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Accurate replication of DNA requires stringent regulation to ensure genome integrity. In human cells, thousands of origins of replication are coordinately activated during S phase, and the velocity of replication forks is adjusted to fully replicate DNA in pace with the cell cycle 1 . Replication stress induces fork stalling and fuels genome instability 2 . The mechanistic basis of replication stress remains poorly understood despite its emerging role in promoting cancer 2 . Here we show that inhibition of poly(ADP-ribose) polymerase (PARP) increases the speed of fork elongation and does not cause fork stalling, which is in contrast to the accepted model in which inhibitors of PARP induce fork stalling and collapse 3 . Aberrant acceleration of fork progression by 40% above the normal velocity leads to DNA damage. Depletion of the treslin or MTBP proteins, which are involved in origin firing, also increases fork speed above the tolerated threshold, and induces the DNA damage response pathway. Mechanistically, we show that poly(ADP-ribosyl)ation (PARylation) and the PCNA interactor p21 Cip1 (p21) are crucial modulators of fork progression. PARylation and p21 act as suppressors of fork speed in a coordinated regulatory network that is orchestrated by the PARP1 and p53 proteins. Moreover, at the fork level, PARylation acts as a sensor of replication stress. During PARP inhibition, DNA lesions that induce fork arrest and are normally resolved or repaired remain unrecognized by the replication machinery. Conceptually, our results show that accelerated replication fork progression represents a general mechanism that triggers replication stress and the DNA damage response. Our findings contribute to a better understanding of the mechanism of fork speed control, with implications for genomic (in)stability and rational cancer treatment. Inhibition of PARP is shown to accelerate the speed of replication fork elongation, which prevents fork stalling and induces DNA damage, with implications for genomic instability and cancer treatment.

DNA replication is an integral step in the herpes simplex virus type 1 (HSV-1) life cycle that is coordinated with the cellular DNA damage response, repair and recombination of the viral genome, and viral gene transcription. HSV-1 encodes its own DNA replication machinery, including an origin binding protein (UL9), single-stranded DNA binding protein (ICP8), DNA polymerase (UL30), processivity factor (UL42), and a helicase/primase complex (UL5/UL8/UL52). In addition, HSV-1 utilizes a combination of accessory viral and cellular factors to coordinate viral DNA replication with other viral and cellular processes. The purpose of this review is to outline the roles of viral and cellular proteins in HSV-1 DNA replication and replication-coupled processes, and to highlight how HSV-1 may modify and adapt cellular proteins to facilitate productive infection.

Rondinelli et al. show that EZH2-mediated H3K27me3 at stalled replication forks recruits MUS81 nuclease to facilitate fork degradation. Loss of EZH2 contributes to PARPi resistance in BRCA2-deficient tumours. The emergence of resistance to poly-ADP-ribose polymerase inhibitors (PARPi) poses a threat to the treatment of BRCA1 and BRCA2 (BRCA1/2)-deficient tumours 1 . Stabilization of stalled DNA replication forks is a recently identified PARPi-resistance mechanism that promotes genomic stability in BRCA1/2-deficient cancers 2 . Dissecting the molecular pathways controlling genomic stability at stalled forks is critical. Here we show that EZH2 localizes at stalled forks where it methylates Lys27 on histone 3 (H3K27me3), mediating recruitment of the MUS81 nuclease. Low EZH2 levels reduce H3K27 methylation, prevent MUS81 recruitment at stalled forks and cause fork stabilization. As a consequence, loss of function of the EZH2/MUS81 axis promotes PARPi resistance in BRCA2-deficient cells. Accordingly, low EZH2 or MUS81 expression levels predict chemoresistance and poor outcome in patients with BRCA2-mutated tumours. Moreover, inhibition of Ezh2 in a murine Brca2 −/− breast tumour model is associated with acquired PARPi resistance. Our findings identify EZH2 as a critical regulator of genomic stability at stalled forks that couples histone modifications to nuclease recruitment. Our data identify EZH2 expression as a biomarker of BRCA2-deficient tumour response to chemotherapy.

