Chromatin remodeler Fft3 plays a dual role at blocked DNA replication forks

AbstractIn response to DNA damage during S phase, cells slow DNA replication. This slowing is orchestrated by the intra-S checkpoint and involves inhibition of origin firing and reduction of replication fork speed. Slowing of replication allows for tolerance of DNA damage and suppresses genomic instability. Although the mechanisms of origin inhibition by the intra-S checkpoint are understood, major questions remain about how the checkpoint regulates replication forks: Does the checkpoint regulate the rate of fork progression? Does the checkpoint affect all forks, or only those encountering damage? Does the checkpoint facilitate the replication of polymerase-blocking lesions? To address these questions, we have analyzed the checkpoint in the fission yeast Schizosaccharomyces pombe using a single-molecule DNA combing assay, which allows us to unambiguously separate the contribution of origin and fork regulation towards replication slowing, and allows us to investigate the behavior of individual forks. Moreover, we have interrogated the role of forks interacting with individual sites of damage by using three damaging agents—MMS, 4NQO and bleomycin—that cause similar levels of replication slowing with very different frequency of DNA lesions. We find that the checkpoint slows replication by inhibiting origin firing, but not by decreasing fork rates. However, the checkpoint appears to facilitate replication of damaged templates, allowing forks to more quickly pass lesions. Finally, using a novel analytic approach, we rigorously identify fork stalling events in our combing data and show that they play a previously unappreciated role in shaping replication kinetics in response to DNA damage.Author SummaryFaithful duplication of the genome is essential for genetic stability of organisms and species. To ensure faithful duplication, cells must be able to replicate damaged DNA. To do so, they employ checkpoints that regulate replication in response to DNA damage. However, the mechanisms by which checkpoints regulate DNA replication forks, the macromolecular machines that contain the helicases and polymerases required to unwind and copy the parental DNA, is unknown. We have used DNA combing, a single-molecule technique that allows us to monitor the progression of individual replication forks, to characterize the response of fission yeast replication forks to DNA damage that blocks the replicative polymerases. We find that forks pass most lesions with only a brief pause and that this lesion bypass is checkpoint independent. However, at a low frequency, forks stall at lesions, and that the checkpoint is required to prevent these stalls from accumulating single-stranded DNA. Our results suggest that the major role of the checkpoint is not to regulate the interaction of replication forks with DNA damage, per se, but to mitigate the consequences of fork stalling when forks are unable to successfully navigate DNA damage on their own.

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Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.400-406.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 400-406

Author(s):

W P Deng ◽

J A Nickoloff

Keyword(s):

Dna Replication ◽

Mismatch Repair ◽

Mammalian Cells ◽

Strand Break ◽

Wild Type ◽

Heteroduplex Dna ◽

Frameshift Mutations ◽

Single Strand Annealing ◽

Extrachromosomal Recombination ◽

Strand Annealing

Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication.

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Set1-dependent H3K4 methylation becomes critical for DNA replication fork progression in response to changes in S phase dynamics in Saccharomyces cerevisiae

10.1101/2020.10.26.355008 ◽

2020 ◽

Author(s):

