PRIMPOL ensures robust handoff between on-the-fly and post-replicative DNA lesion bypass.

The primase/polymerase PRIMPOL restarts DNA synthesis when replication is arrested by template impediments. However, we do not have a comprehensive view of how PRIMPOL-dependent repriming integrates with the main pathways of damage tolerance, REV1-dependent 'on-the-fly' lesion bypass at the fork and PCNA ubiquitination-dependent post-replicative gap filling. Guided by genome-wide CRISPR/Cas9 screens to survey the genetic interactions of PRIMPOL in a non-transformed and p53-proficient human cell line, we find that PRIMPOL is needed for cell survival following loss of the Y-family polymerases REV1 and POLη in a lesion-dependent manner, while it plays a broader role in promoting survival of cells lacking PCNA K164-dependent post-replicative gap filling. Thus, while REV1- and PCNA K164R-bypass provide two layers of protection to ensure effective damage tolerance, PRIMPOL is required to maximise the effectiveness of the interaction between them. We propose this is through the restriction of post-replicative gap length provided by PRIMPOL-dependent repriming.

Continuous synthesis of E. coli genome sections and Mb-scale human DNA assembly.

Whole-genome synthesis provides a powerful approach for understanding and expanding organism function1-3. To build large genomes rapidly, scalably and in parallel, we need (1) methods for assembling megabases of DNA from shorter precursors and (2) strategies for rapidly and scalably replacing the genomic DNA of organisms with synthetic DNA. Here we develop bacterial artificial chromosome (BAC) stepwise insertion synthesis (BASIS)-a method for megabase-scale assembly of DNA in Escherichia coli episomes. We used BASIS to assemble 1.1 Mb of human DNA containing numerous exons, introns, repetitive sequences, G-quadruplexes, and long and short interspersed nuclear elements (LINEs and SINEs). BASIS provides a powerful platform for building synthetic genomes for diverse organisms. We also developed continuous genome synthesis (CGS)-a method for continuously replacing sequential 100 kb stretches of the E. coli genome with synthetic DNA; CGS minimizes crossovers1,4 between the synthetic DNA and the genome such that the output for each 100 kb replacement provides, without sequencing, the input for the next 100 kb replacement. Using CGS, we synthesized a 0.5 Mb section of the E. coli genome-a key intermediate in its total synthesis1-from five episomes in 10 days. By parallelizing CGS and combining it with rapid oligonucleotide synthesis and episome assembly5,6, along with rapid methods for compiling a single genome from strains bearing distinct synthetic genome sections1,7,8, we anticipate that it will be possible to synthesize entire E. coli genomes from functional designs in less than 2 months.

DNA replication initiation shapes the mutational landscape and expression of the human genome.

The interplay between active biological processes and DNA repair is central to mutagenesis. Here, we show that the ubiquitous process of replication initiation is mutagenic, leaving a specific mutational footprint at thousands of early and efficient replication origins. The observed mutational pattern is consistent with two distinct mechanisms, reflecting the two-step process of origin activation, triggering the formation of DNA breaks at the center of origins and local error-prone DNA synthesis in their immediate vicinity. We demonstrate that these replication initiation-dependent mutational processes exert an influence on phenotypic diversity in humans that is disproportionate to the origins' genomic size: By increasing mutational loads at gene promoters and splice junctions, the presence of an origin significantly influences both gene expression and mRNA isoform usage. Last, we show that mutagenesis at origins not only drives the evolution of origin sequences but also contributes to sculpting regulatory domains of the human genome.

Creation and resolution of non-B-DNA structural impediments during replication.

During replication, folding of the DNA template into non-B-form secondary structures provides one of the most abundant impediments to the smooth progression of the replisome. The core replisome collaborates with multiple accessory factors to ensure timely and accurate duplication of the genome and epigenome. Here, we discuss the forces that drive non-B structure formation and the evidence that secondary structures are a significant and frequent source of replication stress that must be actively countered. Taking advantage of recent advances in the molecular and structural biology of the yeast and human replisomes, we examine how structures form and how they may be sensed and resolved during replication.

Mechanisms of APOBEC3 mutagenesis in human cancer cells.

