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Leading-strand template aberrations cause helicase–polymerase uncoupling and impede replication fork progression, but the details of how uncoupled forks are restarted remain uncertain. Using purified proteins from Saccharomyces cerevisiae, we have reconstituted translesion synthesis (TLS)-mediated restart of a eukaryotic replisome following collision with a cyclobutane pyrimidine dimer. We find that TLS functions ‘on the fly’ to promote resumption of rapid replication fork rates, despite lesion bypass occurring uncoupled from the Cdc45-MCM-GINS (CMG) helicase. Surprisingly, the main lagging-strand polymerase, Pol δ, binds the leading strand upon uncoupling and inhibits TLS. Pol δ is also crucial for efficient recoupling of leading-strand synthesis to CMG following lesion bypass. Proliferating cell nuclear antigen monoubiquitination positively regulates TLS to overcome Pol δ inhibition. We reveal that these mechanisms of negative and positive regulation also operate on the lagging strand. Our observations have implications for both fork restart and the division of labor during leading-strand synthesis generally.In vitro reconstitution of translesion synthesis–mediated replication fork restart shows how DNA Pol η is recruited to bypass a CPD lesion on the leading strand in the context of the yeast replisome.

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.

Key Points Ataxia telangiectasia and Rad3-related (ATR) is an essential kinase that is active in S phase, senses stressed replication forks and orchestrates a multifaceted response to DNA replication stress. This response helps ensure completion of DNA replication and maintains the integrity of the genome. ATR and its binding partner, ATR-interacting protein (ATRIP), are recruited to stalled forks through direct interactions with the replication protein A–single-stranded DNA (RPA–ssDNA) complex that forms at stressed replication forks. When bound to ssDNA, the kinase activity of ATR is stimulated by the ATR-activating domains of topoisomerase II binding protein 1 (TOPBP1) or Ewing tumour-associated antigen 1 (ETAA1), which are independently recruited to junctions between ssDNA and double-stranded DNA (dsDNA) and to RPA–ssDNA, respectively. ATR activity can be amplified by generating more ssDNA–dsDNA junctions at individual replication forks, through feed-forward signalling loops and by post-translational modifications of the signalling complexes. When activated, ATR directs the replication stress response to arrest the cell cycle, block origin of replication firing and stabilize and repair stalled replication forks. ATR and its effector, checkpoint kinase 1 (CHK1), are active both during an unperturbed S phase, to prevent excessive origin firing, and in response to replication stress, to slow DNA replication. However, this negative regulation of replication initiation does not prevent the firing of dormant origins within a replication domain, which can rescue replication completion without requiring the damaged fork to restart. ATR phosphorylates numerous replisome proteins and repair factors that prevent fork collapse and the formation of DNA breaks. These post-translational modifications regulate the remodelling of replication forks and subsequent nuclease-dependent cleavage and/or resection of forks. They also regulate pathways needed to repair stalled forks and restart DNA synthesis. Replication stress is controlled by the kinase ataxia telangiectasia and Rad3-related (ATR), which senses and resolves threats to DNA integrity. ATR activation is complex and involves a core set of components that recruit ATR to stressed replication forks, stimulate its kinase activity and amplify downstream signalling to maintain the stability of replication forks. One way to preserve a rare book is to lock it away from all potential sources of damage. Of course, an inaccessible book is also of little use, and the paper and ink will continue to degrade with age in any case. Like a book, the information stored in our DNA needs to be read, but it is also subject to continuous assault and therefore needs to be protected. In this Review, we examine how the replication stress response that is controlled by the kinase ataxia telangiectasia and Rad3-related (ATR) senses and resolves threats to DNA integrity so that the DNA remains available to read in all of our cells. We discuss the multiple data that have revealed an elegant yet increasingly complex mechanism of ATR activation. This involves a core set of components that recruit ATR to stressed replication forks, stimulate kinase activity and amplify ATR signalling. We focus on the activities of ATR in the control of cell cycle checkpoints, origin firing and replication fork stability, and on how proper regulation of these processes is crucial to ensure faithful duplication of a challenging genome.

