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Double-strand breaks (DSBs) are genotoxic DNA lesions that pose significant threats to genomic stability, necessitating precise and efficient repair mechanisms to prevent cell death or mutations. DSBs are repaired through nonhomologous end-joining (NHEJ) or homology-directed repair (HDR), which includes homologous recombination (HR) and single-strand annealing (SSA). CtIP and Rif1 are conserved proteins implicated in DSB repair pathway choice, possibly through redundant roles in promoting DNA end-resection required for HDR. Although the roles of these proteins have been well-established in other organisms, the role of Rif1 and its potential redundancies with CtIP in Drosophila melanogaster remain elusive. To examine the roles of DmCtIP and DmRif1 in DSB repair, this study employed the direct repeat of white (DR- white) assay, tracking across indels by decomposition (TIDE) analysis, and P { wIw_2 kb 3 ′} assay to track repair outcomes in HR, NHEJ, and SSA, respectively. These experiments were performed in DmCtIPΔ/Δ single mutants, DmRif1 Δ/Δ single mutants, and DmRif1 Δ/Δ ; DmCtIPΔ/Δ double mutants. This work demonstrates significant defects in both HR and SSA repair in DmCtIPΔ/Δ and DmRif1 Δ/Δ single mutants. However, experiments in DmRif1 Δ/Δ ; DmCtIPΔ/Δ double mutants reveal that DmCtIP is epistatic to DmRif1 in promoting HDR. Overall, this study concludes that DmRif1 and DmCtIP do not perform their activities in a redundant pathway, but rather DmCtIP is the main driver in promoting HR and SSA, most likely through its role in end resection. [ABSTRACT FROM AUTHOR]

The measurement of dynamic changes in protein level and localization throughout the cell cycle is of major relevance to studies of cellular processes tightly coordinated with the cycle, such as replication, transcription, DNA repair, and checkpoint control. Currently available methods include biochemical assays of cells in bulk following synchronization, which determine protein levels with poor temporal and no spatial resolution. Taking advantage of genetic engineering and live-cell microscopy, we performed time-lapse imaging of cells expressing fluorescently tagged proteins under the control of their endogenous regulatory elements in order to follow their levels throughout the cell cycle. We effectively discern between cell cycle phases and S subphases based on fluorescence intensity and distribution of co-expressed proliferating cell nuclear antigen (PCNA)-mCherry. This allowed us to precisely determine and compare the levels and distribution of multiple replication-associated factors, including Rap1-interacting factor 1 (RIF1), minichromosome maintenance complex component 6 (MCM6), origin recognition complex subunit 1 (ORC1, and Claspin, with high spatiotemporal resolution in HeLa Kyoto cells. Combining these data with available mass spectrometry-based measurements of protein concentrations reveals the changes in the concentration of these proteins throughout the cell cycle. Our approach provides a practical basis for a detailed interrogation of protein dynamics in the context of the cell cycle. [ABSTRACT FROM AUTHOR]

Ovarian cancer, more precisely high-grade serous ovarian cancer, is one of the most lethal age-independent gynecologic malignancies in women worldwide, regardless of age. There is mounting evidence that there is a link between telomeres and the RIF1 protein and the proliferation of cancer cells. Telomeres are hexameric (TTAGGG) tandem repeats at the tip of chromosomes that shorten as somatic cells divide, limiting cell proliferation and serving as an important barrier in preventing cancer. RIF1 (Replication Time Regulation Factor 1) plays, among other factors, an important role in the regulation of telomere length. Interestingly, RIF1 appears to influence the DNA double-strand break (DSB) repair pathway. However, detailed knowledge regarding the interplay between RIF1 and telomeres and their degree of engagement in epithelial ovarian cancer (EOC) is still elusive, despite the fact that such knowledge could be of relevance in clinical practice to find novel biomarkers. In this review, we provide an update of recent literature to elucidate the relation between telomere biology and the RIF1 protein during the development of ovarian cancer in women. [ABSTRACT FROM AUTHOR]

Polytene chromosomes in Drosophila serve as a classical model for cytogenetic studies. However, heterochromatic regions of chromosomes are typically under-replicated, hindering their analysis. Mutations in the Rif1 gene lead to additional replication of heterochromatic sequences, including satellite DNA, in salivary gland cells. Here, we investigated the impact of the Rif1 mutation on heterochromatin in polytene chromosomes formed in ovarian germ cells due to the otu gene mutation. By the analysis of otu11; Rif11 double mutants, we found that, in the presence of the Rif1 mutation, ovarian cells undergo additional polytenization of pericentromeric regions. This includes the formation of large chromatin blocks composed of satellite DNA. Thus, the effects of the Rif1 mutation are similar in salivary gland and germ cells. The otu11; Rif11 system opens new possibilities for studying factors associated with heterochromatin during oogenesis. [ABSTRACT FROM AUTHOR]

HeLa cells are a cell line with two unique cellular features: a short-shouldered survival curve and two peaks of radioresistance during the cell cycle phase, while their underlying mechanisms remain unclear. We herein proposed that these radiobiological features are due to a common mechanism by which radiation suppresses homologous recombination repair (HRR) in a dose-dependent manner. This radio-suppression of HRR is mediated by an intra-S checkpoint and reduces survivals of cells in S phase, especially early S phase, resulting in both short shoulder and radioresistance with two peaks in the cell cycle. This new explanation may not be limited to HeLa cells since a similar close association of these features is also observed in other type of cells. [ABSTRACT FROM AUTHOR]

