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• W2885440056 abstract "Article13 August 2018Open Access Transparent process Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks Shin-ichiro Hiraga Corresponding Author Shin-ichiro Hiraga [email protected] orcid.org/0000-0002-8722-3869 Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK Search for more papers by this author Chandre Monerawela Chandre Monerawela Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK Search for more papers by this author Yuki Katou Yuki Katou Institute for Quantitative Biosciences, University of Tokyo, Tokyo, Japan Search for more papers by this author Sophie Shaw Sophie Shaw orcid.org/0000-0003-2367-2670 Centre for Genome-Enabled Biology and Medicine, University of Aberdeen, Aberdeen, UK Search for more papers by this author Kate RM Clark Kate RM Clark Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK Search for more papers by this author Katsuhiko Shirahige Katsuhiko Shirahige Institute for Quantitative Biosciences, University of Tokyo, Tokyo, Japan Search for more papers by this author Anne D Donaldson Corresponding Author Anne D Donaldson [email protected] orcid.org/0000-0001-7842-8136 Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK Search for more papers by this author Shin-ichiro Hiraga Corresponding Author Shin-ichiro Hiraga [email protected] orcid.org/0000-0002-8722-3869 Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK Search for more papers by this author Chandre Monerawela Chandre Monerawela Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK Search for more papers by this author Yuki Katou Yuki Katou Institute for Quantitative Biosciences, University of Tokyo, Tokyo, Japan Search for more papers by this author Sophie Shaw Sophie Shaw orcid.org/0000-0003-2367-2670 Centre for Genome-Enabled Biology and Medicine, University of Aberdeen, Aberdeen, UK Search for more papers by this author Kate RM Clark Kate RM Clark Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK Search for more papers by this author Katsuhiko Shirahige Katsuhiko Shirahige Institute for Quantitative Biosciences, University of Tokyo, Tokyo, Japan Search for more papers by this author Anne D Donaldson Corresponding Author Anne D Donaldson [email protected] orcid.org/0000-0001-7842-8136 Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK Search for more papers by this author Author Information Shin-ichiro Hiraga *,1, Chandre Monerawela1, Yuki Katou2, Sophie Shaw3, Kate RM Clark1, Katsuhiko Shirahige2 and Anne D Donaldson *,1 1Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK 2Institute for Quantitative Biosciences, University of Tokyo, Tokyo, Japan 3Centre for Genome-Enabled Biology and Medicine, University of Aberdeen, Aberdeen, UK *Corresponding author. Tel: +44 1224 437317; E-mail: [email protected] *Corresponding author. Tel: +44 1224 437316; E-mail: [email protected] EMBO Reports (2018)19:e46222https://doi.org/10.15252/embr.201846222 Correction(s) for this article Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks26 June 2019 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 Despite its evolutionarily conserved function in controlling DNA replication, the chromosomal binding sites of the budding yeast Rif1 protein are not well understood. Here, we analyse genome-wide binding of budding yeast Rif1 by chromatin immunoprecipitation, during G1 phase and in S phase with replication progressing normally or blocked by hydroxyurea. Rif1 associates strongly with telomeres through interaction with Rap1. By comparing genomic binding of wild-type Rif1 and truncated Rif1 lacking the Rap1-interaction domain, we identify hundreds of Rap1-dependent and Rap1-independent chromosome interaction sites. Rif1 binds to centromeres, highly transcribed genes and replication origins in a Rap1-independent manner, associating with both early and late-initiating origins. Interestingly, Rif1 also binds around activated origins when replication progression is blocked by hydroxyurea, suggesting association with blocked forks. Using nascent DNA labelling and DNA combing techniques, we find that in cells treated with hydroxyurea, yeast Rif1 stabilises recently synthesised DNA. Our results indicate