Recognition and repair of damaged replication forks are essential to maintain genome stability and are coordinated by the combined action of the Fanconi anemia and homologous recombination pathways. These pathways are vital to protect stalled replication forks from uncontrolled nucleolytic activity, which otherwise causes irreparable genomic damage. Here, we identify BOD1L as a component of this fork protection pathway, which safeguards genome stability after replication stress. Loss of BOD1L confers exquisite cellular sensitivity to replication stress and uncontrolled resection of damaged replication forks, due to a failure to stabilize RAD51 at these forks. Blocking DNA2-dependent resection, or downregulation of the helicases BLM and FBH1, suppresses both catastrophic fork processing and the accumulation of chromosomal damage in BOD1L-deficient cells. Thus, our work implicates BOD1L as a critical regulator of genome integrity that restrains nucleolytic degradation of damaged replication forks.

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication, and protect, repair and restart damaged forks. Here we identify DONSON as a novel fork protection factor, and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilises forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATR-dependent signalling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity, and potentiating chromosomal instability. Hypomorphic mutations substantially reduce DONSON protein levels and impair fork stability in patient cells, consistent with defective DNA replication underlying the disease phenotype. In summary, we identify mutations in DONSON as a common cause of microcephalic dwarfism, and establish DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability.

PARP1 regulates the repair of DNA single-strand breaks generated directly, or during base excision repair (BER). However, the role of PARP2 in these and other repair mechanisms is unknown. Here, we report a requirement for PARP2 in stabilising replication forks that encounter BER intermediates through Fbh1-dependent regulation of Rad51. Whereas PARP2 is dispensable for tolerance of cells to SSBs or homologous recombination dysfunction, it is redundant with PARP1 in BER. Therefore, combined disruption of PARP1 and PARP2 leads to defective BER, resulting in elevated levels of replication-associated DNA damage owing to an inability to stabilise Rad51 at damaged replication forks and prevent uncontrolled DNA resection. Together, our results demonstrate how PARP1 and PARP2 regulate two independent, but intrinsically linked aspects of DNA base damage tolerance by promoting BER directly, and by stabilising replication forks that encounter BER intermediates.

SummaryComponents of the Fanconi anemia and homologous recombination pathways play a vital role in protecting newly replicated DNA from uncontrolled nucleolytic degradation, safeguarding genome stability. Here we report that histone methylation by the lysine methyltransferase SETD1A is crucial for protecting stalled replication forks from deleterious resection. Depletion of SETD1A sensitizes cells to replication stress and leads to uncontrolled DNA2-dependent resection of damaged replication forks. The ability of SETD1A to prevent degradation of these structures is mediated by its ability to catalyze methylation on Lys4 of histone H3 (H3K4) at replication forks, which enhances FANCD2-dependent histone chaperone activity. Suppressing H3K4 methylation or expression of a chaperone-defective FANCD2 mutant leads to loss of RAD51 nucleofilament stability and severe nucleolytic degradation of replication forks. Our work identifies epigenetic modification and histone mobility as critical regulatory mechanisms in maintaining genome stability by restraining nucleases from irreparably damaging stalled replication forks.

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism/Seckel syndrome. We establish that TRAIP relocalizes to sites of DNA damage where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to UV irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a novel component of the DNA damage response to replication-blocking DNA lesions.

Chronic infection by hepatitis C virus (HCV) is a major risk factor for the onset and development of hepatocellular carcinoma (HCC), although the underlying mechanisms are unclear. The c-Myc oncogene contributes to the genesis of many types of cancers, including HCC, partly via the induction of genetic damage and the inhibition of the cellular response to genotoxic stress. Here, we show a previously undiscovered mechanistic link between HCV infection and enhanced c-Myc expression. c-Myc expression was augmented in non-tumoral liver tissues from HCV-infected individuals with or without HCC and in hepatocyte cell lines harboring an HCV replicon and the infectious HCV strain JFH1. Increased c-Myc expression was confirmed in vivo in a transgenic murine model expressing the entire HCV open reading frame, demonstrating a direct role for HCV protein expression in c-Myc induction. Mechanistically, activation of Akt by the HCV non-structural protein NS5A, and the subsequent stabilization of the transcription factor β-catenin, was demonstrated to be responsible for activation of the c-Myc promoter, and for increased c-Myc transcription. β-Catenin-dependent c-Myc expression in this context led to increased production of reactive oxygen species, mitochondrial perturbation, enhanced DNA damage and aberrant cell-cycle arrest. Together, these data provide a novel insight into the mechanisms involved in HCV-associated HCCs, strongly suggesting that c-Myc has a crucial contributory role in this process.

Chronic hepatitis C virus (HCV) infection is associated with altered lipid metabolism and hepatocellular steatosis. Virusinduced steatosis is a cytopathic effect of HCV replication. The goal of this study was to examine the mechanisms underlying HCV-induced lipid metabolic defects in a transgenic mouse model expressing the full HCV protein repertoire at levels corresponding to the human infection. In this model, expression of the HCV full-length open reading frame was associated with hepatocellular steatosis and reduced plasma triglyceride levels. Triglyceride secretion was impaired while lipogenesis was activated. Increased lipogenic enzyme transcription resulted from activation at the maturation step and nuclear translocation of sterol regulatory element binding protein 1c (SREBP1c). No ER-stress marker was expressed at significantly higher levels in HCV transgenic mice than in their wild-type counterparts, suggesting that SREBP1c proteolytic cleavage was independent of ER stress. In conclusion, transgenic mice expressing the HCV full-length polyprotein at low, physiological levels, have decreased plasma triglyceride levels and develop hepatocellular steatosis in the same way as HCV-infected patients. In these mice, de novo triglyceride synthesis is induced by direct SREBP1c activation by one or several HCV proteins through induction of the lipogenic pathway, independently of ER stress, while triglyceride secretion is simultaneously reduced.

Summary As transcription and replication use DNA as substrate, conflicts between transcription and replication can occur, leading to genome instability with direct consequences for human health. To determine how the two processes are coordinated throughout S phase, we characterize both processes together at high resolution. We find that transcription occurs during DNA replication, with transcription start sites (TSSs) not fully replicated along with surrounding regions and remaining under-replicated until late in the cell cycle. TSSs undergo completion of DNA replication specifically when cells enter mitosis, when RNA polymerase II is removed. Intriguingly, G2/M DNA synthesis occurs at high frequency in unperturbed cell culture, but it is not associated with increased DNA damage and is fundamentally separated from mitotic DNA synthesis. TSSs duplicated in G2/M are characterized by a series of specific features, including high levels of antisense transcription, making them difficult to duplicate during S phase.