Background: RAP1 interacting factor 1 (Rif1) is highly expressed in mice embryos and mouse embryonic stem cells (mESCs). It plays critical roles in telomere length homeostasis, DNA damage, DNA replication timing and ERV silencing. However, whether Rif1 regulates early differentiation of mESC is still unclear. Methods: In this study, we generated a Rif1 conditional knockout mouse embryonic stem (ES) cell line based on Cre-loxP system. Western blot, flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR), RNA high-throughput sequencing (RNA-Seq), chromatin immunoprecipitation followed high-throughput sequencing (ChIP-Seq), chromatin immunoprecipitation quantitative PCR (ChIP-qPCR), immunofluorescence, and immunoprecipitation were employed for phenotype and molecular mechanism assessment. Results: Rif1 plays important roles in self-renewal and pluripotency of mESCs and loss of Rif1 promotes mESC differentiation toward the mesendodermal germ layers. We further show that Rif1 interacts with histone H3K27 methyltransferase EZH2, a subunit of PRC2, and regulates the expression of developmental genes by directly binding to their promoters. Rif1 deficiency reduces the occupancy of EZH2 and H3K27me3 on mesendodermal gene promoters and activates ERK1/2 activities. Conclusion: Rif1 is a key factor in regulating the pluripotency, self-renewal, and lineage specification of mESCs. Our research provides new insights into the key roles of Rif1 in connecting epigenetic regulations and signaling pathways for cell fate determination and lineage specification of mESCs. [ABSTRACT FROM AUTHOR]

To examine the established link between DNA replication and telomere length, we tested whether firing of telomeric origins would cause telomere lengthening. We found that RIF1 mutants that block Protein Phosphatase 1 (PP1) binding activated telomeric origins but did not elongate telomeres. In a second approach, we found overexpression of ∆N-Dbf4 and Cdc7 increased DDK activity and activated telomeric origins, yet telomere length was unchanged. We tested a third mechanism to activate origins using the sld3-A mcm5-bob1 mutant that de-regulates the pre-replication complex, and again saw no change in telomere length. Finally, we tested whether mutations in RIF1 that cause telomere elongation would affect origin firing. We found that neither rif1-∆1322 nor rif1HOOK affected firing of telomeric origins. We conclude that telomeric origin firing does not cause telomere elongation, and the role of Rif1 in regulating origin firing is separable from its role in regulating telomere length.

Abstract Mouse embryonic stem cells (mESCs) cycle in and out of a transient 2-cell (2C)-like totipotent state, driven by a complex genetic circuit involves both the coding and repetitive sections of the genome. While a vast array of regulators, including the multi-functional protein Rif1, has been reported to influence the switch of fate potential, how they act in concert to achieve this cellular plasticity remains elusive. Here, by modularizing the known totipotency regulatory factors, we identify an unprecedented functional connection between Rif1 and the non-canonical polycomb repressive complex PRC1.6. Downregulation of the expression of either Rif1 or PRC1.6 subunits imposes similar impacts on the transcriptome of mESCs. The LacO-LacI induced ectopic colocalization assay detects a specific interaction between Rif1 and Pcgf6, bolstering the intactness of the PRC1.6 complex. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) analysis further reveals that Rif1 is required for the accurate targeting of Pcgf6 to a group of genomic loci encompassing many genes involved in the regulation of the 2C-like state. Depletion of Rif1 or Pcgf6 not only activates 2C genes such as Zscan4 and Zfp352, but also derepresses a group of the endogenous retroviral element MERVL, a key marker for totipotency. Collectively, our findings discover that Rif1 can serve as a novel auxiliary component in the PRC1.6 complex to restrain the genetic circuit underlying totipotent fate potential, shedding new mechanistic insights into its function in regulating the cellular plasticity of embryonic stem cells.

53BP1 (also known as TP53BP1) is a key mediator of the nonhomologous end joining (NHEJ) DNA repair pathway, which is the primary repair pathway in interphase cells. However, the mitotic functions of 53BP1 are less well understood. Here, we describe 53BP1 mitotic stress bodies (MSBs) formed in cancer cell lines in response to delayed mitosis. These bodies displayed liquid--liquid phase separation characteristics, were close to centromeres, and included lamin A/C and the DNA repair protein RIF1. After release from mitotic arrest, 53BP1 MSBs decreased in number and moved away from the chromatin. Using GFP fusion constructs, we found that the 53BP1 oligomerization domain region was required for MSB formation, and that inclusion of the 53BP1 N terminus increased MSB size. Exogenous expression of 53BP1 did not increase MSB size or number but did increase levels of MSB-free 53BP1. This was associated with slower mitotic progression, elevated levels of DNA damage and increased apoptosis, which is consistent with MSBs suppressing a mitotic surveillance by 53BP1 through sequestration. The 53BP1 MSBs, which were also found spontaneously in a subset of normally dividing cancer cells but not in non-transformed cells (ARPE-19), might facilitate the survival of cancer cells following aberrant mitoses. [ABSTRACT FROM AUTHOR]

Abstract Background Maternal obesity impairs embryonic developmental potential and significantly increases the risks of metabolic disorders in offspring. However, the epigenetic transmission mechanism of maternal metabolic abnormalities is still poorly understood. Methods We established an obesity model in female mice by high-fat diet (HFD) feeding. The effects of the HFD on the developmental potential of oocytes and embryos, the metabolic phenotype, and epigenetic modifications were investigated. The efficacy of metformin administration was assessed. Finally, the regulatory pathway of epigenetic remodeling during zygotic genome activation (ZGA) was explored. Results Maternal HFD consumption significantly impaired glucose tolerance and increased the risk of metabolic disorders in F0 and F1 mice. Maternal HFD consumption also decreased embryonic developmental potential, increased reactive oxygen species (ROS) and γH2AX levels, and reduced the mitochondrial membrane potential (MMP) within oocytes, causing high levels of oxidative stress damage and DNA damage. Starting with this clue, we observed significantly increased RIF1 levels and shortened telomeres in obese mice. Moreover, significant abnormal DNA methylation and histone modification remodeling were observed during ZGA in obese mice, which may be coregulated by RIF1 and the ZGA marker gene MuERV-L. Metformin treatment reduced RIF1 levels, and partially improved ZGA activation status by rescuing epigenetic modification remodeling in oocytes and preimplantation embryos of obese mice. RIF1 knockdown experiments employing Trim-Away methods showed that RIF1 degradation altered the H3K4me3 and H3K9me3 enrichment and then triggered the MuERV-L transcriptional activation. Moreover, ChIP-seq data analysis of RIF1 knockouts also showed that RIF1 mediates the transcriptional regulation of MuERV-L by changing the enrichment of H3K4me3 and H3K9me3 rather than by altered DNA methylation. Conclusion Elevated RIF1 in oocytes caused by maternal obesity may mediate abnormal embryonic epigenetic remodeling and increase metabolic risk in offspring by regulating histone modifications on MuERV-L, which can be partially rescued by metformin treatment.