that, in addition to controlling DNA replication initiation, budding yeast Rif1 plays an ongoing role after initiation and controls events at blocked replication forks. Synopsis This study identifies genome-wide Rif1 bindings sites in budding yeast. Rif1 binds both replication origins and stalled replication forks and protects nascent DNA from degradation. ChIP-Seq analysis revealed Rap1-dependent and –independent budding yeast Rif1 chromosomal binding sites. Rif1 binds early and late origins without apparent preference. Rif1 binds stalled replication forks or post-replicative chromatin under replication stress, and protects nascent DNA from degradation. Rif1 associates with centromeres in S phase. Introduction Chromosomes are highly dynamic, and chromatin changes its structural composition during functional processes and at different cell cycle stages. For example, replication or transcription fork passage requires the disassembly then reassembly of nucleosomes. During mitosis, chromatin is compacted and must withstand the physical tension occurring during sister chromatid segregation. Additionally, spontaneous and replication-associated damage to chromosomes must be repaired in a timely way during the cell cycle. Failure or incomplete execution of any of these processes can cause genome instability. Rif1 is an evolutionarily conserved protein involved in multiple genome integrity pathways. Rif1 was originally identified in the budding yeast Saccharomyces cerevisiae as a component of telomeric chromatin that regulates telomere length 1, 2. Rif1 associates with telomeres, and with the MAT locus and mating cassettes, mainly through interaction with the transcription factor Rap1 3. Rap1 recognises a TG-rich motif present upstream of genes it regulates. This recognition motif also occurs within the telomeric terminal TG repeat sequences, and multiple copies of Rap1 bind telomeres 4. While originally isolated for its role in binding Rap1 at telomeres, Rif1 (Rap1-Interacting Factor 1) has recently been identified as an important regulator of DNA replication initiation, in a function conserved from yeast to human 5-11. Despite replication control being one of its conserved functions, it however proved difficult for many years to demonstrate binding of Rif1 at replication origin sites. Rif1 has been implicated in additional pathways of genome integrity, in particular directing double-strand break repair pathway choice 12-18, and suppressing or resolving mitotic chromosome entanglements 19, 20. A critical step in replication initiation is executed by Dbf4-dependent protein kinase (DDK), which promotes DNA replication initiation by phosphorylating the MCM complex to activate it as the replicative helicase. In DNA replication control, Rif1 counteracts the function of DDK by directing Protein Phosphatase 1 (PP1) to dephosphorylate MCM proteins and oppose replication initiation. Notably, this action of Rif1 as a substrate-targeting subunit for PP1 is evolutionarily conserved, with Rif1 also controlling replication initiation in mammalian cells 8-10, 24-26. Budding yeast Rif1 and PP1 bound to the chromosome ends specify the late replication timing of origins in the vicinity of telomeres 8, 10, 24-26. However, in S. cerevisiae, the importance of Rif1 for the replication timing programme at sites other than telomeres appears to be fairly minor in an unimpeded S phase 26. Although its contribution to specification of the replication temporal programme occurs primarily at telomeres, budding yeast Rif1 does nonetheless clearly affect initiation at numerous replication origins genome-wide, since when available DDK is limited, deletion of RIF1 permits replication initiation to occur throughout large tracts of the genome 9. Also, when replication is blocked by hydroxyurea (HU), additional replication origins show initiation in a rif1∆ mutant when compared to wild type 11. These observations suggest that Rif1 does play a critical role in controlling origin firing, especially under replication stress, even though the role of S. cerevisiae Rif1 in normal replication timing control otherwise appears largely limited to telomeric regions. Our understanding of the effects of Rif1 in replication control