Chk1 requirement for high global rates of replication fork progression during normal vertebrate S phase

Chk1 protein kinase maintains replication fork stability in metazoan cells in response to DNA damage and DNA replication inhibitors. Here, we have employed DNA fiber labeling to quantify, for the first time, the extent to which Chk1 maintains global replication fork rates during normal vertebrate S phase. We report that replication fork rates in Chk1¿/¿ chicken DT40 cells are on average half of those observed with wild-type cells. Similar results were observed if Chk1 was inhibited or depleted in wild-type DT40 cells or HeLa cells by incubation with Chk1 inhibitor or small interfering RNA. In addition, reduced rates of fork extension were observed with permeabilized Chk1¿/¿ cells in vitro. The requirement for Chk1 for high fork rates during normal S phase was not to suppress promiscuous homologous recombination at replication forks, because inhibition of Chk1 similarly slowed fork progression in XRCC3¿/¿ DT40 cells. Rather, we observed an increased number of replication fibers in Chk1¿/¿ cells in which the nascent strand is single-stranded, supporting the idea that slow global fork rates in unperturbed Chk1¿/¿ cells are associated with the accumulation of aberrant replication fork structure

PrimPol bypasses UV photoproducts during eukaryotic chromosomal DNA replication

DNA damage can stall the DNA replication machinery, leading to genomic instability. Thus, numerous mechanisms exist to complete genome duplication in the absence of a pristine DNA template, but identification of the enzymes involved remains incomplete. Here, we establish that Primase-Polymerase (PrimPol; CCDC111), an archaeal-eukaryotic primase (AEP) in eukaryotic cells, is involved in chromosomal DNA replication. PrimPol is required for replication fork progression on ultraviolet (UV) lightdamaged DNA templates, possibly mediated by its ability to catalyze translesion synthesis (TLS) of these lesions. This PrimPol UV lesion bypass pathway is not epistatic with the Pol h-dependent pathway and, as a consequence, protects xeroderma pigmentosum variant (XP-V) patient cells from UV-induced cytotoxicity. In addition, we establish that PrimPol is also required for efficient replication fork progression during an unperturbed S phase. These and other findings indicate that PrimPol is an important player in replication fork progression in eukaryotic cells

Gaps and forks in DNA replication: Rediscovering old models

Most current models for replication past damaged lesions envisage that translesion synthesis occurs at the replication fork. However older models suggested that gaps were left opposite lesions to allow the replication fork to proceed, and these gaps were subsequently sealed behind the replication fork. Two recent articles lend support to the idea that bypass of the damage occurs behind the fork. In the first paper, electron micrographs of DNA replicated in UV-irradiated yeast cells show regions of single-stranded DNA both at the replication forks and behind the fork, the latter being consistent with the presence of gaps in the daughter-strands opposite lesions. The second paper describes an in vitro DNA replication system reconstituted from purified bacterial proteins. Repriming of synthesis downstream from a blocked fork occurred not only on the lagging strand as expected, but also on the leading strand, demonstrating that contrary to widely accepted beliefs, leading strand synthesis does not need to be continuous

A requirement for STAG2 in replication fork progression creates a targetable synthetic lethality in cohesin-mutant cancers.

Cohesin is a multiprotein ring that is responsible for cohesion of sister chromatids and formation of DNA loops to regulate gene expression. Genomic analyses have identified that the cohesin subunit STAG2 is frequently inactivated by mutations in cancer. However, the reason STAG2 mutations are selected during tumorigenesis and strategies for therapeutically targeting mutant cancer cells are largely unknown. Here we show that STAG2 is essential for DNA replication fork progression, whereby STAG2 inactivation in non-transformed cells leads to replication fork stalling and collapse with disruption of interaction between the cohesin ring and the replication machinery as well as failure to establish SMC3 acetylation. As a consequence, STAG2 mutation confers synthetic lethality with DNA double-strand break repair genes and increased sensitivity to select cytotoxic chemotherapeutic agents and PARP or ATR inhibitors. These studies identify a critical role for STAG2 in replication fork procession and elucidate a potential therapeutic strategy for cohesin-mutant cancers

A dual role of BRCA1 in two distinct homologous recombination mediated repair in response to replication arrest

Homologous recombination (HR) is a major mechanism utilized to repair blockage of DNA replication forks. Here, we report that a sister chromatid exchange (SCE) generated by crossover-associated HR efficiently occurs in response to replication fork stalling before any measurable DNA double-strand breaks (DSBs). Interestingly, SCE produced by replication fork collapse following DNA DSBs creation is specifically suppressed by ATR, a central regulator of the replication checkpoint. BRCA1 depletion leads to decreased RPA2 phosphorylation (RPA2-P) following replication fork stalling but has no obvious effect on RPA2-P following replication fork collapse. Importantly, we found that BRCA1 promotes RAD51 recruitment and SCE induced by replication fork stalling independent of ATR. In contrast, BRCA1 depletion leads to a more profound defect in RAD51 recruitment and SCE induced by replication fork collapse when ATR is depleted. We concluded that BRCA1 plays a dual role in two distinct HR-mediated repair upon replication fork stalling and collapse. Our data established a molecular basis for the observation that defective BRCA1 leads to a high sensitivity to agents that cause replication blocks without being associated with DSBs, and also implicate a novel mechanism by which loss of cell cycle checkpoints promotes BRCA1-associated tumorigenesis via enhancing HR defect resulting from BRCA1 deficiency

JAK2V617F promotes replication fork stalling with disease-restricted impairment of the intra-S checkpoint response

