Clinical success with poly (ADP-ribose) polymerase inhibitors (PARPi) is impeded by inevitable resistance and associated cytotoxicity. Depletion of Amplified in Liver Cancer 1 (ALC1), a chromatin-remodeling enzyme, can overcome these limitations by hypersensitizing BReast CAncer genes 1/2 (BRCA1/2) mutant cells to PARPi. Here, we demonstrate that PARPi hypersensitivity upon ALC1 loss is reliant on its role in promoting the repair of chromatin buried abasic sites.
View Article and Find Full Text PDFBRCA1/2 proteins function in genome stability by promoting repair of double-stranded DNA breaks through homologous recombination and by protecting stalled replication forks from nucleolytic degradation. In BRCA1/2-deficient cancer cells, extensively degraded replication forks can be rescued through distinct fork recovery mechanisms that also promote cell survival. Here, we identified a novel pathway mediated by the E3 ubiquitin ligase RAD18, the E2-conjugating enzyme UBC13, the recombination factor PALB2, the E3 ubiquitin ligase RNF168 and PCNA ubiquitination that promotes fork recovery in BRCA1- but not BRCA2-deficient cells.
View Article and Find Full Text PDFMutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown.
View Article and Find Full Text PDFThe ATR kinase safeguards genomic integrity during S phase, but how ATR protects DNA replication forks remains incompletely understood. Here, we combine four distinct assays to analyze ATR functions at ongoing and newly assembled replication forks upon replication inhibition by hydroxyurea. At ongoing forks, ATR inhibitor (ATRi) increases MRE11- and EXO1-mediated nascent DNA degradation from PrimPol-generated, single-stranded DNA (ssDNA) gaps.
View Article and Find Full Text PDFDNA combing and DNA spreading are two central approaches for studying DNA replication fork dynamics genome-wide at single-molecule resolution by distributing labeled genomic DNA on coverslips or slides for immunodetection. Perturbations in DNA replication fork dynamics can differentially affect either leading or lagging strand synthesis, for example in instances where replication is blocked by a lesion or obstacle on only one of the two strands. Thus, we sought to investigate whether the DNA combing and/or spreading approaches are suitable for resolving adjacent sister chromatids during DNA replication, thereby enabling the detection of DNA replication dynamics within individual nascent strands.
View Article and Find Full Text PDFThe primary method for probing DNA replication dynamics is DNA fiber analysis, which utilizes thymidine analog incorporation into nascent DNA, followed by immunofluorescent microscopy of DNA fibers. Besides being time-consuming and prone to experimenter bias, it is not suitable for studying DNA replication dynamics in mitochondria or bacteria, nor is it adaptable for higher-throughput analysis. Here, we present mass spectrometry-based analysis of nascent DNA (MS-BAND) as a rapid, unbiased, quantitative alternative to DNA fiber analysis.
View Article and Find Full Text PDFDNA topoisomerase 1 (TOP11) inhibitors are mainstays of anticancer therapy. These drugs trap TOP1 on DNA, stabilizing the TOP1-cleavage complex (TOP1-cc). The accumulation of TOP1-ccs perturbs DNA replication fork progression, leading to DNA breaks and cell death.
View Article and Find Full Text PDFHigh-fidelity DNA replication is critical for the faithful transmission of genetic information to daughter cells. Following genotoxic stress, specialized DNA damage tolerance pathways are activated to ensure replication fork progression. These pathways include translesion DNA synthesis, template switching and repriming.
View Article and Find Full Text PDFDNA replication forks are tightly controlled by a large protein network consisting of well-known core regulators and many accessory factors which remain functionally undefined. In this study, we report previously unknown nuclear functions of the actin-binding factor profilin-1 (PFN1) in DNA replication, which occur in a context-dependent fashion and require its binding to poly-L-proline (PLP)-containing proteins instead of actin. In unperturbed cells, PFN1 increases DNA replication initiation and accelerates fork progression by binding and stimulating the PLP-containing nucleosome remodeler SNF2H.
