Fork Stalling and Recombination of the Nascent DNA: Addressing the Limitations

AbstractMutant KRAS is a common tumor driver and frequently confers resistance to anti-cancer treatments such as radiation. DNA replication stress in these tumors may constitute a therapeutic liability but is poorly understood. Here, using single-molecule DNA fiber analysis, we first characterized baseline replication stress in a panel of unperturbed isogenic and non-isogenic cancer cell lines. Correlating with the observed enhanced replication stress we found increased levels of cytosolic double-stranded DNA in KRAS mutant compared to wild-type cells. Yet, despite this phenotype replication stress-inducing agents failed to selectively impact KRAS mutant cells, which were protected by CHK1. Similarly, most exogenous stressors studied did not differentially augment cytosolic DNA accumulation in KRAS mutant compared to wild-type cells. However, we found that proton radiation was able to slow fork progression and preferentially induce fork stalling in KRAS mutant cells. Proton treatment also partly reversed the radioresistance associated with mutant KRAS. The cellular effects of protons in the presence of KRAS mutation clearly contrasted that of other drugs affecting replication, highlighting the unique nature of the underlying DNA damage caused by protons. Taken together, our findings provide insight into the replication stress response associated with mutated KRAS, which may ultimately yield novel therapeutic opportunities.

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Saccharomyces cerevisiae Rrm3p DNA Helicase Promotes Genome Integrity by Preventing Replication Fork Stalling: Viability of rrm3 Cells Requires the Intra-S-Phase Checkpoint and Fork Restart Activities

Molecular and Cellular Biology ◽

10.1128/mcb.24.8.3198-3212.2004 ◽

2004 ◽

Vol 24 (8) ◽

pp. 3198-3212 ◽

Cited By ~ 97

Author(s):

Jorge Z. Torres ◽

Sandra L. Schnakenberg ◽

Virginia A. Zakian

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Replication Fork ◽

Opportunity To Learn ◽

Normal Growth ◽

Dna Helicase ◽

S Phase ◽

Exogenous Dna ◽

Fork Stalling ◽

S Phase Checkpoint

ABSTRACT Rrm3p is a 5′-to-3′ DNA helicase that helps replication forks traverse protein-DNA complexes. Its absence leads to increased fork stalling and breakage at over 1,000 specific sites located throughout the Saccharomyces cerevisiae genome. To understand the mechanisms that respond to and repair rrm3-dependent lesions, we carried out a candidate gene deletion analysis to identify genes whose mutation conferred slow growth or lethality on rrm3 cells. Based on synthetic phenotypes, the intra-S-phase checkpoint, the SRS2 inhibitor of recombination, the SGS1/TOP3 replication fork restart pathway, and the MRE11/RAD50/XRS2 (MRX) complex were critical for viability of rrm3 cells. DNA damage checkpoint and homologous recombination genes were important for normal growth of rrm3 cells. However, the MUS81/MMS4 replication fork restart pathway did not affect growth of rrm3 cells. These data suggest a model in which the stalled and broken forks generated in rrm3 cells activate a checkpoint response that provides time for fork repair and restart. Stalled forks are converted by a Rad51p-mediated process to intermediates that are resolved by Sgs1p/Top3p. The rrm3 system provides a unique opportunity to learn the fate of forks whose progress is impaired by natural impediments rather than by exogenous DNA damage.

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Mechanism for inverted-repeat recombination induced by a replication fork barrier

Nature Communications ◽

10.1038/s41467-021-27443-w ◽

2022 ◽

Vol 13 (1) ◽

Author(s):

Léa Marie ◽

Lorraine S. Symington

Keyword(s):