The APOBEC3 family of cytosine deaminases has been implicated in some of the most prevalent mutational signatures in cancer1-3. However, a causal link between endogenous APOBEC3 enzymes and mutational signatures in human cancer genomes has not been established, leaving the mechanisms of APOBEC3 mutagenesis poorly understood. Here, to investigate the mechanisms of APOBEC3 mutagenesis, we deleted implicated genes from human cancer cell lines that naturally generate APOBEC3-associated mutational signatures over time4. Analysis of non-clustered and clustered signatures across whole-genome sequences from 251 breast, bladder and lymphoma cancer cell line clones revealed that APOBEC3A deletion diminished APOBEC3-associated mutational signatures. Deletion of both APOBEC3A and APOBEC3B further decreased APOBEC3 mutation burdens, without eliminating them. Deletion of APOBEC3B increased APOBEC3A protein levels, activity and APOBEC3A-mediated mutagenesis in some cell lines. The uracil glycosylase UNG was required for APOBEC3-mediated transversions, whereas the loss of the translesion polymerase REV1 decreased overall mutation burdens. Together, these data represent direct evidence that endogenous APOBEC3 deaminases generate prevalent mutational signatures in human cancer cells. Our results identify APOBEC3A as the main driver of these mutations, indicate that APOBEC3B can restrain APOBEC3A-dependent mutagenesis while contributing its own smaller mutation burdens and dissect mechanisms that translate APOBEC3 activities into distinct mutational signatures.

In vitro evolution predicts emerging SARS-CoV-2 mutations with high affinity for ACE2 and cross-species binding.

Emerging SARS-CoV-2 variants are creating major challenges in the ongoing COVID-19 pandemic. Being able to predict mutations that could arise in SARS-CoV-2 leading to increased transmissibility or immune evasion would be extremely valuable in development of broad-acting therapeutics and vaccines, and prioritising viral monitoring and containment. Here we use in vitro evolution to seek mutations in SARS-CoV-2 receptor binding domain (RBD) that would substantially increase binding to ACE2. We find a double mutation, S477N and Q498H, that increases affinity of RBD for ACE2 by 6.5-fold. This affinity gain is largely driven by the Q498H mutation. We determine the structure of the mutant-RBD:ACE2 complex by cryo-electron microscopy to reveal the mechanism for increased affinity. Addition of Q498H to SARS-CoV-2 RBD variants is found to boost binding affinity of the variants for human ACE2 and confer a new ability to bind rat ACE2 with high affinity. Surprisingly however, in the presence of the common N501Y mutation, Q498H inhibits binding, due to a clash between H498 and Y501 side chains. To achieve an intermolecular bonding network, affinity gain and cross-species binding similar to Q498H alone, RBD variants with the N501Y mutation must acquire instead the related Q498R mutation. Thus, SARS-CoV-2 RBD can access large affinity gains and cross-species binding via two alternative mutational routes involving Q498, with route selection determined by whether a variant already has the N501Y mutation. These mutations are now appearing in emerging SARS-CoV-2 variants where they have the potential to influence human-to-human and cross-species transmission.

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 while unreplicated DNA remains '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 early 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.

Distinct roles for PARP-1 and PARP-2 in c-Myc-driven B-cell lymphoma in mice.

Dysregulation of the c-Myc oncogene occurs in a wide variety of hematologic malignancies, and its overexpression has been linked with aggressive tumor progression. Here, we show that poly (ADP-ribose) polymerase 1 (PARP-1) and PARP-2 exert opposing influences on progression of c-Myc-driven B-cell lymphoma. PARP-1 and PARP-2 catalyze the synthesis and transfer of ADP-ribose units onto amino acid residues of acceptor proteins in response to DNA strand breaks, playing a central role in the response to DNA damage. Accordingly, PARP inhibitors have emerged as promising new cancer therapeutics. However, the inhibitors currently available for clinical use are not able to discriminate between individual PARP proteins. We found that genetic deletion of PARP-2 prevents c-Myc-driven B-cell lymphoma, whereas PARP-1 deficiency accelerates lymphomagenesis in the Eµ-Myc mouse model of aggressive B-cell lymphoma. Loss of PARP-2 aggravates replication stress in preleukemic Eµ-Myc B cells, resulting in accumulation of DNA damage and concomitant cell death that restricts the c-Myc-driven expansion of B cells, thereby providing protection against B-cell lymphoma. In contrast, PARP-1 deficiency induces a proinflammatory response and an increase in regulatory T cells, likely contributing to immune escape of B-cell lymphoma, resulting in an acceleration of lymphomagenesis. These findings pinpoint specific functions for PARP-1 and PARP-2 in c-Myc-driven lymphomagenesis with antagonistic consequences that may help inform the design of new PARP-centered therapeutic strategies, with selective PARP-2 inhibition potentially representing a new therapeutic approach for the treatment of c-Myc-driven tumors.