View Article and Find Full Text PDFDNA topoisomerases resolve topological stress by introducing transient single- or double-strand breaks into the DNA duplex. This reaction requires the covalent binding of topoisomerases to DNA while the topological stress is being released. This transient intermediate is known as topoisomerase-covalent complex and represents the target of many anti-cancer drugs.
View Article and Find Full Text PDFSingle-stranded DNA gaps are frequent structures that accumulate on newly synthesized DNA under conditions of replication stress. The identification of these single-stranded DNA gaps has been instrumental to uncover the mechanisms that allow the DNA replication machinery to skip intrinsic replication obstacles or DNA lesions. DNA fiber assays provide an essential tool for detecting perturbations in DNA replication fork dynamics genome-wide at single molecule resolution along with identifying the presence of single-stranded gaps when used in combination with S1 nuclease.
View Article and Find Full Text PDFCentral to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious.
View Article and Find Full Text PDFOver 80% of women with high-grade serous ovarian cancer (HGSOC) develop tumor resistance to chemotherapy and die of their disease. There are currently no FDA-approved agents to improve sensitivity to first-line platinum- and taxane-based chemotherapy or to PARP inhibitors. Here, we tested the hypothesis that expression of growth arrest-specific 6 (GAS6), the ligand of receptor tyrosine kinase AXL, is associated with chemotherapy response and that sequestration of GAS6 with AVB-S6-500 (AVB-500) could improve tumor response to chemotherapy and PARP inhibitors.
View Article and Find Full Text PDFLamin A/C provides a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. Lamin A/C dysfunction is associated with cancer, aging, and degenerative diseases. The mechanisms whereby lamin A/C regulates genome stability remain poorly understood.
View Article and Find Full Text PDFPRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition.
View Article and Find Full Text PDFRAD51 facilitates replication fork reversal and protects reversed forks from nuclease degradation. Although potentially a useful replication stress response mechanism, unregulated fork reversal can cause genome instability. Here we show that RADX, a single-strand DNA binding protein that binds to and destabilizes RAD51 nucleofilaments, can either inhibit or promote fork reversal depending on replication stress levels.
View Article and Find Full Text PDFIt is well established that short telomeres activate an ATM-driven DNA damage response that leads to senescence in terminally differentiated cells. However, technical limitations have hampered our understanding of how telomere shortening is signaled in human stem cells. Here, we show that telomere attrition induces ssDNA accumulation (G-strand) at telomeres in human pluripotent stem cells (hPSCs), but not in their differentiated progeny.
View Article and Find Full Text PDFAccurate DNA replication is constantly threatened by DNA lesions arising from endogenous and exogenous sources. Specialized DNA replication stress response pathways ensure replication fork progression in the presence of DNA lesions with minimal delay in fork elongation. These pathways broadly include translesion DNA synthesis, template switching, and replication fork repriming.
View Article and Find Full Text PDFDNA replication forks use multiple mechanisms to deal with replication stress, but how the choice of mechanisms is made is still poorly understood. Here, we show that CARM1 associates with replication forks and reduces fork speed independently of its methyltransferase activity. The speeding of replication forks in CARM1-deficient cells requires RECQ1, which resolves reversed forks, and RAD18, which promotes translesion synthesis.
View Article and Find Full Text PDFDNA replication forks are constantly challenged by DNA lesions induced by endogenous and exogenous sources. DNA damage tolerance mechanisms ensure that DNA replication continues with minimal effects on replication fork elongation either by using specialized DNA polymerases, which have the ability to replicate through the damaged template, or by skipping the damaged DNA, leaving it to be repaired after replication. These mechanisms are evolutionarily conserved in bacteria, yeast, and higher eukaryotes, and are paramount to ensure timely and faithful duplication of the genome.
View Article and Find Full Text PDFTAR DNA-binding protein 43 (TDP-43; also known as TARDBP) is an RNA-binding protein whose aggregation is a hallmark of the neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43 loss increases DNA damage and compromises cell viability, but the actual function of TDP-43 in preventing genome instability remains unclear. Here, we show that loss of TDP-43 increases R-loop formation in a transcription-dependent manner and results in DNA replication stress.
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