Replication Fork ◽

Repetitive Sequences ◽

Replication Stress ◽

Yeast Genome ◽

Physical Analysis ◽

Srs2 Helicase ◽

Fork Stalling ◽

Strand Annealing ◽

Recombination Pathway ◽

Stalled Replication Forks

AbstractReplication stress and abundant repetitive sequences have emerged as primary conditions underlying genomic instability in eukaryotes. To gain insight into the mechanism of recombination between repeated sequences in the context of replication stress, we used a prokaryotic Tus/Ter barrier designed to induce transient replication fork stalling near inverted repeats in the budding yeast genome. Our study reveals that the replication fork block stimulates a unique recombination pathway dependent on Rad51 strand invasion and Rad52-Rad59 strand annealing activities, Mph1/Rad5 fork remodelers, Mre11/Exo1/Dna2 resection machineries, Rad1-Rad10 nuclease and DNA polymerase δ. Furthermore, we show recombination at stalled replication forks is limited by the Srs2 helicase and Mus81-Mms4/Yen1 nucleases. Physical analysis of the replication-associated recombinants revealed that half are associated with an inversion of sequence between the repeats. Based on our extensive genetic characterization, we propose a model for recombination of closely linked repeats that can robustly generate chromosome rearrangements.

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MCM7 Ubiquitination Regulates CMG Helicase Disassembly in Human

10.21203/rs.3.rs-692464/v2 ◽

2021 ◽

Author(s):

Qianqian Sun ◽

Kun Liu ◽

Fangzhou Li ◽

Bingquan Qiu ◽

Zhisong Fu ◽

...

Keyword(s):

Cell Cycle ◽

Genome Stability ◽

Human Tumor ◽

Cell Fractionation ◽

Minichromosome Maintenance ◽

E3 Ligases ◽

Minichromosome Maintenance Protein ◽

Fork Stalling

Abstract BackgroundThe disassembly of the replisome plays an essential role in maintaining genome stability at the termination of DNA replication. However, the mechanism of replisome disassembly remains unknown in human. In this study, we screened E3 ligases and deubiquitinases (DUBs) for the ubiquitination of minichromosome maintenance protein (MCM) 7 and provided evidence of this process driving CMG helicase disassembly in human tumor cells. MethodsSILAC-MS/MS was analyzed to identify ubiquitinated proteins in HeLa cells. The ubiquitination/deubiquitylation assay in vitro and in vivo were detected by Western blot. Thymidine and HU were implied to synchronized cell cycle，and detect the role of ubiquitinated MCM7 in cell cycle. Cell fractionation assay was used to detect the function of ubiquitination of MCM7 in chromatin and non-chromatin. Aphidicolin、Etoposide、ICRF-193 and IR were applied to cause replication fork stalling. MG-132 and NMS-873 were used to inhibit the proteasome degradation and p97 segregase. Flow cytometer and FlowJo flow cytometry software were used to cell cycle analysis.ResultsIn our study, we found that the ubiquitin ligase RNF8 catalyzes the k63-linked poly-ubiquitination of MCM7 both in vivo and in vitro, and lysine 145 of MCM7 is the primary ubiquitination site. Moreover, the poly-ubiquitination of MCM7 mainly exists in the chromatin, which is dynamically regulated by the cell cycle, mainly occurs in the late S phase. And DNA damage can significantly reduce the poly-ubiquitylation of MCM7 in the late S phage. Furthermore, the proteasome, p97 segregase, USP29 and ATXN3 are required for the removal of MCM7 ubiquitination to promote the disassembly of CMG on chromatin. ConclusionsIn the late S phage of cell cycle, RNF8 catalyzes the poly-ubiquitination of MCM7, and then initiates the disassembly of CMG helicase from chromatin, which is mediated by p97, proteasome, USP29 and ATXN3 in human. We reveal the novel function of the poly-ubiquitylation of MCM7, which is a regulatory signal to control CMG complex unloading at replication termination sites.

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Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo

The EMBO Journal ◽

10.1038/sj.emboj.7601155 ◽

2006 ◽

Vol 25 (11) ◽

pp. 2596-2604 ◽

Cited By ~ 81

Author(s):

Christophe Possoz ◽

Sergio R Filipe ◽

Ian Grainge ◽

David J Sherratt

Keyword(s):

Escherichia Coli ◽

Replication Fork ◽

Replication Fork Stalling ◽

Fork Stalling

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Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication

Science Advances ◽

10.1126/sciadv.abc0330 ◽

2020 ◽

Vol 6 (38) ◽

pp. eabc0330 ◽

Cited By ~ 1

Author(s):

D. T. Gruszka ◽

S. Xie ◽

H. Kimura ◽

H. Yardimci

Keyword(s):