has been impeded by the fact that it has been difficult to detect Rif1 interacting directly with replication origins, even in S. cerevisiae which has the best understood replication origin sites of any eukaryote 27, 28. No high-resolution chromatin immunoprecipitation (ChIP) analysis has been described for S. cerevisiae Rif1, and until now, the available information describing chromosome association of Rif1 in budding yeast has been limited to a few very specific sites, including the telomeres, the MAT locus, and mating type cassettes—interactions all mediated mainly through interaction with Rap1 3. One impediment to genome-wide analysis of Rif1 binding has been the very strong preference displayed by Rif1 for these specific, repeated chromosomal loci, which tended to obscure binding to other loci in microarray analyses of ChIP experiments. To obtain a better understanding of Rif1 function in DNA replication and genome maintenance, we examined the chromatin association patterns of Rif1 by next-generation sequencing analysis of ChIP samples (ChIP-Seq), which provides an improved dynamic range of analysis compared to microarrays. As well as wild-type Rif1, we examined a mutated version of Rif1 that lacks the Rap1-interaction domain, to distinguish Rap1-dependent and Rap1-independent binding sites. Our results reveal Rif1 interaction with several new classes of chromosome loci. We find clear association of Rif1 with replication origins throughout the yeast genome. We additionally detect Rif1 localised to various other types of genomic site, including blocked replication forks, highly transcribed genes and centromeric sequences. These observations suggest potential new roles for Rif1 in modulating chromosome transactions. Investigating in particular the role of Rif1 at blocked replication forks, we find that at forks whose progression is blocked by hydroxyurea treatment, Rif1 is crucial to protect newly replicated DNA. Results The C-terminal portion of yeast Rif1 is dispensable for opposing DDK in replication control Rif1 associates with telomeric regions, consistent with its function in controlling telomere length and replication timing near telomeres 1, 2, 8, 11, 25. Rif1 additionally binds the HML, HMR and MAT loci, interacting with Rap1 at these sites as at telomeres 3, 29. However, although it impacts on replication control more broadly, the association of budding yeast Rif1 protein with other chromosomal loci has not been reported. We suspected that its strong preference for telomeric regions might have hindered the detection of Rif1 at non-telomeric regions in previous microarray studies. Structural studies revealed two domains within Rif1 that interact with Rap1: the Rap1-binding motif (RBM) and a C-terminal domain (CTD; Fig 1A) 30. To explore the behaviour and physiological functions of Rif1 independent of Rap1, we used a truncated version of Rif1 lacking the RBM and CTD (Fig 1A) 10. This C-terminally truncated RIF1 allele, rif1-∆C594, retains the ability to control DNA replication by counteracting DDK, since it represses growth of a cdc7-1 mutant strain at 30°C, like wild-type RIF1+. A rif1∆ allele in contrast permits growth of a cdc7-1 mutant at 30°C (Fig 1B), as previously described. The repressive effect of rif1-∆C594 on cdc7-1 growth is consistent with previous observations that the C-terminal region of Rif1 is dispensable for replication control 9, 10. We designate the truncated protein Rif1-∆C594. Figure 1. The C-terminus of yeast Rif1 is dispensable for control of DNA replication Structure of budding yeast Rif1 and C-terminally truncated mutant Rif1-∆C594. RBM, Rap1-binding motif; CTD, C-terminal domain. Rif1-∆C594 retains function to control DNA replication. Growth of a cdc7-1 rif1-∆C594 mutant was compared with growth of cdc7-1 RIF1 and cdc7-1 rif1∆ strains at 23°C (permissive temperature for cdc7-1 allele), 26°C (mild restrictive temperature) and 30°C (strict restrictive temperature). Download figure Download PowerPoint ChIP-Seq analysis identifies Rif1 genomic binding site dependent on Rap1 Since next-generation sequencing provides an increased dynamic range compared to microarray-based methods, we investigated whether ChIP-Seq analysis could reveal