Christophe de La Roche Saint-André ◽

Vincent Géli

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Dna Replication ◽

Replication Fork ◽

Genetic Material ◽

S Phase ◽

Replication Forks ◽

H3k4 Methylation ◽

Origin Firing ◽

Phase Dynamics

AbstractDNA replication is a highly regulated process that occurs in the context of chromatin structure and is sensitive to several histone post-translational modifications. In Saccharomyces cerevisiae, the histone methylase Set1 is responsible for the transcription-dependent deposition of H3K4 methylation (H3K4me) throughout the genome. Here we show that a combination of a hypomorphic replication mutation (orc5-1) with the absence of Set1 (set1Δ) compromises the progression through S phase, and this is associated with a large increase in DNA damage. The ensuing DNA damage checkpoint activation, in addition to that of the spindle assembly checkpoint, restricts the growth of orc5-1 set1Δ. Interestingly, orc5-1 set1Δ is sensitive to the lack of RNase H activity while a reduction of histone levels is able to counterbalance the loss of Set1. We propose that the recently described Set1-dependent mitigation of transcription-replication conflicts becomes critical for growth when the replication forks accelerate due to decreased origin firing in the orc5-1 background. Furthermore, we show that an increase of reactive oxygen species (ROS) levels, likely a consequence of the elevated DNA damage, is partly responsible for the lethality in orc5-1 set1Δ.Author summaryDNA replication, that ensures the duplication of the genetic material, starts at discrete sites, termed origins, before proceeding at replication forks whose progression is carefully controlled in order to avoid conflicts with the transcription of genes. In eukaryotes, DNA replication occurs in the context of chromatin, a structure in which DNA is wrapped around proteins, called histones, that are subjected to various chemical modifications. Among them, the methylation of the lysine 4 of histone H3 (H3K4) is carried out by Set1 in Saccharomyces cerevisiae, specifically at transcribed genes. We report that, when the replication fork accelerates in response to a reduction of active origins, the absence of Set1 leads to accumulation of DNA damage. Because H3K4 methylation was recently shown to slow down replication at transcribed genes, we propose that the Set1-dependent becomes crucial to limit the occurrence of conflicts between replication and transcription caused by replication fork acceleration. In agreement with this model, stabilization of transcription-dependent structures or reduction histone levels, to limit replication fork velocity, respectively exacerbates or moderates the effect of Set1 loss. Last, but not least, we show that the oxidative stress associated to DNA damage is partly responsible for cell lethality.

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cGAS suppresses genomic instability as a decelerator of replication forks

Science Advances ◽

10.1126/sciadv.abb8941 ◽

2020 ◽

Vol 6 (42) ◽

pp. eabb8941 ◽

Cited By ~ 1

Author(s):

Hao Chen ◽

Jiamin Zhang ◽

Yumin Wang ◽

Antoine Simoneau ◽

...

Keyword(s):

Immune Response ◽

Dna Damage ◽

Dna Replication ◽

Cancer Cells ◽

Genomic Instability ◽

Cyclic Gmp ◽

Replication Fork ◽

Innate Immune ◽

Dependent Manner ◽

Replication Forks

The cyclic GMP-AMP synthase (cGAS), a sensor of cytosolic DNA, is critical for the innate immune response. Here, we show that loss of cGAS in untransformed and cancer cells results in uncontrolled DNA replication, hyperproliferation, and genomic instability. While the majority of cGAS is cytoplasmic, a fraction of cGAS associates with chromatin. cGAS interacts with replication fork proteins in a DNA binding–dependent manner, suggesting that cGAS encounters replication forks in DNA. Independent of cGAMP and STING, cGAS slows replication forks by binding to DNA in the nucleus. In the absence of cGAS, replication forks are accelerated, but fork stability is compromised. Consequently, cGAS-deficient cells are exposed to replication stress and become increasingly sensitive to radiation and chemotherapy. Thus, by acting as a decelerator of DNA replication forks, cGAS controls replication dynamics and suppresses replication-associated DNA damage, suggesting that cGAS is an attractive target for exploiting the genomic instability of cancer cells.

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The Fml1-MHF complex suppresses inter-fork strand annealing in fission yeast

eLife ◽

10.7554/elife.49784 ◽

2019 ◽

Vol 8 ◽

Author(s):

Io Nam Wong ◽

Jacqueline PS Neo ◽

Judith Oehler ◽

Sophie Schafhauser ◽

Fekret Osman ◽

...

Keyword(s):

Catalytic Activity ◽

Dna Replication ◽

Fission Yeast ◽

Replication Fork ◽

Dna Helicase ◽

Genomic Deletions ◽

Terminal Domain ◽

Plausible Mechanism ◽

Strand Annealing

Previously we reported that a process called inter-fork strand annealing (IFSA) causes genomic deletions during the termination of DNA replication when an active replication fork converges on a collapsed fork (Morrow et al., 2017). We also identified the FANCM-related DNA helicase Fml1 as a potential suppressor of IFSA. Here, we confirm that Fml1 does indeed suppress IFSA, and show that this function depends on its catalytic activity and ability to interact with Mhf1-Mhf2 via its C-terminal domain. Finally, a plausible mechanism of IFSA suppression is demonstrated by the finding that Fml1 can catalyse regressed fork restoration in vitro.

