Replication origins drive genetic and phenotypic variation in humans

DNA replication starts with the activation of the replicative helicases, polymerases and associated factors at thousands of origins per S-phase. Due to local torsional constraints generated during licensing and the switch between polymerases of distinct fidelity and proofreading ability following firing, origin activation has the potential to induce DNA damage and mutagenesis. However, whether sites of replication initiation exhibit a specific mutational footprint has not yet been established. Here we demonstrate that mutagenesis is increased at early and highly efficient origins. The elevated mutation rate observed at these sites is caused by two distinct mutational processes consistent with formation of DNA breaks at the origin itself and local error-prone DNA synthesis in the immediate vicinity of the origin. We demonstrate that these replication-dependent mutational processes create the skew in base composition observed at human replication origins. Further, we show that mutagenesis associated with replication initiation exerts an influence on phenotypic diversity in human populations disproportionate to the origins genomic footprint: by increasing mutational loads at gene promoters and splice junctions the presence of an origin influences both gene expression and mRNA isoform usage. These findings have important implications for our understanding of the mutational processes that sculpt the human genome.

Cell-Cycle—Dependent Chromatin Dynamics at Replication Origins

Origins of DNA replication are specified by the ordered recruitment of replication factors in a cell-cycle—dependent manner. The assembly of the pre-replicative complex in G1 and the pre-initiation complex prior to activation in S phase are well characterized; however, the interplay between the assembly of these complexes and the local chromatin environment is less well understood. To investigate the dynamic changes in chromatin organization at and surrounding replication origins, we used micrococcal nuclease (MNase) to generate genome-wide chromatin occupancy profiles of nucleosomes, transcription factors, and replication proteins through consecutive cell cycles in Saccharomyces cerevisiae. During each G1 phase of two consecutive cell cycles, we observed the downstream repositioning of the origin-proximal +1 nucleosome and an increase in protected DNA fragments spanning the ARS consensus sequence (ACS) indicative of pre-RC assembly. We also found that the strongest correlation between chromatin occupancy at the ACS and origin efficiency occurred in early S phase, consistent with the rate-limiting formation of the Cdc45–Mcm2-7–GINS (CMG) complex being a determinant of origin activity. Finally, we observed nucleosome disruption and disorganization emanating from replication origins and traveling with the elongating replication forks across the genome in S phase, likely reflecting the disassembly and assembly of chromatin ahead of and behind the replication fork, respectively. These results provide insights into cell-cycle–regulated chromatin dynamics and how they relate to the regulation of origin activity.

Determination of human DNA replication origin position and efficiency reveals principles of initiation zone organisation

Replication of the human genome initiates within broad zones of ~ 150 kb. The extent to which firing of individual DNA replication origins within initiation zones is spatially stochastic or localised at defined sites remains a matter of debate. A thorough characterisation of the dynamic activation of origins within initiation zones is hampered by the lack of a high-resolution map of both their position and efficiency. To address this shortcoming, we describe a modification of initiation site sequencing (ini-seq) based on density substitution. Newly-replicated DNA is rendered heavy-light (HL) by incorporation of BrdUTP, unreplicated DNA remaining light-light (LL). Replicated HL-DNA is separated from unreplicated LL-DNA by equilibrium density gradient centrifugation, then both fractions are subjected to massive parallel sequencing. This allows precise mapping of 23,905 replication origins simultaneously with an assignment of a replication initiation efficiency score to each. We show that origin firing within initiation zones is not randomly distributed. Rather, origins are arranged hierarchically with a set of very highly efficient origins marking zone boundaries. We propose that these origins explain much of the early firing activity arising within initiation zones, helping to unify the concept of replication initiation zones with the identification of discrete replication origin sites.

Cell cycle-dependent chromatin dynamics at replication origins

Origins of DNA replication are specified by the ordered recruitment of replication factors in a cell cycle dependent manner. The assembly of the pre-replicative complex in G1 and the pre-initiation complex prior to activation in S-phase are well characterized; however, the interplay between the assembly of these complexes and the local chromatin environment is less well understood. To investigate the dynamic changes in chromatin organization at and surrounding replication origins, we used micrococcal nuclease (MNase) to generate genome-wide chromatin occupancy profiles of nucleosomes, transcription factors and replication proteins through consecutive cell cycles in Saccharomyces cerevisiae. During each G1 phase of two consecutive cell cycles, we observed the downstream repositioning of the origin-proximal +1 nucleosome and an increase in protected DNA fragments spanning the ARS consensus sequence (ACS) indicative of pre-RC assembly. We also found that the strongest correlation between the chromatin occupancy at the ACS and origin efficiency occurred in early S-phase consistent with the rate limiting formation of the Cdc45-Mcm2-7-GINS (CMG) complex being a determinant of origin activity. Finally, we observed nucleosome disruption and disorganization emanating from replication origins and traveling with the elongating replication forks across the genome in S-phase, likely reflecting the disassembly and assembly of chromatin ahead of and behind the replication fork, respectively. These results provide insights into cell cycle-regulated chromatin dynamics and how they relate to the regulation of origin activity.

RNF4 Regulates the BLM Helicase in Recovery From Replication Fork Collapse

Sumoylation is an important enhancer of responses to DNA replication stress and the SUMO-targeted ubiquitin E3 ligase RNF4 regulates these responses by ubiquitylation of sumoylated DNA damage response factors. The specific targets and functional consequences of RNF4 regulation in response to replication stress, however, have not been fully characterized. Here we demonstrated that RNF4 is required for the restart of DNA replication following prolonged hydroxyurea (HU)-induced replication stress. Contrary to its role in repair of γ-irradiation-induced DNA double-strand breaks (DSBs), our analysis revealed that RNF4 does not significantly impact recognition or repair of replication stress-associated DSBs. Rather, using DNA fiber assays, we found that the firing of new DNA replication origins, which is required for replication restart following prolonged stress, was inhibited in cells depleted of RNF4. We also provided evidence that RNF4 recognizes and ubiquitylates sumoylated Bloom syndrome DNA helicase BLM and thereby promotes its proteosome-mediated turnover at damaged DNA replication forks. Consistent with it being a functionally important RNF4 substrate, co-depletion of BLM rescued defects in the firing of new replication origins observed in cells depleted of RNF4 alone. We concluded that RNF4 acts to remove sumoylated BLM from collapsed DNA replication forks, which is required to facilitate normal resumption of DNA synthesis after prolonged replication fork stalling and collapse.