non-telomeric chromosome association sites of Rif1. We also tested binding of Rif1-∆C594, to distinguish Rap1-dependent and Rap1-independent chromosome association sites. Myc-tagged versions of Rif1 and Rif1-∆C594 were utilised to enable ChIP-Seq analysis of chromatin association. Binding was analysed during G1 phase (cells blocked with α-factor), 60 and 90 min after release from α-factor at 16°C, and in cells released from α-factor into hydroxyurea (HU) to block replication fork progression. As expected 3, full-length Rif1 showed strong binding to telomeres (Fig 2A blue plots), as well as mating type cassettes (Fig EV1A blue plots). Rif1-∆C594, in contrast, showed greatly reduced association with telomere and subtelomeric sequences (Fig 2A red plots) and virtually no association with mating type loci (Fig EV1A red plots). These effects on association with telomeres and mating loci are as expected, since both modes of binding depend to a large extent on interaction with Rap1. Figure 2. Full-length and C-terminally truncated Rif1 proteins bind distinct chromosomal loci Specimen overview of chromosome VI-left region showing results of ChIP-Seq analysis of Rif1 and Rif1-∆C594 proteins. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for chromosome VI coordinates 1–80,000. Y-axis shows enrichment values (linear scale, range is 0–3.5). Values below 1 are shown in grey, and values above 1 (i.e. sequences enriched in ChIP samples) are coloured blue (Rif1) and red (Rif1-∆C594). Plots show ChIP analysis results from cells arrested by α-factor (G1), released from α-factor at 16°C for 60 and 90 min, or released from α-factor into 0.2 M HU for 60 min at 23°C. Rap1-dependent association of Rif1 with the promoter regions of Rap1-controlled genes. ChIP enrichment around PAU3 (left) and MAM3 (right), both genes whose transcription is controlled by Rap1. Values above 1 (i.e. enriched) shown by overlaid blue and red histograms for Rif1 and Rif1-∆C594, respectively. Values below 1 shown in grey. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Rap1-dependent association of Rif1, but not Rif1-∆C, with telomeres, MAT locus and mating type cassettes Enrichment of ChIP signal relative to Input DNA of Rif1 (blue) and Rif1-∆C594 (red) along entire chromosome III. Y-axis scale is 0–35. Same data with Y-axis scale 0–3.5. Download figure Download PowerPoint As well as associating with telomeres, Rap1 regulates multiple genes as a promoter-bound transcription factor. At these sites of Rap1 transcriptional control, we detected binding of Rif1 dependent on its C-terminal Rap1 interaction domain. For example, full-length Rif1 bound the promoter regions of the Rap1-regulated genes PAU3 and MAM3 (Fig 2B, blue plots) 31-33. In contrast, Rif1-∆C594 protein did not bind these promoters (Fig 2B, red plots). Our results therefore clearly demonstrate that Rif1-∆C594 is defective for Rap1-dependent association, both with telomeres and sites of Rap1-mediated transcription regulation. ChIP-Seq analysis identifies multiple Rap1-independent Rif1 genomic binding sites In addition to the expected Rap1-dependent associations, we observed that full-length Rif1 and Rif1-∆C594 bind hundreds of additional sites, with binding intensity often appearing higher for the Rif1-∆C594 protein. Both full-length Rif1 and Rif1-∆C594 associate with many DNA replication origins (Figs 2A, and 3A and B), including both telomere-proximal origins and origins distant from telomeres, as discussed in more detail below. Figure 3. Rif1 and Rif1-∆C bind replication origins, centromeres and tRNA genes ChIP-Seq analysis of Rif1 and Rif1-∆C594 proteins shows tRNA gene and origin binding, with widened peaks at early origins ARS606 and ARS607 in HU block. Plots show chromosome VI genome coordinates 160,000–210,000. Plot colours here and in following Figures are as in Fig 2B. Widened peaks are not observed in unperturbed S phase samples, or for Rif1-∆C594. Association of Rif1-∆C594 at replication origins is enhanced in HU block. ChIP enrichment of Rif1 and Rif1-∆C594 around early origin ARS1426 (left) and late origin ARS1412 (right). Differential association of Rif1 and Rif1-∆C594 to centromeres. ChIP enrichment of Rif1 and Rif1-∆C594 around