Nearly 70 years after the proposal of semiconservative replication of generic material by Watson and Crick, we now understand many of the proteins involved in the replication of host chromosomes and how they operate. The initiator and replicator, proposed in the replicon hypothesis, are now well defined in both prokaryotes and eukaryotes. On the other hand, studies in prokaryotes and Archaea indicate alternative modes of initiation, which may not depend on an initiator. Here I summarize recent progress in the field of DNA replication and discuss the evolution of replication systems. [ABSTRACT FROM AUTHOR]

The pericentromeric heterochromatin is largely composed of repetitive sequences, making it difficult to analyze with standard molecular biological methods. At the same time, it carries many functional elements with poorly understood mechanisms of action. The search for new experimental models for the analysis of heterochromatin is an urgent task. In this work, we used the Rif1 mutation, which suppresses the underreplication of all types of repeated sequences, to analyze heterochromatin regions in polytene chromosomes of Drosophila melanogaster. In the Rif1 background, we discovered and described in detail a new inversion, In(1)19EHet, which arose on a chromosome already carrying the In(1)sc8 inversion and transferred a large part of X chromosome heterochromatin, including the nucleolar organizer to a new euchromatic environment. Using nanopore sequencing and FISH, we have identified the eu- and heterochromatin breakpoints of In(1)19EHet. The combination of the new inversion and the Rif1 mutation provides a promising tool for studies of X chromosome heterochromatin structure, nucleolar organization, and the nucleolar dominance phenomenon. In particular, we found that, with the complete polytenization of rDNA repeats, the nucleolus consists of a cloud-like structure corresponding to the classical nucleolus of polytene chromosomes, as well as an unusual intrachromosomal structure containing alternating transcriptionally active and inactive regions. [ABSTRACT FROM AUTHOR]

In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using Xenopus egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signalling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. Furthermore, we show that either Yap or Rif1 depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. In Xenopus embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that either Yap or Rif1 depletion triggers an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as brakes to control the DNA replication program in early embryos and post-embryonic stem cells.

Ewing sarcoma is a cancer of bone and soft tissue in children and young adults primarily driven by the EWS-FLI1 fusion oncoprotein, which has been undruggable. Here, we report that Ewing sarcoma depends on secreted sphingomyelin phosphodiesterase 1 (SMPD1), a ceramide-generating enzyme, and ceramide. We find that G-protein-coupled receptor 64 (GPR64)/adhesion G-protein-coupled receptor G2 (ADGRG2) responds to ceramide and mediates critical growth signaling in Ewing sarcoma. We show that ceramide induces the cleavage of the C-terminal intracellular domain of GPR64, which translocates to the nucleus and restrains the protein levels of RIF1 in a manner dependent on SPOP, a substrate adaptor of the Cullin3-RING E3 ubiquitin ligase. We demonstrate that both SMPD1 and GPR64 are transcriptional targets of EWS-FLI1, indicating that SMPD1 and GPR64 are EWS-FLI1-induced cytokine-receptor dependencies. These results reveal the SMPD1-ceramide-GPR64 pathway, which drives Ewing sarcoma growth and is amenable to therapeutic intervention. [Display omitted] • Ewing sarcoma depends on secreted SMPD1, a ceramide-generating enzyme, and ceramide • GPR64 responds to ceramide and mediates critical growth signaling in Ewing sarcoma • Ceramide induces the cleavage of the C-terminal intracellular domain (ICD) of GPR64 • GPR64 ICD restrains the protein levels of RIF1 via a SPOP-dependent mechanism Ceramide exhibits a variety of biological activities, but its role in receptor-mediated signaling is unknown. Suvarna et al. report that GPR64 responds to ceramide and mediates critical growth signaling in Ewing sarcoma. Ceramide induces the cleavage of the GPR64 intracellular domain, which serves as a platform for RIF1 degradation. [ABSTRACT FROM AUTHOR]

In the metazoan S phase, coordinated firing of clusters of origins replicates different parts of the genome in a temporal program. Despite advances, neither the mechanism controlling timing nor that coordinating firing of multiple origins is fully understood. Rif1, an evolutionarily conserved inhibitor of DNA replication, recruits protein phosphatase 1 (PP1) and counteracts firing of origins by S-phase kinases. During the midblastula transition (MBT) in Drosophila embryos, Rif1 forms subnuclear hubs at each of the large blocks of satellite sequences and delays their replication. Each Rif1 hub disperses abruptly just prior to the replication of the associated satellite sequences. Here, we show that the level of activity of the S-phase kinase, DDK, accelerated this dispersal program, and that the level of Rif1-recruited PP1 retarded it. Further, Rif1-recruited PP1 supported chromatin association of nearby Rif1. This influence of nearby Rif1 can create a "community effect" counteracting kinase-induced dissociation such that an entire hub of Rif1 undergoes switch-like dispersal at characteristic times that shift in response to the balance of Rif1-PP1 and DDK activities. We propose a model in which the spatiotemporal program of late replication in the MBT embryo is controlled by self-stabilizing Rif1-PP1 hubs, whose abrupt dispersal synchronizes firing of associated late origins. [ABSTRACT FROM AUTHOR]

RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular feature that enables the genome-protective function of RIF1 during DNA replication stress.