Dna Replication ◽

Real Time ◽

Single Molecule ◽

Replication Fork ◽

Epigenetic Inheritance ◽

Replication Forks ◽

Replication Fork Progression ◽

Egg Extracts ◽

Fork Stalling ◽

The Moment

During replication, nucleosomes are disrupted ahead of the replication fork, followed by their reassembly on daughter strands from the pool of recycled parental and new histones. However, because no previous studies have managed to capture the moment that replication forks encounter nucleosomes, the mechanism of recycling has remained unclear. Here, through real-time single-molecule visualization of replication fork progression in Xenopus egg extracts, we determine explicitly the outcome of fork collisions with nucleosomes. Most of the parental histones are evicted from the DNA, with histone recycling, nucleosome sliding, and replication fork stalling also occurring but at lower frequencies. Critically, we find that local histone recycling becomes dominant upon depletion of endogenous histones from extracts, revealing that free histone concentration is a key modulator of parental histone dynamics at the replication fork. The mechanistic details revealed by these studies have major implications for our understanding of epigenetic inheritance.

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Ongoing replication forks delay nuclear envelope breakdown upon mitotic entry

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015142 ◽

2020 ◽

pp. jbc.RA120.015142

Author(s):

Yoshitami Hashimoto ◽

Hirofumi Tanaka

Keyword(s):

Dna Replication ◽

Nuclear Envelope ◽

Dependent Manner ◽

Repair Synthesis ◽

Nuclear Envelope Breakdown ◽

M Phase ◽

Egg Extracts ◽

Fork Stalling ◽

Dna Repair Synthesis ◽

Xenopus Egg Extracts

DNA replication is a major contributor to genomic instability and protection against DNA replication perturbation is essential for normal cell division. Certain types of replication stress agents, such as aphidicolin and hydroxyurea, have been shown to cause reversible replication fork stalling, wherein replisome complexes are stably maintained with competence to restart in the S-phase of the cell cycle. If these stalled forks persist into the M-phase without a replication restart, replisomes are disassembled in a p97-dependent pathway and under-replicated DNA is subjected to mitotic DNA repair synthesis. Here, using Xenopus egg extracts, we investigated the consequences that arise when stalled forks are released simultaneously with the induction of mitosis. Ara-cytidine-5’-triphosphate (Ara-CTP)-induced stalled forks were able to restart with the addition of excess dCTPduring early mitosis before the nuclear envelope breakdown (NEB). However, stalled forks could no longer restart efficiently after NEB. Although replisome complexes were finally disassembled in a p97-dependent manner during mitotic progression whether or not fork stalling was relieved, the timing of NEB was delayed with the ongoing forks, rather than the stalled forks, and the delay was dependent on Wee1/Myt1 kinase activities. Thus, ongoing DNA replication was found to be directly linked to the regulation of Wee1/Myt1 kinases to modulate cyclin-dependent kinase (CDK) activities, owing to which DNA replication and mitosis occur in a mutually exclusive and sequential manner.

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Synergistic targeting and resistance to PARP inhibition in DNA damage repair-deficient pancreatic cancer

Gut ◽

10.1136/gutjnl-2019-319970 ◽

2020 ◽

pp. gutjnl-2019-319970 ◽

Cited By ~ 2

Author(s):

Johann Gout ◽

Lukas Perkhofer ◽

Mareen Morawe ◽

Frank Arnold ◽

Michaela Ihle ◽

...

Keyword(s):