the CEN4 locus. Download figure Download PowerPoint Unexpectedly, we also found Rif1 and Rif1-∆C594 association with the coding regions of highly transcribed genes, for example ACT1, RPL22B and HAC1 genes as shown in Fig 2A, and tRNA genes as illustrated by Fig 3A. Since Rif1-∆C594 binds to these sites with generally similar intensity to full-length Rif1, association with highly transcribed loci appears to be independent of Rap1 interaction. We also noticed association of Rif1 with centromeres (Figs 3C and EV2A). Full-length Rif1 binds some centromeres fairly weakly during G1 phase, but binds much more strongly when cells enter S phase (Fig 3C), and to virtually all centromeres in HU-blocked cells (Fig EV2A). Rif1-∆C594 on the other hand showed slightly higher association with centromeres during G1 phase than the full-length protein, but reduced association under conditions of HU blockage (Fig EV2A), suggesting that Rif1 association with centromeres is largely dependent on its C-terminus. This association is, however, unlikely to occur through Rap1 interaction, because previous genome-wide ChIP study did not find Rap1 associated with centromeres 33. Click here to expand this figure. Figure EV2. Association of Rif1 and Rif1-∆C594 with centromeres and DNA replication origins Association of Rif1 protein with centromeres. Top. Average ChIP profiles at all 16 centromeres of Rif1 and Rif1-∆C594 in G1 phase and HU-arrested cells. Bottom. Heat maps showing Rif1 and Rif1-∆C594 ChIP enrichment at individual centromeres. Association of Rif1 and Rif1-∆C594 with replication origins. Locations of ChIP peaks, identified by peak-calling algorithm MACS2, were compared with locations of origins of known replication timing (shown as “Crabbe”) 34. Subtelomeric origins (< 15 kb from chromosome ends) were excluded from this analysis. Distributions of peaks detected at early and late origins were statistically tested against distribution of total origins with known replication timing, using hypergeometric distribution. The P-values obtained are shown above. Download figure Download PowerPoint Rif1 associates with replication origins We observed binding of Rif1 and Rif1-∆C594 to many replication origin sites genome-wide, with typical patterns observed illustrated by Figs 2A, and 3A and B). Rif1 associated with both early-activated (e.g. ARS607, Fig 3A and ARS1426, Fig 3B left) and late-activated (e.g. ARS1412, Fig 3B right) origins, before and after origin initiation (e.g. at ARS1412, 90 min after release from α-factor). Rif1-∆C594 was observed more frequently at origins than full-length Rif1 (e.g. at ARS603 in Fig 2A), presumably reflecting increased availability of the truncated protein for binding to non-telomeric sites, caused by its release from telomeres. Results of the ChIP-Seq analysis were confirmed at individual origins by ChIP followed by real-time quantitative PCR (ChIP-qPCR; Fig 4A). For example, using ChIP-qPCR we observed clear association of Rif1-∆C594 with late origin ARS1412 during G1 phase, association that was further increased at an HU block (Fig 4A right panel). The full-length Rif1 protein showed somewhat weaker association with ARS1412, again occurring in both G1 phase and HU-arrested cells (Fig 4A right panel). This association pattern is consistent with the ChIP-Seq result at the same locus (Fig 3B right). Rif1 and Rif1-∆C594 showed similar binding patterns at an early origin (ARS1426, Fig 4A left), again consistent with the ChIP-Seq result (Fig 3B left). Although there was considerable scatter in the data (as is typical for ChIP-qPCR results close to the detection threshold), the results consistently revealed above-background binding and confirm that the ChIP-Seq profiles represent the genome-wide binding efficiencies of Rif1 and Rif1-∆C594 reasonably well. These ChIP-qPCR analyses do also generally suggest higher binding levels of the ∆594 protein than wild type, possibly reflecting increased availability of the truncated protein due to its release from telomeres. By ChIP-Seq Rif1-∆C594 also appears to show higher binding than full-length Rif1 at many loci (e.g. at ARS1412). However, peak heights in ChIP-Seq data may not provide an accurate measure of occupancy, due