Complete and accurate DNA replication requires the progression of replication forks through DNA damage, actively transcribed regions, structured DNA and compact chromatin. Recent studies have revealed a remarkable plasticity of the replication process in dealing with these obstacles, which includes modulation of replication origin firing, of the architecture of replication forks, and of the functional organization of the replication machinery in response to replication stress. However, these specialized mechanisms also expose cells to potentially dangerous transactions while replicating DNA. In this Review, we discuss how replication forks are actively stalled, remodelled, processed, protected and restarted in response to specific types of stress. We also discuss adaptations of the replication machinery and the role of chromatin modifications during these transactions. Finally, we discuss interesting recent data on the relevance of replication fork plasticity to human health, covering its role in tumorigenesis, its crosstalk with innate immunity responses and its potential as an effective cancer therapy target.Different obstacles can stall the progression of replication forks. Recent studies have revealed that stalled forks are remarkably diverse in their composition and architecture. This plasticity enables fork remodelling, processing and restart in response to specific types of replication stress, thereby influencing tumorigenesis and innate immunity.

The division of labour among DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequencing (Pu-seq) in HCT116 cells and mapped Polε and Polα usage genome wide. The polymerase usage profiles show Polε synthesises the leading strand and Polα contributes mainly to lagging strand synthesis. Combining the Polε and Polα profiles, we accurately predict the genome-wide pattern of fork directionality plus zones of replication initiation and termination. We confirm that transcriptional activity contributes to the pattern of initiation and termination and, by separately analysing the effect of transcription on co-directional and converging forks, demonstrate that coupled DNA synthesis of leading and lagging strands is compromised by transcription in both co-directional and convergent forks. Polymerase uncoupling is particularly evident in the vicinity of large genes, including the two most unstable common fragile sites, FRA3B and FRA3D, thus linking transcription-induced polymerase uncoupling to chromosomal instability. Together, our result demonstrated that Pu-seq in human cells provides a powerful and straightforward methodology to explore DNA polymerase usage and replication fork dynamics. Profiling of human DNA polymerase Polε and Polα demonstrates their roles in leading and lagging strand DNA synthesis, and their independent measures allowed accurate predictions of replication dynamics and effects of transcription.

Genotoxins cause nascent strand degradation (NSD) and fork reversal during DNA replication. NSD and fork reversal are crucial for genome stability and are exploited by chemotherapeutic approaches. However, it is unclear how NSD and fork reversal are triggered. Additionally, the fate of the replicative helicase during these processes is unknown. We developed a biochemical approach to study synchronous, localized NSD and fork reversal using Xenopus egg extracts and validated this approach with experiments in human cells. We show that replication fork uncoupling stimulates NSD of both nascent strands and progressive conversion of uncoupled forks to reversed forks. Notably, the replicative helicase remains bound during NSD and fork reversal. Unexpectedly, NSD occurs before and after fork reversal, indicating that multiple degradation steps take place. Overall, our data show that uncoupling causes NSD and fork reversal and elucidate key events that precede fork reversal. Kavlashvili et al. use a new in vitro approach to show that uncoupled replication forks can cause fork reversal and nascent strand degradation. Both processes occur without loss of the replisome from DNA and degradation involves multiple steps.

Key Points Activation of initiation of DNA replication occurs at only a subset of replication origins that were previously assembled in the G1 phase of the cell cycle. This is achieved through a highly regulated sequential two-step process: origin licensing in the G1 phase and origin activation during the S phase. DNA replication origins from Saccharomyces cerevisiae have a sequence consensus, whereas metazoan origins are more plastic and are determined by both sequence preferences, such as G-rich elements, and epigenetic features. Chromosomal environment and transcriptional status also influence the activation of origins. Origins selected in adjacent replication units are synchronously activated and form replication domains, which are activated at specific times during the S phase. Replication timing domains correlate with topologically associated domains (TADs). Replication timing is regulated by specific proteins and chromatin marks. The activation of DNA replication origins is developmentally controlled. Specific checkpoints regulate the initiation of DNA replication in response to replication stress. During the G1–S phase transition of the cell cycle, a variable subset of previously 'licensed' origins of replication is activated to initiate DNA synthesis. Insight is being gained into the mechanisms underlying which origins are activated and when; these mechanisms are associated with nuclear organization, cell differentiation and replication stress. DNA replication begins with the assembly of pre-replication complexes (pre-RCs) at thousands of DNA replication origins during the G1 phase of the cell cycle. At the G1–S-phase transition, pre-RCs are converted into pre-initiation complexes, in which the replicative helicase is activated, leading to DNA unwinding and initiation of DNA synthesis. However, only a subset of origins are activated during any S phase. Recent insights into the mechanisms underlying this choice reveal how flexibility in origin usage and temporal activation are linked to chromosome structure and organization, cell growth and differentiation, and replication stress.

BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from replication stress and genotoxicity, but the specific mechanism(s) by which this is achieved to prevent early oncogenesis remains unclear. Here, we provide evidence that BRCA2 acts as a critical suppressor of head-on transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNase H2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results provide a mechanistic basis for how BRCA2 shields against genomic instability by preventing HO-TRCs through both direct and indirect means occurring at predetermined genomic sites based on the pre-cancer transcriptome. BRCA2 has essential roles in suppressing genome instability at stalled replication forks, but how this is achieved remains unclear. Here, the authors apply Okazaki-fragment sequencing to predict sites of genomic instability caused by head-on transcription-replication conflicts upon BRCA2 loss.

Chromosome replication is performed by a complex and intricate ensemble of proteins termed the replisome, where the DNA polymerases Polδ and Polε, DNA polymerase α-primase (Polα) and accessory proteins including AND-1, CLASPIN and TIMELESS–TIPIN (respectively known as Ctf4, Mrc1 and Tof1–Csm3 in Saccharomyces cerevisiae ) are organized around the CDC45–MCM–GINS (CMG) replicative helicase 1 – 7 . Because a functional human replisome has not been reconstituted from purified proteins, how these factors contribute to human DNA replication and whether additional proteins are required for optimal DNA synthesis are poorly understood. Here we report the biochemical reconstitution of human replisomes that perform fast and efficient DNA replication using 11 purified human replication factors made from 43 polypeptides. Polε, but not Polδ, is crucial for optimal leading-strand synthesis. Unexpectedly, Polε-mediated leading-strand replication is highly dependent on the sliding-clamp processivity factor PCNA and the alternative clamp loader complex CTF18–RFC. We show how CLASPIN and TIMELESS–TIPIN contribute to replisome progression and demonstrate that, in contrast to the budding yeast replisome 8 , AND-1 directly augments leading-strand replication. Moreover, although AND-1 binds to Polα 9 , 10 , the interaction is dispensable for lagging-strand replication, indicating that Polα is functionally recruited via an AND-1-independent mechanism for priming in the human replisome. Collectively, our work reveals how the human replisome achieves fast and efficient leading-strand and lagging-strand DNA replication, and provides a powerful system for future studies of the human replisome and its interactions with other DNA metabolic processes. A biochemical reconstitution of human replisomes that provides a system for future studies of DNA metabolic processes.

Genome-wide analyses of S. cerevisiae replisome mobility reveal overlapping roles of Pif1 and Rrm3 helicases in alleviating replication-fork arrest at tRNA genes. Saccharomyces cerevisiae expresses two Pif1-family helicases—Pif1 and Rrm3—which have been reported to play distinct roles in numerous nuclear processes. Here, we systematically characterized the roles of Pif1 helicases in replisome progression and lagging-strand synthesis in S. cerevisiae . We demonstrate that either Pif1 or Rrm3 redundantly stimulates strand displacement by DNA polymerase δ during lagging-strand synthesis. By analyzing replisome mobility in pif1 and rrm3 mutants, we show that Rrm3, with a partially redundant contribution from Pif1, suppresses widespread terminal arrest of the replisome at tRNA genes. Although both head-on and codirectional collisions induce replication-fork arrest at tRNA genes, head-on collisions arrest a higher proportion of replisomes. In agreement with this observation, we found that head-on collisions between tRNA transcription and replication are under-represented in the S. cerevisiae genome. We demonstrate that tRNA-mediated arrest is R-loop independent and propose that replisome arrest and DNA damage are mechanistically separable.