Successful duplication of the genome requires the accurate replication of billions of base pairs of DNA within a relatively short time frame. Failure to accurately replicate the genome results in genomic instability and a host of diseases. To faithfully and rapidly replicate the genome, DNA replication must be tightly regulated and coordinated with many other nuclear processes. These regulations, however, must also be flexible as replication kinetics can change through development and differentiation. Exactly how DNA replication is regulated and how this regulation changes through development is an active field of research. One aspect of genome duplication where much remains to be discovered is replication timing (RT), which dictates when each segment of the genome is replicated during S phase. All organisms display some level of RT, yet the precise mechanisms that govern RT remain are not fully understood. The study of Rif1, a protein that actively regulates RT from yeast to humans, provides a key to unlock the underlying molecular mechanisms controlling RT. The paradigm for Rif1 function is to delay helicase activation within certain regions of the genome, causing these regions to replicate late in S phase. Many questions, however, remain about the intricacies of Rif1 function. Here, we review the current models for the activity of Rif1 with the goal of trying to understand how Rif1 functions to establish the RT program. [ABSTRACT FROM AUTHOR]

Background: Maternal obesity impairs embryonic developmental potential and significantly increases the risks of metabolic disorders in offspring. However, the epigenetic transmission mechanism of maternal metabolic abnormalities is still poorly understood. Methods: We established an obesity model in female mice by high-fat diet (HFD) feeding. The effects of the HFD on the developmental potential of oocytes and embryos, the metabolic phenotype, and epigenetic modifications were investigated. The efficacy of metformin administration was assessed. Finally, the regulatory pathway of epigenetic remodeling during zygotic genome activation (ZGA) was explored. Results: Maternal HFD consumption significantly impaired glucose tolerance and increased the risk of metabolic disorders in F0 and F1 mice. Maternal HFD consumption also decreased embryonic developmental potential, increased reactive oxygen species (ROS) and γH2AX levels, and reduced the mitochondrial membrane potential (MMP) within oocytes, causing high levels of oxidative stress damage and DNA damage. Starting with this clue, we observed significantly increased RIF1 levels and shortened telomeres in obese mice. Moreover, significant abnormal DNA methylation and histone modification remodeling were observed during ZGA in obese mice, which may be coregulated by RIF1 and the ZGA marker gene MuERV-L. Metformin treatment reduced RIF1 levels, and partially improved ZGA activation status by rescuing epigenetic modification remodeling in oocytes and preimplantation embryos of obese mice. RIF1 knockdown experiments employing Trim-Away methods showed that RIF1 degradation altered the H3K4me3 and H3K9me3 enrichment and then triggered the MuERV-L transcriptional activation. Moreover, ChIP-seq data analysis of RIF1 knockouts also showed that RIF1 mediates the transcriptional regulation of MuERV-L by changing the enrichment of H3K4me3 and H3K9me3 rather than by altered DNA methylation. Conclusion: Elevated RIF1 in oocytes caused by maternal obesity may mediate abnormal embryonic epigenetic remodeling and increase metabolic risk in offspring by regulating histone modifications on MuERV-L, which can be partially rescued by metformin treatment. [ABSTRACT FROM AUTHOR]

Rif1 is a large multifaceted protein involved in various processes of DNA metabolism – from telomere length regulation and replication to double-strand break repair. The mechanistic details of its action, however, are often poorly understood. Here, we report functional characterization of the Rif1 homologue from methylotrophic thermotolerant budding yeast Hansenula polymorpha DL-1. We show that, similar to other yeast species, H. polymorpha Rif1 suppresses telomerase-dependent telomere elongation. We uncover two novel modes of Rif1 recruitment at H. polymorpha telomeres: via direct DNA binding and through the association with the Ku heterodimer. Both of these modes (at least partially) require the intrinsically disordered N-terminal extension – a region of the protein present exclusively in yeast species. We also demonstrate that Rif1 binds Stn1 and promotes its accumulation at telomeres in H. polymorpha.

The onset of random X chromosome inactivation in mouse requires the switch from a symmetric to an asymmetric state, where the identities of the future inactive and active X chromosomes are assigned. This process is known as X chromosome choice. Here, we show that RIF1 and KAP1 are two fundamental factors for the definition of this transcriptional asymmetry. We found that at the onset of differentiation of mouse embryonic stem cells (mESCs), biallelic up‐regulation of the long non‐coding RNA Tsix weakens the symmetric association of RIF1 with the Xist promoter. The Xist allele maintaining the association with RIF1 goes on to up‐regulate Xist RNA expression in a RIF1‐dependent manner. Conversely, the promoter that loses RIF1 gains binding of KAP1, and KAP1 is required for the increase in Tsix levels preceding the choice. We propose that the mutual exclusion of Tsix and RIF1, and of RIF1 and KAP1, at the Xist promoters establish a self‐sustaining loop that transforms an initially stochastic event into a stably inherited asymmetric X‐chromosome state. SYNOPSIS: Random X chromosome inactivation regulated by Xist and Tsix lncRNAs involves switching from a symmetric to an asymmetric state. Here, this process is shown to be differentially controlled by chromatin factors RIF1 and KAP1 in mouse embryonic stem cells. Tsix RNA levels increase at the onset of differentiation.Tsix RNA asymmetrically weakens RIF1 association with one Xist promoter.In the absence of RIF1, KAP1 gains access to one of the two Xist promoters/Tsix terminators.KAP1 binding boosts the levels of Tsix, reinforcing RIF1 exclusion from the future active X chromosome.RIF1 marks the future inactive X chromosome by promoting in cis expression of Xist. [ABSTRACT FROM AUTHOR]

DNA damage response mediator protein 53BP1 is a key regulator of non-homologous end-joining (NHEJ) repair. 53BP1 protects DNA broken ends from resection by recruiting two downstream factors, RIF1 (RAP1-interacting factor 1) and PTIP (Pax transactivation domain-interacting protein), to double-stranded breaks (DSBs) via ATM (ataxia telangiectasia mutated)-mediated 53BP1 phosphorylation, and competes with BRCA1-mediated homologous recombination (HR) repair in G1 phase. In contrast, BRCA1 antagonizes 53BP1-direct NHEJ repair in S/G2 phases. We and others have found that BRCA1 prevents the translocation of RIF1 to DSBs in S/G2 phases; however, the underlying mechanism remains unclear. Here we show that efficient ATM-dependent 53BP1 phosphorylation is restricted to the G1 phase of the cell cycle, as a consequence RIF1 and PTIP accumulation at DSB sites only occur in G1 phase. Mechanistically, both BRCT and RING domains of BRCA1 are required for the inhibition of 53BP1 phosphorylation in S and G2 phases. Thus, our findings reveal how BRCA1 antagonizes 53BP1 signaling to ensure that HR repair is the dominant repair pathway in S/G2 phases.