to limitations in the standardisation of ChIP-Seq results. Figure 4. Analysis of origin binding reveals full-length Rif1 binds broad regions near early origins in HU ChIP-qPCR confirmation of Rif1 and Rif1-∆C594 association with replication origins. ChIP was performed using cells arrested in α-factor (open bars) or HU (grey bars), and qPCR analysis performed for early origin ARS1426 (left) and late origin ARS1412 (right). Values shown are “normalised ChIP efficiencies” obtained by subtracting the value obtained at a control locus (see Materials and Methods). Bars indicate the averages of two biological replicates, with values from each replicate shown by open circles. Numbers of early and late origins associated with Rif1 and Rif1-∆C594 peaks. Plot showing numbers of early and late origins bound by Rif1 and Rif1-∆C594 in G1 phase and HU-blocked cells, based on peak-calling results. Replication timing programme is intact in rif1-∆C594 cells. Replication of selected origins at an HU block analysed by BrdU incorporation. Cells were synchronised by α-factor and released into the medium containing 0.2 M HU and 1.13 mM BrdU. Plots show the percentage of total ARS607, ARS422.5 or ARS1412 DNA pulled down by IP with anti-BrdU, calculated from two biological replicate samples. Bars indicate the average of two biological replicates and open circles the values from each replicate. Insets in ARS422,5 and ARS1412 panels show the same data with Y-axis scales adjusted to 0–0.03%. Peaks of Rif1 become broader in HU-arrested cells. Box and whisker plot shows peak widths of full-length Rif1 at early and late origins in G1 phase and HU-blocked cells. Analysis was performed on those origins detected by peak calling as associated with a Rif1 peak. Boxes show the range of 25th to 75th percentiles, with horizontal lines within the boxes representing 50th percentiles. Whiskers represent 95% confidence intervals. Outliers are presented as open circles. Numbers of origins analysed are as follows: 46 early origins in G1, 56 late origins in G1, 85 early origins in HU and 24 late origins in HU. Download figure Download PowerPoint We performed peak-calling analysis on the ChIP-Seq data to allow comparison of the detected peaks with experimentally confirmed replication origins. Of the 410 replication origins that are experimentally confirmed in S. cerevisiae, we used a list of 329 origins that are not telomere proximal (> 15 kb from telomeres) and whose replication timing can be designated as either early or late (based on whether they have initiated in HU-arrested wild-type cells 34). Within this list, 165 origins were assigned as early non-telomeric and 164 as late non-telomeric origins. We observed full-length Rif1 associated with 104 of these 329 replication origins in G1 cells (Fig 4B; see also Fig EV2B); 47 of these were early and 57 late origins, indicating that Rif1 binds with no particular preference for early or late origins (Fig 4B). Rif1-∆C594 associated with a larger number of origins in G1 phase, 174 of the total 329, but similar to the full-length protein showed no clear preference for either early or late. We also observed clear binding of Rif1 proteins to origin sites in cells where replication was blocked by HU. In HU-blocked cells, full-length Rif1 bound to 111 of the origin sites; 86 of these corresponded to early and 25 to late-initiating origins, so that under HU blockage conditions full-length Rif1 shows a clear preference for early over late origin sites (Fig 4B). The highest number of origin sites was bound in Rif1-∆C594 cells blocked with HU, where a large majority of both early and late origins (300 of the total 329) showed association with this truncated Rif1 protein. Overall, while Rif1 and Rif1-∆C594 have somewhat different preferences for origin association, these preferences do not directly reflect the initiation time of origins, or their pre- or post-activation status. Replicating timing is maintained in rif1-∆C594 mutant The preference of full-length Rif1 for early over late origins in HU (Fig 4B) led us to consider the possibility that, after S phase begins, Rif1 can only bind origins that have already initiated. Such a possibility could be consistent with the binding