Monitoring the replication of structured DNA through heritable epigenetic change.

DNA can adopt non-B form structures that create significant blocks to DNA synthesis and seeking understanding of the mechanisms cells use to resolve such impediments continues to be a very active area of research. However, the ability to monitor the stalling of DNA synthesis at specific sites in the genome in living cells, of central importance to elucidating these mechanisms, poses a significant technical challenge. Replisome stalling is often transient with only a small fraction of events leading to detectable genetic changes, making traditional reporter assays insensitive to the stalling event per se. On the other hand, the imprint stalling leaves on the epigenome can be exploited as a form of biological 'tape recorder' that captures episodes of fork stalling as heritable changes in histone modifications and in transcription. Here we describe a detailed protocol for monitoring DNA structure-dependent epigenetic instability of the BU-1 locus in the avian cell line DT40, which has proved a sensitive tool for understanding the mechanisms by which structured DNA is replicated in a vertebrate system.

The intersection of DNA replication with antisense 3' RNA processing in Arabidopsis FLC chromatin silencing.

How noncoding transcription influences chromatin states is still unclear. The Arabidopsis floral repressor gene FLC is quantitatively regulated through an antisense-mediated chromatin silencing mechanism. The FLC antisense transcripts form a cotranscriptional R-loop that is dynamically resolved by RNA 3' processing factors (FCA and FY), and this is linked to chromatin silencing. Here, we investigate this silencing mechanism and show, using single-molecule DNA fiber analysis, that FCA and FY are required for unimpeded replication fork progression across the Arabidopsis genome. We then employ the chicken DT40 cell line system, developed to investigate sequence-dependent replication and chromatin inheritance, and find that FLC R-loop sequences have an orientation-dependent ability to stall replication forks. These data suggest a coordination between RNA 3' processing of antisense RNA and replication fork progression in the inheritance of chromatin silencing at FLC.

A p53-Dependent Checkpoint Induced upon DNA Damage Alters Cell Fate during hiPSC Differentiation.

The ability of human induced pluripotent stem cells (hiPSCs) to differentiate in vitro to each of the three germ layer lineages has made them an important model of early human development and a tool for tissue engineering. However, the factors that disturb the intricate transcriptional choreography of differentiation remain incompletely understood. Here, we uncover a critical time window during which DNA damage significantly reduces the efficiency and fidelity with which hiPSCs differentiate to definitive endoderm. DNA damage prevents the normal reduction of p53 levels as cells pass through the epithelial-to-mesenchymal transition, diverting the transcriptional program toward mesoderm without induction of an apoptotic response. In contrast, TP53-deficient cells differentiate to endoderm with high efficiency after DNA damage, suggesting that p53 enforces a "differentiation checkpoint" in early endoderm differentiation that alters cell fate in response to DNA damage.

DNA polymerase stalling at structured DNA constrains the expansion of short tandem repeats.

BACKGROUND: Short tandem repeats (STRs) contribute significantly to de novo mutagenesis, driving phenotypic diversity and genetic disease. Although highly diverse, their repetitive sequences induce DNA polymerase slippage and stalling, leading to length and sequence variation. However, current studies of DNA synthesis through STRs are restricted to a handful of selected sequences, limiting our broader understanding of their evolutionary behaviour and hampering the characterisation of the determinants of their abundance and stability in eukaryotic genomes. RESULTS: We perform a comprehensive analysis of DNA synthesis at all STR permutations and interrogate the impact of STR sequence and secondary structure on their genomic representation and mutability. To do this, we developed a high-throughput primer extension assay that allows monitoring of the kinetics and fidelity of DNA synthesis through 20,000 sequences comprising all STR permutations in different lengths. By combining these measurements with population-scale genomic data, we show that the response of a model replicative DNA polymerase to variously structured DNA is sufficient to predict the complex genomic behaviour of STRs, including abundance and mutational constraints. We demonstrate that DNA polymerase stalling at DNA structures induces error-prone DNA synthesis, which constrains STR expansion. CONCLUSIONS: Our data support a model in which STR length in eukaryotic genomes results from a balance between expansion due to polymerase slippage at repeated DNA sequences and point mutations caused by error-prone DNA synthesis at DNA structures.

Timeless couples G-quadruplex detection with processing by DDX11 helicase during DNA replication.