The pericentromeric heterochromatin is largely composed of repetitive sequences, making it difficult to analyze with standard molecular biological methods. At the same time, it carries many functional elements with poorly understood mechanisms of action. The search for new experimental models for the analysis of heterochromatin is an urgent task. In this work, we used the Rif1 mutation, which suppresses the underreplication of all types of repeated sequences, to analyze heterochromatin regions in polytene chromosomes of Drosophila melanogaster. In the Rif1 background, we discovered and described in detail a new inversion, In(1)19EHet, which arose on a chromosome already carrying the In(1)sc8 inversion and transferred a large part of X chromosome heterochromatin, including the nucleolar organizer to a new euchromatic environment. Using nanopore sequencing and FISH, we have identified the eu- and heterochromatin breakpoints of In(1)19EHet. The combination of the new inversion and the Rif1 mutation provides a promising tool for studies of X chromosome heterochromatin structure, nucleolar organization, and the nucleolar dominance phenomenon. In particular, we found that, with the complete polytenization of rDNA repeats, the nucleolus consists of a cloud-like structure corresponding to the classical nucleolus of polytene chromosomes, as well as an unusual intrachromosomal structure containing alternating transcriptionally active and inactive regions.

Summary: DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining (NHEJ) or homologous recombination (HR). RIF1 negatively regulates resection through the effector Shieldin, which associates with a short 3′ single-stranded DNA (ssDNA) overhang by the MRN (MRE11-RAD50-NBS1) complex, to prevent further resection and HR repair. In this study, we show that RIF1, but not Shieldin, inhibits the accumulation of CtIP at DSB sites immediately after damage, suggesting that RIF1 has another effector besides Shieldin. We find that protein phosphatase 1 (PP1), a known RIF1 effector in replication, localizes at damage sites dependent on RIF1, where it suppresses downstream CtIP accumulation and limits the resection by the MRN complex. PP1 therefore acts as a RIF1 effector distinct from Shieldin. Furthermore, PP1 deficiency in the context of Shieldin depletion elevates HR immediately after irradiation. We conclude that PP1 inhibits resection before the action of Shieldin to prevent precocious HR in the early phase of the damage response.

Drosophila melanogaster is one of the popular model organisms in DNA replication studies. Since the 1960s, DNA replication of polytene chromosomes has been extensively studied by cytological methods. In the recent two decades, the progress in our understanding of DNA replication was associated with new techniques. Use of fluorescent dyes increased the resolution of cytological methods significantly. High-throughput methods allowed analysis of DNA replication on a genome scale, as well as its correlation with chromatin structure and gene activi ty. Precise mapping of the cytological structures of polytene chromosomes to the genome assembly allowed comparison of replication between polytene chromosomes and chromosomes of diploid cells. New features of replication characteristic for D. melanogaster were described for both diploid and polytene chromosomes. Comparison of genomic replication profiles revealed a significant similarity between Drosophila and other well-studi ed eukaryotic species, such as human. Early replication is often confined to intensely transcribed gene-dense regions characterized by multiple replication initiation sites. Features of DNA replication in Drosophila might be explained by a compact genome. The organization of replication in polytene chromosomes has much in common with the organization of replication in chromosomes in diploid cells. The most important feature of replication in polytene chromosomes is its low rate and the dependence of S-phase duration on many factors: external and internal, local and global. The speed of replication forks in D. melanogaster polytene chromosomes is affected by SUUR and Rif1 proteins. It is not known yet how universal the mechanisms associated with these factors are, but their study is very promising.

To examine the established link between DNA replication and telomere length, we tested whether firing of telomeric origins would cause telomere lengthening. We found that RIF1 mutants that block Protein Phosphatase 1 (PP1) binding activated telomeric origins but did not elongate telomeres. In a second approach, we found overexpression of ∆N-Dbf4 and Cdc7 increased DDK activity and activated telomeric origins, yet telomere length was unchanged. We tested a third mechanism to activate origins using the sld3-A mcm5-bob1 mutant that de-regulates the pre-replication complex, and again saw no change in telomere length. Finally, we tested whether mutations in RIF1 that cause telomere elongation would affect origin firing. We found that neither rif1-∆1322 nor rif1HOOK affected firing of telomeric origins. We conclude that telomeric origin firing does not cause telomere elongation, and the role of Rif1 in regulating origin firing is separable from its role in regulating telomere length.

Abstract Background Human telomerase reverse transcriptase (hTERT) is highly expressed in over 80% of tumors, including human epithelial ovarian cancer (EOC). However, the mechanisms through which hTERT is up-regulated in EOC and promotes tumor progression remain unclear. The aim of this study is to identify RIF1 as a novel molecular target that modulate hTERT signaling and EOC growth. Methods RIF1 expression in ovarian cancer, benign and normal ovarian tissues was examined by immunohistochemistry. The biological role of RIF1 was revealed by MTS, colony formation and sphere formation assays. Luciferase reporter assay and chromatin immunoprecipitation (CHIP) assay were used to verify RIF1 as a novel hTERT promoter-binding protein in EOC cells. The role of RIF1 on tumorigenesis in vivo was detected by the xenograft model. Results RIF1 expression is upregulated in EOC tissues and is closely correlated with FIGO stage and prognosis of EOC patients. Functionally, RIF1 knockdown suppressed the expression and promoter activity of hTERT and consequently inhibited the growth and CSC-like traits of EOC cells. RIF1 knockdown also inhibited tumorigenesis in xenograft model. RIF1 overexpression had the opposite effect. Luciferase reporter assay and ChIP assay verified RIF1 directly bound to hTERT promoter to upregulate its expression. The rescue experiments suggested hTERT overexpression rescued the inhibition of EOC cell growth and CSC-like traits mediated by RIF1 knockdown. Consistently, hTERT knockdown abrogated the RIF1-induced promotion of EOC cell growth and CSC-like traits. Conclusions RIF1 promotes EOC progression by activating hTERT and the RIF1/hTERT pathway may be a potential therapeutic target for EOC patients.

