging agents to a similar degree (Appendix Fig S1A and B). They are also sensitive to DNA-damaging agents when grown at 16°C (Appendix Fig S2A), a temperature used to perform genome-wide Rfa1 ChIP-seq without HU (see below). Furthermore, expression of RTT105 driven by its own promoter in rtt105Δ cells rescues the phenotype of rtt105Δ mutant cells (Appendix Fig S1C). We chose the W303 background, which is our standard yeast background, to perform the remaining experiments. Because RPA protects ssDNA at replication forks, we next explored whether Rtt105 mediates RPA behavior during replication (Wold, 1997). We used chromatin immunoprecipitation (ChIP) to determine whether deletion of RTT105 affects RPA binding at replication forks under two conditions: hydroxyurea (HU)-stalled forks and active forks without HU treatment (Fig 2A). First, G1-phase cells were released into early S phase in the presence of 0.2 M HU for 45 min. HU arrests cells at early S phase and has no apparent effect on initiation of DNA replication from early replication origins. HU treatment also activates the DNA replication checkpoint, which in turn inhibits initiation of DNA replication from late replication origins. Antibodies against Rfa1 or Rfa2 or TAP-specific antibodies (IgG beads) against Rfa1-TAP were used to perform ChIP assays (Figs 2A and EV2A). Quantitative PCR (qPCR) analysis of ChIP DNA revealed that Rfa1 and Rfa2 bound at early replication origins (ARS305 and ARS607), but not at distal sites (ARS607 + 14 kb and ARS305 + 12 kb) that remain unreplicated during early S phase in the presence of HU (Liu et al, 2017; Fig EV2B and C). Deletion of RTT105 reduced the association of both assayed RPA subunits with replicating DNA (Fig EV2B and C). Exogenous expression of full-length Rtt105 rescued the chromatin-binding defects of Rfa1 in rtt105∆ cells, whereas expression of an Rtt105 mutant lacking its C-terminus or harboring the E171L172AA mutation (EL) failed to do so (Fig EV2D). We also analyzed the impact of the rtt105∆ mutation on the association of Rfa1 across the genome using ChIP-seq. Rfa1 ChIP-seq peaks co-localized with almost all early replication origins (Figs 2B and EV2E). A calculation of the average Rfa1 ChIP-seq read density across all fired origins revealed that the binding of Rfa1 at early replication origins was reduced in rtt105∆ cells compared to WT cells at HU-stalled forks (Fig 2C). These results indicate that Rtt105 is required for RPA binding to HU-stalled replication forks genome-wide. Figure 2. RTT105 deletion leads to a reduction in RPA binding at replicating regions genome-wide Schematic for Rfa1 and Rfa2 ChIP-seq (this figure) and BrdU IP-seq (Fig 6). Yeast cells were arrested with α-factor at G1 phase and then released into fresh YPD medium containing BrdU to label newly synthesized DNA as well as to allow cell to enter S phase at two conditions: (i) in medium with 0.2 M HU for 45 min at standard growth temperature (25°C), and (ii) in medium without HU for 72 min at low temperature (16°C) to slow down S-phase progression. Cells were then collected for Rfa1/Rfa2 ChIP or BrdU IP. The resulted DNAs were detected by sequencing. Snapshots of Rfa1 ChIP-seq peak at ARS305 from WT and rtt105∆ cells released into HU medium. The average Rfa1 ChIP-seq read density from cells released into HU medium around ACS sites. ACS, ARS consensus sequence. Snapshots of Rfa2 ChIP-seq peaks at ARS305 from cells released at 16°C for 72 min. The average Rfa2 ChIP-seq read density surrounding ACS sites from cells released at 16°C for 72 min. Rfa1 occupancy analysis of cells progression through S phase. G1-arrested cells were released into fresh YPD medium to allow cell progression through S phase at standard growth temperature (25°C). Equal amounts of cells were collected at each time point. Rfa1 ChIP was performed using IgG beads for TAP tag. ARS305 and ARS607, early fired replication origins; ARS607 + 14 kb, a corresponding 14-kb away region of ARS607. The mean and standard deviation (SD) of three biological replicates are shown. The single-tailed nonparametric Wilcoxon test was performed as described in Materials and Methods (**0.001 ≤ P-value < 0.01). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Rtt105 is important for the association of RPA with replication forks A. An outline for ChIP assays for Rfa1 and Rfa2 subunits. G1 yeast cells were released into fresh YPD medium containing 0.2 M HU to arrest cells at early S phase. Equal amounts of cells were collected prior to G1 (0 min) or at different time points following release. Rfa1 and Rfa2 ChIP was performed using IgG beads for TAP-tagged strains, or anti-Rfa2 antibody. B, C. RPA binding was dramatically decreased at HU-stalled replication forks upon deletion of RTT105. ChIP DNA and input DNA were analyzed by quantitative real-time PCR using primers against the replication origins ARS607 and ARS305 and their corresponding distal regions (ARS607 + 14 kb and ARS305 + 12 kb), and the percentage of ChIP DNA over the input DNA was calculated. The mean and standard error (SE) of at least two biological replicates are shown, with P-values derived from two-way analysis of variance (ANOVA; **0.001 ≤ P-value < 0.01, ***P-value ≤ 0.001). D. The C-terminus of Rtt105 is important for RPA binding at replication forks. pRS313-based plasmids expressing Flag-tagged WT Rtt105 and mutants (Δ4 and EL) were transformed into the rtt105Δ strain. ChIP was performed using Rfa2 antibodies. Vc: pRS313 vector; WT: pRS313-Rtt105-Flag; Δ4: pRS313-Rtt105 (Δ155–208)-Flag; EL: pRS313-Rtt105E171AL172A-Flag. The mean and standard error (SE) of two biological replicates are shown. Statistical significance was evaluated based on Student's t-tests (**0.001 ≤ P-value < 0.01, ***0.0001 ≤ P-value < 0.001; ****P-value < 0.0001). E. Snapshot of Rfa1 ChIP-seq at chromosome III under HU synchronized cells is shown. Yeast cells were synchronized at G1 phase and then released into fresh medium containing 0.2 M HU and 400 μg/ml BrdU for 45 min at 25°C. Equal amounts of cells were collected just prior to G1 (0 min) or at 45 min following release into early S phase in the presence of HU. Rfa1 ChIP DNA was processed for sequencing. The sequencing reads were ma