Dna Damage ◽

Epithelial Mesenchymal Transition ◽

Synthetic Lethality ◽

Parp Inhibitor ◽

Copy Number Variations ◽

Ductal Adenocarcinoma ◽

Parp Inhibition ◽

Independent Manner ◽

Mesenchymal Transition ◽

Fork Stalling

ObjectiveATM serine/threonine kinase (ATM) is the most frequently mutated DNA damage response gene, involved in homologous recombination (HR), in pancreatic ductal adenocarcinoma (PDAC).DesignCombinational synergy screening was performed to endeavour a genotype-tailored targeted therapy.ResultsSynergy was found on inhibition of PARP, ATR and DNA-PKcs (PAD) leading to synthetic lethality in ATM-deficient murine and human PDAC. Mechanistically, PAD-induced PARP trapping, replication fork stalling and mitosis defects leading to P53-mediated apoptosis. Most importantly, chemical inhibition of ATM sensitises human PDAC cells toward PAD with long-term tumour control in vivo. Finally, we anticipated and elucidated PARP inhibitor resistance within the ATM-null background via whole exome sequencing. Arising cells were aneuploid, underwent epithelial-mesenchymal-transition and acquired multidrug resistance (MDR) due to upregulation of drug transporters and a bypass within the DNA repair machinery. These functional observations were mirrored in copy number variations affecting a region on chromosome 5 comprising several of the upregulated MDR genes. Using these findings, we ultimately propose alternative strategies to overcome the resistance.ConclusionAnalysis of the molecular susceptibilities triggered by ATM deficiency in PDAC allow elaboration of an efficient mutation-specific combinational therapeutic approach that can be also implemented in a genotype-independent manner by ATM inhibition.

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Ctf18-RFC and DNA Pol ϵ form a stable leading strand polymerase/clamp loader complex required for normal and perturbed DNA replication

Nucleic Acids Research ◽

10.1093/nar/gkaa541 ◽

2020 ◽

Vol 48 (14) ◽

pp. 8128-8145 ◽

Cited By ~ 2

Author(s):

Katy Stokes ◽

Alicja Winczura ◽

Boyuan Song ◽

Giacomo De Piccoli ◽

Daniel B Grabarczyk

Keyword(s):

Structural Biology ◽

Replication Fork ◽

Catalytic Domain ◽

Replication Forks ◽

Yeast Genetics ◽

Clamp Loader ◽

Checkpoint Signaling ◽

Chromatid Cohesion ◽

Leading Strand ◽

Fork Stalling

Abstract The eukaryotic replisome must faithfully replicate DNA and cope with replication fork blocks and stalling, while simultaneously promoting sister chromatid cohesion. Ctf18-RFC is an alternative PCNA loader that links all these processes together by an unknown mechanism. Here, we use integrative structural biology combined with yeast genetics and biochemistry to highlight the specific functions that Ctf18-RFC plays within the leading strand machinery via an interaction with the catalytic domain of DNA Pol ϵ. We show that a large and unusually flexible interface enables this interaction to occur constitutively throughout the cell cycle and regardless of whether forks are replicating or stalled. We reveal that, by being anchored to the leading strand polymerase, Ctf18-RFC can rapidly signal fork stalling to activate the S phase checkpoint. Moreover, we demonstrate that, independently of checkpoint signaling or chromosome cohesion, Ctf18-RFC functions in parallel to Chl1 and Mrc1 to protect replication forks and cell viability.

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Replication fork stalling elicits chromatin compaction for the stability of stalling replication forks

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1821475116 ◽

2019 ◽

Vol 116 (29) ◽

pp. 14563-14572 ◽

Cited By ~ 3

Author(s):

Gang Feng ◽

Yue Yuan ◽

Zeyang Li ◽

Lu Wang ◽

Bo Zhang ◽

...

Keyword(s):

Replication Fork ◽

Cellular Mechanism ◽

Eukaryotic Cells ◽

Replication Forks ◽

Physical Separation ◽

Replicative Helicase ◽

Chromatin Compaction ◽

Acetylation Site ◽

Fork Stalling ◽

The Stability

DNA replication forks in eukaryotic cells stall at a variety of replication barriers. Stalling forks require strict cellular regulations to prevent fork collapse. However, the mechanism underlying these cellular regulations is poorly understood. In this study, a cellular mechanism was uncovered that regulates chromatin structures to stabilize stalling forks. When replication forks stall, H2BK33, a newly identified acetylation site, is deacetylated and H3K9 trimethylated in the nucleosomes surrounding stalling forks, which results in chromatin compaction around forks. Acetylation-mimic H2BK33Q and its deacetylase clr6-1 mutations compromise this fork stalling-induced chromatin compaction, cause physical separation of replicative helicase and DNA polymerases, and significantly increase the frequency of stalling fork collapse. Furthermore, this fork stalling-induced H2BK33 deacetylation is independent of checkpoint. In summary, these results suggest that eukaryotic cells have developed a cellular mechanism that stabilizes stalling forks by targeting nucleosomes and inducing chromatin compaction around stalling forks. This mechanism is named the “Chromsfork” control: Chromatin Compaction Stabilizes Stalling Replication Forks.

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