In eukaryotes, DNA replication progresses through a finely orchestrated temporal and spatial program. The 3D genome structure and nuclear architecture have recently emerged as fundamental determinants of the replication program. Factors with established roles in replication have been recognized as genome organization regulators. Exploiting paradigms from yeasts and mammals, we discuss how DNA replication is regulated in time and space through DNA-associated trans -acting factors, diffusible limiting replication initiation factors, higher-order chromatin folding, dynamic origin localization, and specific nuclear microenvironments. We present an integrated model for the regulation of DNA replication in 3D and highlight the importance of accurate spatio-temporal regulation of DNA replication in physiology and disease. 3D chromatin structure, nuclear architecture, and subnuclear origin localization are critical determinants of the replication program. Opposing activities of the rate limiting Dbf4-dependent kinase and protein phosphatase 1 drive timely origin activation. Bridging proteins recruit positive and negative replication regulators and establish long-range interactions shaping origin positioning and coregulation in 3D. Activity gradients and local enrichments of diffusible initiation factors couple nuclear architecture to the replication program. Subnuclear compartments act as specialized microenvironments providing the appropriate conditions for the execution of timely DNA replication. Aberrations in the DNA replication program affect single nucleotide and copy number alterations along the genome and are linked to cancer and progeria. [ABSTRACT FROM AUTHOR]

G-quadrupex is now known to play crucial roles in various biological reactions. However, direct evidence for its presence in cells has been limited, due to the lack of versatile and non-biased methodology. We use Rif1 binding sites on the fission yeast genome, which has been shown to adopt G4 structures, as a model to prove that Rif1 BS indeed adopt G4 structure in cells. We take advantage of the presence of a single-stranded loop in the G4 structure. Rif1BS is unique in that they contain unusually long loop sequences, and we replace them with a 18 bp I-SceI restriction site. We show in vitro that I-SceI in the loop is not cleaved when G4 is formed on duplex Rif1BS DNA, but is cleaved when G4 is not formed due to a mutation in the G-tracts. This is observed both heat-induced and transcription-induced G4 structure, and gives proof of evidence for this procedure. We apply this strategy for detection of a G4 structure at the same Rif1BS in fission yeast cells. We present evidence that in vivo cleavage of I-SceI can be a measure for the presence of G4 at the target sequence in cells as well. The method described here gives a platform strategy for genome-wide analyses of cellular G4 and their dynamic formation and disruption. • G4 formation in vitro could be examined by the resistance to restriction digestion in the modified loop of G4-forming sequences. • G4 generated on duplex DNA by heat denaturation or transcription equally exhibited restriction enzyme resistance. • This method was successfully applied to detect G4 formation at the cellular model G4 sequences. [ABSTRACT FROM AUTHOR]

Replication initiation in eukaryotic cells occurs asynchronously throughout S phase, yielding early- and late-replicating regions of the genome, a process known as replication timing (RT). RT changes during development to ensure accurate genome duplication and maintain genome stability. To understand the relative contributions that cell lineage, cell cycle, and replication initiation regulators have on RT, we utilized the powerful developmental systems available in Drosophila melanogaster. We generated and compared RT profiles from mitotic cells of different tissues and from mitotic and endocycling cells of the same tissue. Our results demonstrate that cell lineage has the largest effect on RT, whereas switching from a mitotic to an endoreplicative cell cycle has little to no effect on RT. Additionally, we demonstrate that the RT differences we observed in all cases are largely independent of transcriptional differences. We also employed a genetic approach in these same cell types to understand the relative contribution the eukaryotic RT control factor, Rif1, has on RT control. Our results demonstrate that Rif1 can function in a tissue-specific manner to control RT. Importantly, the Protein Phosphatase 1 (PP1) binding motif of Rif1 is essential for Rif1 to regulate RT. Together, our data support a model in which the RT program is primarily driven by cell lineage and is further refined by Rif1/PP1 to ultimately generate tissue-specific RT programs. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks (DSBs) are repaired by either the homology-directed repair (HDR) or the non-homologous end-joining (NHEJ) pathway. RIF1 (RAP1-interacting factor homolog) was recently shown to stimulate NHEJ through an interaction with 53BP1 (p53-binding protein 1) phosphorylated at S/TQ sites, but the molecular mechanism underlying pathway choice remains unclear. Here, we show that SCAI (suppressor of cancer cell invasion) binds to 53BP1 phosphorylated at S/TP sites and facilitates HDR. Upon DNA damage, RIF1 immediately accumulates at damage sites and then gradually dissociates from 53BP1 and is subsequently replaced with SCAI. Depletion of SCAI reduces both the accumulation of HDR factors, including BRCA1 (breast cancer susceptibility gene 1), at damage sites and the efficiency of HDR, as detected by a reporter assay system. These data suggest that SCAI inhibits RIF1 function to allow BRCA1-mediated repair, which possibly includes alt-NHEJ and resection-dependent NHEJ in G1, as well as HDR in S/G2.

Dbf4-dependent kinases (DDKs) are required for the initiation of DNA replication, their essential targets being the MCM2-7 proteins. We show that, in Xenopus laevis egg extracts and human cells, hyper-phosphorylation of DNA-bound Mcm4, but not phosphorylation of Mcm2, correlates with DNA replication. These phosphorylations are differentially affected by the DDK inhibitors PHA-767491 and XL413. We show that DDK-dependent MCM phosphorylation is reversed by protein phosphatase 1 (PP1) targeted to chromatin by Rif1. Loss of Rif1 increased MCM phosphorylation and the rate of replication initiation and also compromised the ability of cells to block initiation when challenged with replication inhibitors. We also provide evidence that Rif1 can mediate MCM dephosphorylation at replication forks and that the stability of dephosphorylated replisomes strongly depends on Chk1 activity. We propose that both replication initiation and replisome stability depend on MCM phosphorylation, which is maintained by a balance of DDK-dependent phosphorylation and Rif1-mediated dephosphorylation.