Cancers result from the accumulation of genetic lesions, but the cellular consequences of driver mutations remain unclear, especially during the earliest stages of malignancy. The V617F mutation in the JAK2 non-receptor tyrosine kinase (JAK2V617F) is present as an early somatic event in most patients with myeloproliferative neoplasms (MPNs), and the study of these chronic myeloid malignancies provides an experimentally tractable approach to understanding early tumorigenesis. Introduction of exogenous JAK2V617F impairs replication fork progression and is associated with activation of the intra-S checkpoint, with both effects mediated by phosphatidylinositide 3-kinase (PI3K) signaling. Analysis of clonally derived JAK2V617F-positive erythroblasts from MPN patients also demonstrated impaired replication fork progression accompanied by increased levels of replication protein A (RPA)-containing foci. However, the associated intra-S checkpoint response was impaired in erythroblasts from polycythemia vera (PV) patients, but not in those from essential thrombocythemia (ET) patients. Moreover, inhibition of p53 in PV erythroblasts resulted in more gamma-H2Ax (γ-H2Ax)–marked double-stranded breaks compared with in like-treated ET erythroblasts, suggesting the defective intra-S checkpoint function seen in PV increases DNA damage in the context of attenuated p53 signaling. These results demonstrate oncogene-induced impairment of replication fork progression in primary cells from MPN patients, reveal unexpected disease-restricted differences in activation of the intra-S checkpoint, and have potential implications for the clonal evolution of malignancies

Correlation Between Chromatid Deletion Production and Progression of the DNA Replication Fork in UV-Irradiated S Phase Xenopus Cells

Experimentation was performed primarily to determine whether progression of the DNA replication fork along segments of S phase Xenopus chromosomes, which contain UV-induced pre-aberrational lesions, plays a significant role in conversion of these lesions into chromatid deletions. Specifically, a Xenopus chromosome that was both easy to identify and that possessed a single DNA replication fork in one arm was found and used to conduct the experimentation. This chromosome was exposed to UV in early S phase and a Bromodeoxyuridine/Giemsa differential staining technique was applied in conjunction with conventional aberrational techniques to correlate progression of the DNA replication fork through segments of this arm with chromatid deletion production in these segments. The results point to direct evidence for the role of the DNA replication fork in converting some UV-induced pre-aberrational DNA damage into chromosomal deletions

The NuA4 acetyltransferase and histone H4 acetylation promote replication recovery after topoisomerase I-poisoning

BACKGROUND: Histone acetylation plays an important role in DNA replication and repair because replicating chromatin is subject to dynamic changes in its structures. However, its precise mechanism remains elusive. In this report, we describe roles of the NuA4 acetyltransferase and histone H4 acetylation in replication fork protection in the fission yeast Schizosaccharomyces pombe. RESULTS: Downregulation of NuA4 subunits renders cells highly sensitive to camptothecin, a compound that induces replication fork breakage. Defects in NuA4 function or mutations in histone H4 acetylation sites lead to impaired recovery of collapsed replication forks and elevated levels of Rad52 DNA repair foci, indicating the role of histone H4 acetylation in DNA replication and fork repair. We also show that Vid21 interacts with the Swi1-Swi3 replication fork protection complex and that Swi1 stabilizes Vid21 and promotes efficient histone H4 acetylation. Furthermore, our genetic analysis demonstrates that loss of Swi1 further sensitizes NuA4 and histone H4 mutant cells to replication fork breakage. CONCLUSION: Considering that Swi1 plays a critical role in replication fork protection, our results indicate that NuA4 and histone H4 acetylation promote repair of broken DNA replication forks

Checkpoint-Dependent Regulation of Origin Firing and Replication Fork Movement in Response to DNA Damage in Fission Yeast

To elucidate the checkpoint mechanism responsible for slowing passage through S phase when fission yeast cells are treated with the DNA-damaging agent methyl methanesulfonate (MMS), we carried out two-dimensional gel analyses of replication intermediates in cells synchronized by cdc10 block (in G1) followed by release into synchronous S phase. The results indicated that under these conditions early-firing centromeric origins were partially delayed but late-firing telomeric origins were not delayed. Replication intermediates persisted in MMS-treated cells, suggesting that replication fork movement was inhibited. These effects were dependent on the Cds1 checkpoint kinase and were abolished in cells overexpressing the Cdc25 phosphatase, suggesting a role for the Cdc2 cyclin-dependent kinase. We conclude that both partial inhibition of the firing of a subset of origins and inhibition of replication fork movement contribute to the slowing of S phase in MMS-treated fission yeast cells

Checkpoints are blind to replication restart and recombination intermediates that result in gross chromosomal rearrangements

Replication fork inactivation can be overcome by homologous recombination, but this can cause gross chromosomal rearrangements that subsequently missegregate at mitosis, driving further chromosome instability. It is unclear when the chromosome rearrangements are generated and whether individual replication problems or the resulting recombination intermediates delay the cell cycle. Here we have investigated checkpoint activation during HR-dependent replication restart using a site-specific replication fork-arrest system. Analysis during a single cell cycle shows that HR-dependent replication intermediates arise in S phase, shortly after replication arrest, and are resolved into acentric and dicentric chromosomes in G2. Despite this, cells progress into mitosis without delay. Neither the DNA damage nor the intra-S phase checkpoints are activated in the first cell cycle, demonstrating that these checkpoints are blind to replication and recombination intermediates as well as to rearranged chromosomes. The dicentrics form anaphase bridges that subsequently break, inducing checkpoint activation in the second cell cycle