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Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq

PLoS Biology ◽

10.1371/journal.pbio.3000886 ◽

2021 ◽

Vol 19 (3) ◽

pp. e3000886

Author(s):

Neesha Kara ◽

Felix Krueger ◽

Peter Rugg-Gunn ◽

Jonathan Houseley

Keyword(s):

Dna Replication ◽

Mammalian Cells ◽

Replication Fork ◽

Strand Break ◽

Double Strand Break ◽

Replication Forks ◽

Dna Breaks ◽

Genome Wide ◽

Fork Stalling ◽

End Resection

Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3′ ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3′ ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

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Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq

10.1101/2020.08.10.243931 ◽

2020 ◽

Author(s):

Neesha Kara ◽

Felix Krueger ◽

Peter Rugg-Gunn ◽

Jonathan Houseley

Keyword(s):

Dna Replication ◽

Mammalian Cells ◽

Replication Fork ◽

High Sensitivity ◽

Strand Break ◽

Double Strand Break ◽

Replication Forks ◽

Dna Breaks ◽

Biochemical Purification ◽

Genome Wide

AbstractUnderstanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods sensitive to both changes in replication fork structure and the formation of recombinogenic DNA ends. Here we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single stranded DNA 3’ ends genome-wide and with base pair resolution. TrAEL-seq labels DNA breaks, and profiles both stalled and processive replication forks in yeast and mammalian cells. Replication forks stalling at defined barriers and expressed genes are detected by TrAEL-seq with exceptional signal-to-noise, most likely through labelling of DNA 3’ ends exposed during fork reversal. TrAEL-seq also labels unperturbed processive replication forks to yield maps of replication fork direction similar to those obtained by Okazaki fragment sequencing, however TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3’ ends also allows accurate detection of double strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double strand break hotspots in a dmc1Δ mutant. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

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A ruthenium polypyridyl intercalator stalls DNA replication forks, radiosensitizes human cancer cells and is enhanced by Chk1 inhibition

Scientific Reports ◽

10.1038/srep31973 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 22

Author(s):

Martin R. Gill ◽

Siti Norain Harun ◽

Swagata Halder ◽

Ramon A. Boghozian ◽

Kristijan Ramadan ◽

...

Keyword(s):

Cell Proliferation ◽

Dna Damage ◽

Dna Replication ◽

Cancer Cells ◽

Replication Fork ◽

Human Cancer ◽

Cell Signalling ◽

Ionising Radiation ◽

Replication Forks ◽

Replication Fork Progression

Abstract Ruthenium(II) polypyridyl complexes can intercalate DNA with high affinity and prevent cell proliferation; however, the direct impact of ruthenium-based intercalation on cellular DNA replication remains unknown. Here we show the multi-intercalator [Ru(dppz)2(PIP)]2+ (dppz = dipyridophenazine, PIP = 2-(phenyl)imidazo[4,5-f][1,10]phenanthroline) immediately stalls replication fork progression in HeLa human cervical cancer cells. In response to this replication blockade, the DNA damage response (DDR) cell signalling network is activated, with checkpoint kinase 1 (Chk1) activation indicating prolonged replication-associated DNA damage, and cell proliferation is inhibited by G1-S cell-cycle arrest. Co-incubation with a Chk1 inhibitor achieves synergistic apoptosis in cancer cells, with a significant increase in phospho(Ser139) histone H2AX (γ-H2AX) levels and foci indicating increased conversion of stalled replication forks to double-strand breaks (DSBs). Normal human epithelial cells remain unaffected by this concurrent treatment. Furthermore, pre-treatment of HeLa cells with [Ru(dppz)2(PIP)]2+ before external beam ionising radiation results in a supra-additive decrease in cell survival accompanied by increased γ-H2AX expression, indicating the compound functions as a radiosensitizer. Together, these results indicate ruthenium-based intercalation can block replication fork progression and demonstrate how these DNA-binding agents may be combined with DDR inhibitors or ionising radiation to achieve more efficient cancer cell killing.

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STAG2 Loss Gives Rise to Therapeutically Targetable DNA Damage Repair Defects and Altered Replication Fork Dynamics in Acute Myeloid Leukaemia

Blood ◽

10.1182/blood-2019-129828 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1255-1255

Author(s):