Telomere shortening results in cellular senescence and the regulatory mechanisms remain unclear. Here, we report that the sub-telomere regions facilitate telomere lengthening by homologous recombination, thereby attenuating senescence in yeast Saccharomyces cerevisiae. The telomere protein complex Sir3/4 represses, whereas Rif1 promotes, the sub-telomere Y′ element recombination. Genetic disruption of SIR4 increases Y′ element abundance and rescues telomere-shortening-induced senescence in a Rad51-dependent manner, indicating a sub-telomere regulatory switch in regulating organismal senescence by DNA recombination. Inhibition of the sub-telomere recombination requires Sir4 binding to perinuclear protein Mps3 for telomere perinuclear localization and transcriptional repression of the telomeric repeat-containing RNA TERRA. Furthermore, Sir4 repression of Y′ element recombination is negatively regulated by Rif1 that mediates senescence-evasion induced by Sir4 deficiency. Thus, our results demonstrate a dual opposing control mechanism of sub-telomeric Y′ element recombination by Sir3/4 and Rif1 in the regulation of telomere shortening and cell senescence.

DNA damage-induced signaling by ATR and CHK1 inhibits DNA replication, stabilizes stalled and collapsed replication forks, and mediates the repair of multiple classes of DNA lesions. We and others have shown that ATR kinase inhibitors, three of which are currently undergoing clinical trials, induce excessive origin firing during unperturbed DNA replication, indicating that ATR kinase activity limits replication initiation in the absence of damage. However, the origins impacted and the underlying mechanism(s) have not been described. Here, we show that unperturbed DNA replication is associated with a low level of ATR and CHK1 kinase signaling and that inhibition of this signaling induces dormant origin firing at sites of ongoing replication throughout the S phase. We show that ATR and CHK1 kinase inhibitors induce RIF1 Ser2205 phosphorylation in a CDK1-dependent manner, which disrupts an interaction between RIF1 and PP1 phosphatase. Thus, ATR and CHK1 signaling suppresses CDK1 kinase activity throughout the S phase and stabilizes an interaction between RIF1 and PP1 in replicating cells. PP1 dephosphorylates key CDC7 and CDK2 kinase substrates to inhibit the assembly and activation of the replicative helicase. This mechanism limits origin firing during unperturbed DNA replication in human cells. [ABSTRACT FROM AUTHOR]

In buddying yeast, like all eukaryotes examined so far, DNA replication is under temporal control, such that some origins fire early and some late during S phase. This replication timing program is established in G1 phase, where chromatin states are thought to prevent binding of key-limiting initiation factors at late-firing origins. Although many factors are involved in replication initiation, a new player, Rif1, has recently entered the scene, with a spate of papers revealing a global role for the protein in the control of replication initiation timing from yeasts to humans. Since budding yeast Rif1 was known to bind only to telomeric and silent mating loci regions, it remained controversial whether Rif1 acts directly at replication origins or instead influences origin activity indirectly. In this perspective, we discuss our recent finding that Rif1 binds directly to the replication origins that it controls. In this study, we also found that Rif1's regulatory activity at origins is best revealed by an assay (sort-seq) that measures replication in unperturbed, freely cycling cultures, as opposed to commonly used protocols in which cells are first blocked in the G1 phase of the cell cycle by mating pheromone, then released into a synchronous S phase. Finally, we discuss how the sequestration of Rif1 at telomeres, through an interaction with the arrays of Rap1 molecules bound there, plays an important role in limiting Rif1's action primarily to telomere-proximal replication origins. [ABSTRACT FROM AUTHOR]

Clearance of entangled DNA from the anaphase mid-region must accurately proceed in order for chromosomes to segregate with high fidelity. Loss of Taz1 (fission yeast ortholog of human TRF1/TRF2) leads to stalled telomeric replication forks that trigger telomeric entanglements; the resolution of these entanglements fails at ≤20°C. Here, we investigate these entanglements and their promotion by the conserved replication/repair protein Rif1. Rif1 plays no role in taz1Δ fork stalling. Rather, Rif1 localizes to the anaphase mid-region and regulates the resolution of persisting DNA structures. This anaphase role for Rif1 is genetically separate from the role of Rif1 in S/G2, though both roles require binding to PP1 phosphatase, implying spatially and temporally distinct Rif1-regulated phosphatase substrates. Rif1 thus acts as a double-edged sword. Although it inhibits the resolution of taz1Δ telomere entanglements, it promotes the resolution of non-telomeric ultrafine anaphase bridges at ≤20°C. We suggest a unifying model for Rif1’s seemingly diverse roles in chromosome segregation in eukaryotes.

In Drosophila salivary gland polytene chromosomes, a substantial portion of heterochromatin is underreplicated. The combination of mutations SuURES and Su(var)3-906 results in the polytenization of a substantial fraction of unique and moderately repeated sequences but has almost no effect on satellite DNA replication. The Rap1 interacting factor 1 (Rif) protein is a conserved regulator of replication timing, and in Drosophila, it affects underreplication in polytene chromosomes. We compared the morphology of pericentromeric regions and labeling patterns of in situ hybridization of heterochromatin-specific DNA probes between wild-type salivary gland polytene chromosomes and the chromosomes of Rif1 mutants and SuUR Su(var)3-906 double mutants. We show that, despite general similarities, heterochromatin zones exist that are polytenized only in the Rif1 mutants, and that there are zones that are under specific control of Su(var)3-9. In the Rif1 mutants, we found additional polytenization of the largest blocks of satellite DNA (in particular, satellite 1.688 of chromosome X and simple satellites in chromosomes X and 4) as well as partial polytenization of chromosome Y. Data on pulsed incorporation of 5-ethynyl-2′-deoxyuridine (EdU) into polytene chromosomes indicated that in the Rif1 mutants, just as in the wild type, most of the heterochromatin becomes replicated during the late S phase. Nevertheless, a significantly increased number of heterochromatin replicons was noted. These results suggest that Rif1 regulates the activation probability of heterochromatic origins in the satellite DNA region.