of Rif1-∆C594 to both early and late origin sites in HU, if it were the case that in the rif1-∆C594 mutant the replication timing programme was disrupted, so that almost all origins initiate before the HU block. To investigate this possibility, we tested whether the replication timing programme is intact in the rif1-∆C594 strain by examining bromodeoxyuridine (BrdU) incorporation at early and late origins in HU-blocked cells. As assessed by BrdU incorporation, early origin ARS607 was already activated in HU as expected (Fig 4C left). Two different late origins, ARS422.5 and ARS1412, were inactive in both RIF1+ and also in rif1-∆C594 strains (Fig 4C, middle and right), indicating that these origins remain inactive in HU and the replication timing programme is not lost in the rif1-∆C594 mutant. Both of these late origins showed somewhat increased BrdU incorporation in rif1Δ as expected based on previous analysis 11. Overall therefore, the rif1-∆C594 mutant does not undergo wholesale disruption of the replication timing programme but instead maintains the distinction between early and late origin activation. It moreover appears that Rif1-∆C594 does associate with virtually all origins under HU-arrested condition (Figs 4B and EV2B), irrespective of whether origins have been activated or not. Rif1 protects nascent DNA at HU-blocked replication forks We noticed that peaks of full-length Rif1 at early origins tend to broaden in HU (e.g. ARS606 & ARS607, Fig 3A). This broadened association seems to be specific to the HU-arrested condition, because it was not observed in the unperturbed S phase samples (Fig 3A). Systematic analysis at early origins confirmed an increase in median peak width at early origins from 0.4 kb in G1 phase to 1.6 kb at the HU block (Figs 4D and EV3, heat maps). We did not observe such peak broadening at late origins (Figs 4D and EV3), suggesting that peak broadening requires origin activation, and probably therefore reflects association with replication forks stalled by HU inhibition. In a few cases (e.g. early origin ARS1528, Fig EV4A), we indeed observed peak splitting surrounding the origin site, consistent with the pattern representing association with blocked forks. Interestingly, the Rif1-∆C594 mutant protein did not exhibit this pattern of replication fork association, as evidenced by the fact that peak broadening was not observed around early origins in HU (Fig EV3). Click here to expand this figure. Figure EV3. Average and heat map presentation of Rif1 and Rif1-∆C594 ChIP profiles at replication originsRif1 and Rif1-∆C594 ChIP signals were aligned for the all DNA replication origins with known replication timing and predicted ARS Consensus Sequence (ACS) 34, 63. Origins are centred on the predicted ACS site. Top panels show the average ChIP profiles and heat maps at the 115 early initiating origins, and the bottom panel shows those at the 90 late-initiating origins. The two late origin sites showing particularly strong, broad Rif1 ChIP signals (appearing at top of “Late origins Rif1” panels) correspond to the replication origins located within mating type cassettes. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Association of Rif1 with replication forks and protection of nascent DNA# Split Rif1 ChIP peak observed at ARS1528 in HU. ChIP profiles of Rif1 and Rif1-∆C594 are shown as in Fig 2B. Splitting of Rif1 peak indicated by blue arrows. Position of the predicted ACS for ARS1528 marked by a green dotted line. Rif1 protects nascent DNA from degradation at stalled forks. Protection of nascent DNA from degradation was assayed as in Fig 5, but with slightly longer labelling time (22 min). Cells were blocked in HU for 0, 1 and 2 h af" @default.
• W2885440056 created "2018-08-22" @default.
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• W2885440056 date "2018-08-13" @default.
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• W2885440056 title "Budding yeast Rif1 binds to replication origins and protects <scp>DNA</scp> at blocked replication forks" @default.
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• W2885440056 doi "https://doi.org/10.15252/embr.201846222" @default.
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