Harmony Estelle Black ◽

Satpal Jhujh ◽

Grant S Stewart ◽

Kienan I Savage ◽

Ken I Mills

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Dna Repair ◽

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Replication Fork ◽

Dna Damage Repair ◽

Replication Forks ◽

Damage Repair ◽

Acute Myeloid

Acute Myeloid Leukaemia (AML) is a genetically heterogeneous disease of the blood and bone marrow primarily affecting older populations. Chemotherapy has been the standard of care for four decades, just 10% of elderly patients show a response and it will be curative in only a third of patients aged 18-60. Therefore, it's necessary to identify new therapeutic targets for improved treatment options. The search for novel mutations within the genomes of karyotypically normal AML patients identified Cohesin's STAG2 as a recurrently mutated gene (6%). Cohesin is a ring-shaped multimeric protein complex which has a multitude of roles including maintaining sister chromatid cohesion, DNA replication and repair, gene expression and chromatin architecture. STAG2 remains the most understudied Cohesin gene, how a mutation affects the ability of Cohesin to fulfil certain roles needs elucidation as well as how STAG2 functions as part of Cohesin to repair DNA breaks, promote faithful replication and control gene expression. Therefore, we sought to explore the consequences of loss-of-function STAG2 mutations on DNA repair and replication using a STAG2 knockout (ΔSTAG2) isogenic cell line model created using CRISPR/Cas9. Immunofluorescent staining of DNA damage repair proteins 53BP1 and RAD51 following 2Gy irradiation (IR) suggests ΔSTAG2 cells have a significant DNA repair defect. The average 53BP1 and RAD51 foci counts were significantly higher (p=0.001) in mutant cells which had three times as many foci at 24 hours after IR. In addition, 40% of ΔSTAG2 cells stain positive for 53BP1 and RAD51 respectively at 24 hours post-IR. This damage persists at 48 and 72 hours, whereas wild type (WT) damage returns to basal levels. Given that our data suggests a HR defect, we assessed the response of WT and ΔSTAG2 cells to PARP inhibitors (PARPi). Interestingly, ΔSTAG2 cells showed significantly greater sensitivity than WT cells to highly potent PARPi Talazoparib with an IC50 of 62nM. We next assayed single DNA fibres to evaluate the implications of a STAG2 mutation on the dynamics of individual replication forks. Interestingly, we observed a significantly reduced number of ongoing replication forks compared to WT cells (p=0.01) with a concomitant increase in the number of stalled forks (p=0.01) in the absence of exogeneous DNA damage. Following treatment with hydroxyurea (HU) this phenotype was exacerbated and ΔSTAG2 cells exhibit significantly more stalled replication forks than WTs (p=0.001). This indicates a role for STAG2 in maintaining replication fork stability and promoting fork restart following replication stress. Importantly, our data identifies a potential DNA damage repair deficiency that sensitises ΔSTAG2 cells to PARPi monotherapy as well as unearthing an essential role for STAG2 in replication fork progression and fork stability. This mutation could therefore be exploited therapeutically and represents a novel therapeutic target/approach for AML patients with mutated STAG2. Disclosures No relevant conflicts of interest to declare.

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A Degenerate PCNA-Interacting Peptide (DPIP) box targets RNF168 to replicating DNA to limit 53BP1 signaling

10.1101/2021.03.17.435897 ◽

2021 ◽

Author(s):

Yang Yang ◽

Deepika Jayaprakash ◽

Robert Hollingworth ◽

Steven Chen ◽

Amy Jablonski ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Replication Fork ◽

Strand Break ◽

S Phase ◽

C Terminus ◽

Dna Double Strand Break ◽

Damage Response ◽

Phase Functions

The E3 ligase RNF168 has been suggested to have roles at DNA replication forks in addition to its canonical functions in DNA double-strand break (DSB) signaling. However, the precise role of RNF168 in DNA replication remains unclear. Here we demonstrate that RNF168 is recruited to DNA replication factories independent of the canonical DSB response pathway regulators and identify a degenerate PCNA-Interacting Peptide (DPIP) motif in the C-terminus of RNF168 which mediates its binding to PCNA. An RNF168 mutant harboring substitutions in the DPIP box fails to interact with PCNA and is not recruited to sites of DNA synthesis, yet fully retains its ability to promote DSB-induced 53BP1 foci. Surprisingly, the RNF168 DPIP mutant also retains the ability to support ongoing DNA replication fork movement, demonstrating that PCNA-binding is dispensable for normal S-phase functions. However, replisome-associated RNF168 functions to suppress the DSB-induced 53BP1 DNA damage response during S-phase. Moreover, we show that WT RNF168 can perform PCNA ubiquitylation independently of RAD18 and also synergizes with RAD18 to amplify PCNA ubiquitylation. Taken together, our results identify non-canonical functions of RNF168 at the replication fork and demonstrate new mechanisms of cross talk between the DNA damage and replication stress response pathways.

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