Key Points Termination of DNA replication occurs when two replication forks meet on the same stretch of DNA, during which the following events occur, although not necessarily in this order: forks converge until all intervening DNA is unwound; any remaining gaps are filled and ligated; catenanes are removed; and replication proteins are unloaded. In eukaryotes, most termination sites are determined stochastically by the location of replication initiation sites. In bacteria, termination generally occurs at a specific locus. Replication termination can be a problematic process. Termination of simian virus 40 (SV40) replication involves the stalling of converging forks, and bacterial termination is prone to inducing re-replication. By contrast, fork stalling or re-replication have not been observed during unperturbed termination in eukaryotes. Topological stress accumulates between converging forks and is relieved by the generation of pre-catenanes, which are removed by type II topoisomerases. During bacterial and SV40 termination, type II topoisomerases are required for fork convergence, but in eukaryotes they are dispensable for this purpose. After forks converge, any remaining catenanes are removed by a type II topoisomerase. In eukaryotes, gaps are readily filled by the extension of the leading strands, but in bacteria and SV40 this process is less well-defined. In eukaryotes, a dedicated replisome removal pathway has recently been identified, which operates late during termination, after the DNA is fully replicated. It is unclear whether any comparable pathway exists in bacteria. The termination of DNA replication involves convergence of replication forks, the completion of DNA synthesis, replisome disassembly and the decatenation of daughter DNA molecules. Recent discoveries illustrate how replisome disassembly in eukaryotes is controlled by E3 ubiquitin ligases and how this activity is regulated to avoid genome instability. Genome duplication is carried out by pairs of replication forks that assemble at origins of replication and then move in opposite directions. DNA replication ends when converging replication forks meet. During this process, which is known as replication termination, DNA synthesis is completed, the replication machinery is disassembled and daughter molecules are resolved. In this Review, we outline the steps that are likely to be common to replication termination in most organisms, namely, fork convergence, synthesis completion, replisome disassembly and decatenation. We briefly review the mechanism of termination in the bacterium Escherichia coli and in simian virus 40 (SV40) and also focus on recent advances in eukaryotic replication termination. In particular, we discuss the recently discovered E3 ubiquitin ligases that control replisome disassembly in yeast and higher eukaryotes, and how their activity is regulated to avoid genome instability.

Although DNA replication is a fundamental aspect of biology, it is not known what determines where DNA replication starts and stops in the human genome. We directly identified and quantitatively compared sites of replication initiation and termination in untransformed human cells. We found that replication preferentially initiates at the transcription start site of genes occupied by high levels of RNA polymerase II, and terminates at their polyadenylation sites, thereby ensuring global co-directionality of transcription and replication, particularly at gene 5′ ends. During replication stress, replication initiation is stimulated downstream of genes and termination is redistributed to gene bodies; this globally reorients replication relative to transcription around gene 3′ ends. These data suggest that replication initiation and termination are coupled to transcription in human cells, and propose a model for the impact of replication stress on genome integrity.

In order to maintain genomic integrity, DNA replication must be highly coordinated. Disruptions in this process can cause replication stress which is aberrant in many pathologies including cancer. Despite this, little is known about the mechanisms governing the temporal regulation of DNA replication initiation, thought to be related to the limited copy number of firing factors. Here, we present a high (1-kilobase) resolution stochastic model of Saccharomyces cerevisiae whole-genome replication in which origins compete to associate with limited firing factors. After developing an algorithm to fit this model to replication timing data, we validated the model by reproducing experimental inter-origin distances, origin efficiencies, and replication fork directionality. This suggests the model accurately simulates the aspects of DNA replication most important for determining its dynamics. We also use the model to predict measures of DNA replication dynamics which are yet to be determined experimentally and investigate the potential impacts of variations in firing factor concentrations on DNA replication.