Regions of the genome with the potential to form secondary DNA structures pose a frequent and significant impediment to DNA replication and must be actively managed in order to preserve genetic and epigenetic integrity. How the replisome detects and responds to secondary structures is poorly understood. Here, we show that a core component of the fork protection complex in the eukaryotic replisome, Timeless, harbours in its C-terminal region a previously unappreciated DNA-binding domain that exhibits specific binding to G-quadruplex (G4) DNA structures. We show that this domain contributes to maintaining processive replication through G4-forming sequences, and exhibits partial redundancy with an adjacent PARP-binding domain. Further, this function of Timeless requires interaction with and activity of the helicase DDX11. Loss of both Timeless and DDX11 causes epigenetic instability at G4-forming sequences and DNA damage. Our findings indicate that Timeless contributes to the ability of the replisome to sense replication-hindering G4 formation and ensures the prompt resolution of these structures by DDX11 to maintain processive DNA synthesis.

PRIMPOL-Mediated Adaptive Response Suppresses Replication Fork Reversal in BRCA-Deficient Cells.

Acute treatment with replication-stalling chemotherapeutics causes reversal of replication forks. BRCA proteins protect reversed forks from nucleolytic degradation, and their loss leads to chemosensitivity. Here, we show that fork degradation is no longer detectable in BRCA1-deficient cancer cells exposed to multiple cisplatin doses, mimicking a clinical treatment regimen. This effect depends on increased expression and chromatin loading of PRIMPOL and is regulated by ATR activity. Electron microscopy and single-molecule DNA fiber analyses reveal that PRIMPOL rescues fork degradation by reinitiating DNA synthesis past DNA lesions. PRIMPOL repriming leads to accumulation of ssDNA gaps while suppressing fork reversal. We propose that cells adapt to repeated cisplatin doses by activating PRIMPOL repriming under conditions that would otherwise promote pathological reversed fork degradation. This effect is generalizable to other conditions of impaired fork reversal (e.g., SMARCAL1 loss or PARP inhibition) and suggests a new strategy to modulate cisplatin chemosensitivity by targeting the PRIMPOL pathway.

Replication of G Quadruplex DNA.

A cursory look at any textbook image of DNA replication might suggest that the complex machine that is the replisome runs smoothly along the chromosomal DNA. However, many DNA sequences can adopt non-B form secondary structures and these have the potential to impede progression of the replisome. A picture is emerging in which the maintenance of processive DNA replication requires the action of a significant number of additional proteins beyond the core replisome to resolve secondary structures in the DNA template. By ensuring that DNA synthesis remains closely coupled to DNA unwinding by the replicative helicase, these factors prevent impediments to the replisome from causing genetic and epigenetic instability. This review considers the circumstances in which DNA forms secondary structures, the potential responses of the eukaryotic replisome to these impediments in the light of recent advances in our understanding of its structure and operation and the mechanisms cells deploy to remove secondary structure from the DNA. To illustrate the principles involved, we focus on one of the best understood DNA secondary structures, G quadruplexes (G4s), and on the helicases that promote their resolution.

CtIP-BRCA1 complex and MRE11 maintain replication forks in the presence of chain terminating nucleoside analogs.

Chain-terminating nucleoside analogs (CTNAs), which cannot be extended by DNA polymerases, are widely used as antivirals or anti-cancer agents, and can induce cell death. Processing of blocked DNA ends, like camptothecin-induced trapped-topoisomerase I, can be mediated by TDP1, BRCA1, CtIP and MRE11. Here, we investigated whether the CtIP-BRCA1 complex and MRE11 also contribute to cellular tolerance to CTNAs, including 2',3'-dideoxycytidine (ddC), cytarabine (ara-C) and zidovudine (Azidothymidine, AZT). We show that BRCA1-/-, CtIPS332A/-/- and nuclease-dead MRE11D20A/- mutants display increased sensitivity to CTNAs, accumulate more DNA damage (chromosomal breaks, γ-H2AX and neutral comets) when treated with CTNAs and exhibit significant delays in replication fork progression during exposure to CTNAs. Moreover, BRCA1-/-, CtIPS332A/-/- and nuclease-dead MRE11D20A/- mutants failed to resume DNA replication in response to CTNAs, whereas control and CtIP+/-/- cells experienced extensive recovery of DNA replication. In summary, we provide clear evidence that MRE11 and the collaborative action of BRCA1 and CtIP play a critical role in the nuclease-dependent removal of incorporated ddC from replicating genomic DNA. We propose that BRCA1-CTIP and MRE11 prepare nascent DNA ends, blocked from synthesis by CTNAs, for further repair.