DNA double-strand breaks (DSBs) elicit major chromatin changes. Using super-resolution microscopy in human cells, Ochs et al. unveil that the DSB response protein 53BP1 and its effector RIF1 organize DSB-flanking chromatin into circular micro-domains. These structures control the spatial distribution of DSB repair factors safeguarding genome integrity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Telomeres, the ends of eukaryotic chromosomes, consist of repetitive DNA sequences and their bound proteins that protect the end from the DNA damage response. Short telomeres with fewer repeats are preferentially elongated by telomerase. Tel1, the yeast homolog of human ATM kinase, is preferentially recruited to short telomeres and Tel1 kinase activity is required for telomere elongation. Rif1, a telomere-binding protein, negatively regulates telomere length by forming a complex with two other telomere binding proteins, Rap1 and Rif2, to block telomerase recruitment. Rif1 has 14 SQ/TQ consensus phosphorylation sites for ATM kinases, including 6 in a SQ/TQ Cluster Domain (SCD) similar to other DNA damage response proteins. These 14 sites were analyzed as N-terminal, SCD and C-terminal domains. Mutating some sites to non-phosphorylatable residues increased telomere length in cells lacking Tel1 while a different set of phosphomimetic mutants increased telomere length in cells lacking Rif2, suggesting that Rif1 phosphorylation has both positive and negative effects on length regulation. While these mutations did not alter the sensitivity to DNA damaging agents, inducing telomere-specific damage by growing cells lacking YKU70 at high temperature revealed a role for the SCD. Mass spectrometry of Rif1 from wild type cells or those induced for telomere-specific DNA damage revealed increased phosphorylation in cells with telomere damage at an ATM consensus site in the SCD, S1351, and non-ATM sites S181 and S1637. A phosphomimetic rif1-S1351E mutation caused an increase in telomere length at synthetic telomeres but not natural telomeres. These results indicate that the Rif1 SCD can modulate Rif1 function. As all Rif1 orthologs have one or more SCD domains, these results for yeast Rif1 have implications for the regulation of Rif1 function in humans and other organisms. [ABSTRACT FROM AUTHOR]

Summary The Saccharomyces cerevisiae telomere-binding protein Rif1 plays an evolutionarily conserved role in control of DNA replication timing by promoting PP1-dependent dephosphorylation of replication initiation factors. However, ScRif1 binding outside of telomeres has never been detected, and it has thus been unclear whether Rif1 acts directly on the replication origins that it controls. Here, we show that, in unperturbed yeast cells, Rif1 primarily regulates late-replicating origins within 100 kb of a telomere. Using the chromatin endogenous cleavage ChEC-seq technique, we robustly detect Rif1 at late-replicating origins that we show are targets of its inhibitory action. Interestingly, abrogation of Rif1 telomere association by mutation of its Rap1-binding module increases Rif1 binding and origin inhibition elsewhere in the genome. Our results indicate that Rif1 inhibits replication initiation by interacting directly with origins and suggest that Rap1-dependent sequestration of Rif1 increases its effective concentration near telomeres, while limiting its action at chromosome-internal sites. [ABSTRACT FROM AUTHOR]

Sirtuins (SIRTs) are a family of histone deacetylases, which widely regulate cellular metabolism and are also involved in DNA repair. Rap1-interacting factor 1 (Rif1) and O6-alkylguanine DNA alkyltransferase (MGMT) are DNA-repair enzymes, which may potentially be involved in resistance to treatment of classical Hodgkin lymphoma (HL). We assessed the expression levels of (previously unstudied) SIRT1, SIRT4, SIRT6, Rif1, and MGMT immunohistochemically in 85 patients with untreated classical HL. Aberrant distributions of SIRT1, SIRT4, and SIRT6 were detected in Hodgkin neoplastic Reed-Sternberg (RS) cells compared with reactive elements. Low-level expression of both Rif1 and SIRT6 predicted dismal relapse-free survival in radiotherapy-treated patients (multivariate analysis; HR 8.521; 95% CI 1.714-42.358; p=.0088). Expression levels of SIRT1, 4, and 6 were abnormally distributed in RS cells, suggesting a putative role of aberrant acetylation in classical HL carcinogenesis. Rif1 and SIRT6 may also have substantial prognostic and even predictive roles in classical HL. [ABSTRACT FROM AUTHOR]

Telomere length homeostasis is essential for cell survival. In humans, telomeres shorten as a function of age. Short telomeres are known determinants of cell senescence and longevity. The yeast Saccharomyces cerevisiae expresses telomerase and maintains a strict telomere length homeostasis during vegetative growth. We have previously reported that different environmental signals promote changes in telomere length in S. cerevisiae. In particular, exposure to ethanol induces an extensive telomere elongation response due to a reduction in RAP1 mRNA and protein levels. Here we show that the reduction in Rap1 protein levels disrupts the physical interaction between Rap1 and Rif1, which in turn reduces the recruitment of these two proteins to telomeres during G2-phase. Although elongation of the shortest telomeres has been shown to depend on the Rif2 telomeric protein and on the Tel1(ATM) protein kinase, we show here that the extensive telomere elongation in response to ethanol exposure is Rif1 and Mec1 (ATR)-dependent. Our results fit a model in which Rif1 and Rap1 form a complex that is loaded onto telomeres at the end of S-phase. Reduced levels of the Rap1-Rif1 complex in ethanol lead to continuous telomere elongation in a Mec1-dependent process. [ABSTRACT FROM AUTHOR]

The cellular pathways that restart stalled replication forks are essential for genome stability and tumor prevention. However, how many of these pathways exist in cells and how these pathways are selectively activated remain unclear. Here, we describe two major fork restart pathways, and demonstrate that their selection is governed by 53BP1 and BRCA1, which are known to control the pathway choice to repair double-strand DNA breaks (DSBs). Specifically, 53BP1 promotes a fork cleavage-free pathway, whereas BRCA1 facilitates a break-induced replication (BIR) pathway coupled with SLX-MUS complex-mediated fork cleavage. The defect in the first pathway, but not DSB repair, in a 53BP1 mutant is largely corrected by disrupting BRCA1, and vice versa. Moreover, PLK1 temporally regulates the switch of these two pathways through enhancing the assembly of the SLX-MUS complex. Our results reveal two distinct fork restart pathways, which are antagonistically controlled by 53BP1 and BRCA1 in a DSB repair-independent manner.