Oncogene activation results in firing of ectopic origins of replication within transcribed genes, resulting in replication stress and genome instability. How oncogenes drive genome instability Oncogenes can cause genome instability by inducing replication stress, but the molecular mechanisms that underpin this process were unknown. Morgane Macheret and Thanos Halazonetis demonstrate that oncogene activation in human cancer cells results in firing of ectopic origins of replication within transcribed genes. These origins are normally quiescent, as they are suppressed by transcription. When activated, these intragenic origins lead to conflicts between replication and transcription, resulting in collapsed replication forks, double-stranded breaks and translocations. Oncogene-induced DNA replication stress contributes critically to the genomic instability that is present in cancer 1 , 2 , 3 , 4 . However, elucidating how oncogenes deregulate DNA replication has been impeded by difficulty in mapping replication initiation sites on the human genome. Here, using a sensitive assay to monitor nascent DNA synthesis in early S phase, we identified thousands of replication initiation sites in cells before and after induction of the oncogenes CCNE1 and MYC . Remarkably, both oncogenes induced firing of a novel set of DNA replication origins that mapped within highly transcribed genes. These ectopic origins were normally suppressed by transcription during G1, but precocious entry into S phase, before all genic regions had been transcribed, allowed firing of origins within genes in cells with activated oncogenes. Forks from oncogene-induced origins were prone to collapse, as a result of conflicts between replication and transcription, and were associated with DNA double-stranded break formation and chromosomal rearrangement breakpoints both in our experimental system and in a large cohort of human cancers. Thus, firing of intragenic origins caused by premature S phase entry represents a mechanism of oncogene-induced DNA replication stress that is relevant for genomic instability in human cancer.

In all kingdoms of life, DNA is used to encode hereditary information. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by semiconservative replication prior to cell division to ensure each daughter cell receives the full complement of chromosomes. DNA synthesis of daughter strands starts at discrete sites, termed replication origins, and proceeds in a bidirectional manner until all genomic DNA is replicated. Despite the fundamental nature of these events, organisms have evolved surprisingly divergent strategies that control replication onset. Here, we discuss commonalities and differences in replication origin organization and recognition in the three domains of life.

Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2 -deficient cells from DNA damage and rescues the lethality of Brca2 -deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2 -deficient tumour cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance. Protection of nascent DNA from degradation provides a mechanism that can promote synthetic viability and drug resistance in Brca -deficient cells without restoring homologous recombination at double-strand breaks. Chemoresistance in BRCA cancers The breast cancer susceptibility genes BRCA1 and BRCA2 function to protect the genome from DNA damage. For this reason, DNA-damaging agents are used clinically to treat BRCA -deficient cancers. However, these treatments may have a short window of effectiveness; many cancers develop resistance. André Nussenzweig and colleagues show that cells become drug resistant due to loss of the PTIP protein. In its absence, forks that stall during DNA replication are protected from degradation, and this allows the cells to survive. This work highlights a previously unknown mechanism by which resistance to cancer therapy can arise.

The tumour suppressor breast cancer type 1 susceptibility protein (BRCA1) promotes DNA double-strand break (DSB) repair by homologous recombination and protects DNA replication forks from attrition. BRCA1 partners with BRCA1-associated RING domain protein 1 (BARD1) and other tumour suppressor proteins to mediate the initial nucleolytic resection of DNA lesions and the recruitment and regulation of the recombinase RAD51. The discovery of the opposing functions of BRCA1 and the p53-binding protein 1 (53BP1)-associated complex in DNA resection sheds light on how BRCA1 influences the choice of homologous recombination over non-homologous end joining and potentially other mutagenic pathways of DSB repair. Understanding the functional crosstalk between BRCA1–BARD1 and its cofactors and antagonists will illuminate the molecular basis of cancers that arise from a deficiency or misregulation of chromosome damage repair and replication fork maintenance. Such knowledge will also be valuable for understanding acquired tumour resistance to poly(ADP-ribose) polymerase (PARP) inhibitors and other therapeutics and for the development of new treatments. In this Review, we discuss recent advances in elucidating the mechanisms by which BRCA1–BARD1 functions in DNA repair, replication fork maintenance and tumour suppression, and its therapeutic relevance.BRCA1 and its partner BARD1 support repair of double-strand breaks by homologous recombination and protect replication forks from damage. Recent studies have improved our understanding of the molecular mechanisms of these tumour-suppressive functions of BRCA1–BARD1 and how they are subverted in therapy-resistant cancers.