High levels of origin licensing during Xenopus cleavage divisions ensures complete and timely genome duplication

Cells face several challenges to completing genome duplication. One challenge is the irreversible stalling of converging replication forks (double fork stalls). Cell types that cannot delay mitotic entry must also ensure that no replication origins are too far apart (the random gap problem). We show how these challenges can be met in early Xenopus embryos by the very abundant licensing of replication origins: one MCM2-7 double hexamer every ~250 bp. Licensing does not change nucleosome spacing, consistent with MCM2-7 being assembled onto inter-nucleosomal linker DNA. We show that later embryonic development can occur successfully with a per-cell cycle failure rate of <0.2% in early embryos. The high density of licensed origins in the early embryo reduces cell cycle failures from random gaps and from double fork stalls to levels compatible with subsequent development, suggesting that Xenopus early embryonic cells can ensure complete genome duplication without requiring unconventional replication mechanisms.

A Helicase-tethered ORC Flip Enables Bidirectional Helicase Loading

Replication origins are licensed by loading two Mcm2-7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires ORC, Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with FRET, we investigated interactions between ORC and Mcm2-7 during helicase loading. We demonstrate that a single ORC molecule can recruit both Mcm2-7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between the first and second helicase recruitment, we observe a rapid change in interactions between ORC and the first Mcm2-7. In quick succession ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2-7-ORC-DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly-coordinated series of events through which a single ORC molecule can load two oppositely-oriented helicases.

Detection and characterization of replication origins defined by DNA polymerase epsilon

Human origins of replication (ORI) are recognized by the cellular machinery through mechanisms which are still poorly understood. The process of DNA replication is highly conserved, but the location of ORI may vary considerably from one round of replication to the next, and depends on many factors, making them difficult to map in the genome. We investigated the possibility that at genomic scale, the mutator phenotype associated with DNA polymerase epsilon; exonuclease domain mutation could identify genomic positions of replication origins. Here we report the genome-wide localization of DNA replication origins in POLE-mutated tumors using whole genome sequencing data from The Cancer Genome Atlas project. We show that mutation-based detection of replication origins allows to identify constitutive origins shared between various individuals. We developed a novel method to compare two or more sets of genomic coordinates based on Smith-Waterman-like dynamic programming, which we used to compare replication origin positions obtained using multiple different methods. The comparison allowed us to define a consensus set of replication origins, identified consistently by multiple ORI detection methods. Many DNA features co-localized with ORI, including chromatin loop anchors, G-quadruplexes, S/MARs and CpGs. Among all features, the H2A.Z histone exhibited the most significant association. Our results show that mutation-based detection of replication origins is a viable approach to determining their location and associated sequence features.

Transcription-coupled structural dynamics of topologically associating domains regulate replication origin efficiency

Background Metazoan cells only utilize a small subset of the potential DNA replication origins to duplicate the whole genome in each cell cycle. Origin choice is linked to cell growth, differentiation, and replication stress. Although various genetic and epigenetic signatures have been linked to the replication efficiency of origins, there is no consensus on how the selection of origins is determined. Results We apply dual-color stochastic optical reconstruction microscopy (STORM) super-resolution imaging to map the spatial distribution of origins within individual topologically associating domains (TADs). We find that multiple replication origins initiate separately at the spatial boundary of a TAD at the beginning of the S phase. Intriguingly, while both high-efficiency and low-efficiency origins are distributed homogeneously in the TAD during the G1 phase, high-efficiency origins relocate to the TAD periphery before the S phase. Origin relocalization is dependent on both transcription and CTCF-mediated chromatin structure. Further, we observe that the replication machinery protein PCNA forms immobile clusters around TADs at the G1/S transition, explaining why origins at the TAD periphery are preferentially fired. Conclusion Our work reveals a new origin selection mechanism that the replication efficiency of origins is determined by their physical distribution in the chromatin domain, which undergoes a transcription-dependent structural re-organization process. Our model explains the complex links between replication origin efficiency and many genetic and epigenetic signatures that mark active transcription. The coordination between DNA replication, transcription, and chromatin organization inside individual TADs also provides new insights into the biological functions of sub-domain chromatin structural dynamics.

Dynamics of replication origin over-activation

Safeguards against excess DNA replication are often dysregulated in cancer, and driving cancer cells towards over-replication is a promising therapeutic strategy. We determined DNA synthesis patterns in cancer cells undergoing partial genome re-replication due to perturbed regulatory interactions (re-replicating cells). These cells exhibited slow replication, increased frequency of replication initiation events, and a skewed initiation pattern that preferentially reactivated early-replicating origins. Unlike in cells exposed to replication stress, which activated a novel group of hitherto unutilized (dormant) replication origins, the preferred re-replicating origins arose from the same pool of potential origins as those activated during normal growth. Mechanistically, the skewed initiation pattern reflected a disproportionate distribution of pre-replication complexes on distinct regions of licensed chromatin prior to replication. This distinct pattern suggests that circumventing the strong inhibitory interactions that normally prevent excess DNA synthesis can occur via at least two pathways, each activating a distinct set of replication origins.