Replication stress impairs chromosome segregation and preimplantation development in human embryos.

Human cleavage-stage embryos frequently acquire chromosomal aneuploidies during mitosis due to unknown mechanisms. Here, we show that S phase at the 1-cell stage shows replication fork stalling, low fork speed, and DNA synthesis extending into G2 phase. DNA damage foci consistent with collapsed replication forks, DSBs, and incomplete replication form in G2 in an ATR- and MRE11-dependent manner, followed by spontaneous chromosome breakage and segmental aneuploidies. Entry into mitosis with incomplete replication results in chromosome breakage, whole and segmental chromosome errors, micronucleation, chromosome fragmentation, and poor embryo quality. Sites of spontaneous chromosome breakage are concordant with sites of DNA synthesis in G2 phase, locating to gene-poor regions with long neural genes, which are transcriptionally silent at this stage of development. Thus, DNA replication stress in mammalian preimplantation embryos predisposes gene-poor regions to fragility, and in particular in the human embryo, to the formation of aneuploidies, impairing developmental potential.

Mutational Strand Asymmetries in Cancer Genomes Reveal Mechanisms of DNA Damage and Repair.

Mutational processes constantly shape the somatic genome, leading to immunity, aging, cancer, and other diseases. When cancer is the outcome, we are afforded a glimpse into these processes by the clonal expansion of the malignant cell. Here, we characterize a less explored layer of the mutational landscape of cancer: mutational asymmetries between the two DNA strands. Analyzing whole-genome sequences of 590 tumors from 14 different cancer types, we reveal widespread asymmetries across mutagenic processes, with transcriptional ("T-class") asymmetry dominating UV-, smoking-, and liver-cancer-associated mutations and replicative ("R-class") asymmetry dominating POLE-, APOBEC-, and MSI-associated mutations. We report a striking phenomenon of transcription-coupled damage (TCD) on the non-transcribed DNA strand and provide evidence that APOBEC mutagenesis occurs on the lagging-strand template during DNA replication. As more genomes are sequenced, studying and classifying their asymmetries will illuminate the underlying biological mechanisms of DNA damage and repair.

Error-prone repair of stalled replication forks drives mutagenesis and loss of heterozygosity in haploinsufficient BRCA1 cells.

Germline mutations in the BRCA genes are associated with a higher risk of carcinogenesis, which is linked to an increased mutation rate and loss of the second unaffected BRCA allele (loss of heterozygosity, LOH). However, the mechanisms triggering mutagenesis are not clearly understood. The BRCA genes contain high numbers of repetitive DNA sequences. We detected replication forks stalling, DNA breaks, and deletions at these sites in haploinsufficient BRCA cells, thus identifying the BRCA genes as fragile sites. Next, we found that stalled forks are repaired by error-prone pathways, such as microhomology-mediated break-induced replication (MMBIR) in haploinsufficient BRCA1 breast epithelial cells. We detected MMBIR mutations in BRCA1 tumor cells and noticed deletions-insertions (>50 bp) at the BRCA1 genes in BRCA1 patients. Altogether, these results suggest that under stress, error-prone repair of stalled forks is upregulated and induces mutations, including complex genomic rearrangements at the BRCA genes (LOH), in haploinsufficient BRCA1 cells.

Genetic variation in human DNA replication timing.

Genomic DNA replicates in a choreographed temporal order that impacts the distribution of mutations along the genome. We show here that DNA replication timing is shaped by genetic polymorphisms that act in cis upon megabase-scale DNA segments. In genome sequences from proliferating cells, read depth along chromosomes reflected DNA replication activity in those cells. We used this relationship to analyze variation in replication timing among 161 individuals sequenced by the 1000 Genomes Project. Genome-wide association of replication timing with genetic variation identified 16 loci at which inherited alleles associate with replication timing. We call these "replication timing quantitative trait loci" (rtQTLs). rtQTLs involved the differential use of replication origins, exhibited allele-specific effects on replication timing, and associated with gene expression variation at megabase scales. Our results show replication timing to be shaped by genetic polymorphism and identify a means by which inherited polymorphism regulates the mutability of nearby sequences.

Human DDK rescues stalled forks and counteracts checkpoint inhibition at unfired origins to complete DNA replication.

Eukaryotic genomes replicate via spatially and temporally regulated origin firing. Cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) promote origin firing, whereas the S phase checkpoint limits firing to prevent nucleotide and RPA exhaustion. We used chemical genetics to interrogate human DDK with maximum precision, dissect its relationship with the S phase checkpoint, and identify DDK substrates. We show that DDK inhibition (DDKi) leads to graded suppression of origin firing and fork arrest. S phase checkpoint inhibition rescued origin firing in DDKi cells and DDK-depleted Xenopus egg extracts. DDKi also impairs RPA loading, nascent-strand protection, and fork restart. Via quantitative phosphoproteomics, we identify the BRCA1-associated (BRCA1-A) complex subunit MERIT40 and the cohesin accessory subunit PDS5B as DDK effectors in fork protection and restart. Phosphorylation neutralizes autoinhibition mediated by intrinsically disordered regions in both substrates. Our results reveal mechanisms through which DDK controls the duplication of large vertebrate genomes.

Genomic signature of Fanconi anaemia DNA repair pathway deficiency in cancer.

Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage1-3. The FA repair pathway protects against endogenous and exogenous carcinogenic aldehydes4-7. Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas8 (SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or infection with human papillomavirus9 (HPV). Here, by sequencing genomes and exomes of FA SCCs, we demonstrate that the primary genomic signature of FA repair deficiency is the presence of high numbers of structural variants. Structural variants are enriched for small deletions, unbalanced translocations and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not in the context of HPV infection, and lead to somatic copy-number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte-intrinsic inflammatory signalling, which would contribute to the aggressive nature of FA SCCs. We propose that the genomic instability in sporadic HPV-negative HNSCC may arise as a result of the FA repair pathway being overwhelmed by DNA interstrand crosslink damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA-crosslinking damage.

Whole-genome sequencing in autism identifies hot spots for de novo germline mutation.

De novo mutation plays an important role in autism spectrum disorders (ASDs). Notably, pathogenic copy number variants (CNVs) are characterized by high mutation rates. We hypothesize that hypermutability is a property of ASD genes and may also include nucleotide-substitution hot spots. We investigated global patterns of germline mutation by whole-genome sequencing of monozygotic twins concordant for ASD and their parents. Mutation rates varied widely throughout the genome (by 100-fold) and could be explained by intrinsic characteristics of DNA sequence and chromatin structure. Dense clusters of mutations within individual genomes were attributable to compound mutation or gene conversion. Hypermutability was a characteristic of genes involved in ASD and other diseases. In addition, genes impacted by mutations in this study were associated with ASD in independent exome-sequencing data sets. Our findings suggest that regional hypermutation is a significant factor shaping patterns of genetic variation and disease risk in humans.

The evolution of the human DNA replication timing program.

DNA is replicated according to a defined spatiotemporal program that is linked to both gene regulation and genome stability. The evolutionary forces that have shaped replication timing programs in eukaryotic species are largely unknown. Here, we studied the molecular causes and consequences of replication timing evolution across 94 humans, 95 chimpanzees, and 23 rhesus macaques. Replication timing differences recapitulated the species' phylogenetic tree, suggesting continuous evolution of the DNA replication timing program in primates. Hundreds of genomic regions had significant replication timing variation between humans and chimpanzees, of which 66 showed advances in replication origin firing in humans, while 57 were delayed. Genes overlapping these regions displayed correlated changes in expression levels and chromatin structure. Many human-chimpanzee variants also exhibited interindividual replication timing variation, pointing to ongoing evolution of replication timing at these loci. Association of replication timing variation with genetic variation revealed that DNA sequence evolution can explain replication timing variation between species. Taken together, DNA replication timing shows substantial and ongoing evolution in the human lineage that is driven by sequence alterations and could impact regulatory evolution at specific genomic sites.

Integrated genome and transcriptome analyses reveal the mechanism of genome instability in ataxia with oculomotor apraxia 2.

Mutations in the SETX gene, which encodes Senataxin, are associated with the progressive neurodegenerative diseases ataxia with oculomotor apraxia 2 (AOA2) and amyotrophic lateral sclerosis 4 (ALS4). To identify the causal defect in AOA2, patient-derived cells and SETX knockouts (human and mouse) were analyzed using integrated genomic and transcriptomic approaches. A genome-wide increase in chromosome instability (gains and losses) within genes and at chromosome fragile sites was observed, resulting in changes to gene-expression profiles. Transcription stress near promoters correlated with high GCskew and the accumulation of R-loops at promoter-proximal regions, which localized with chromosomal regions where gains and losses were observed. In the absence of Senataxin, the Cockayne syndrome protein CSB was required for the recruitment of the transcription-coupled repair endonucleases (XPG and XPF) and RAD52 recombination protein to target and resolve transcription bubbles containing R-loops, leading to genomic instability. These results show that transcription stress is an important contributor to SETX mutation-associated chromosome fragility and AOA2.

Comprehensive analysis of DNA replication timing across 184 cell lines suggests a role for MCM10 in replication timing regulation.

Cellular proliferation depends on the accurate and timely replication of the genome. Several genetic diseases are caused by mutations in key DNA replication genes; however, it remains unclear whether these genes influence the normal program of DNA replication timing. Similarly, the factors that regulate DNA replication dynamics are poorly understood. To systematically identify trans-acting modulators of replication timing, we profiled replication in 184 cell lines from three cell types, encompassing 60 different gene knockouts or genetic diseases. Through a rigorous approach that considers the background variability of replication timing, we concluded that most samples displayed normal replication timing. However, mutations in two genes showed consistently abnormal replication timing. The first gene was RIF1, a known modulator of replication timing. The second was MCM10, a highly conserved member of the pre-replication complex. Cells from a single patient carrying MCM10 mutations demonstrated replication timing variability comprising 46% of the genome and at different locations than RIF1 knockouts. Replication timing alterations in the mutated MCM10 cells were predominantly comprised of replication delays and initiation site gains and losses. Taken together, this study demonstrates the remarkable robustness of the human replication timing program and reveals MCM10 as a novel candidate modulator of DNA replication timing.

Delayed DNA replication in haploid human embryonic stem cells.

Haploid human embryonic stem cells (ESCs) provide a powerful genetic system but diploidize at high rates. We hypothesized that diploidization results from aberrant DNA replication. To test this, we profiled DNA replication timing in isogenic haploid and diploid ESCs. The greatest difference was the earlier replication of the X Chromosome in haploids, consistent with the lack of X-Chromosome inactivation. We also identified 21 autosomal regions that had delayed replication in haploids, extending beyond the normal S phase and into G2/M. Haploid-delays comprised a unique set of quiescent genomic regions that are also underreplicated in polyploid placental cells. The same delays were observed in female ESCs with two active X Chromosomes, suggesting that increased X-Chromosome dosage may cause delayed autosomal replication. We propose that incomplete replication at the onset of mitosis could prevent cell division and result in re-entry into the cell cycle and whole genome duplication.

Positive and Negative Regulation of DNA Replication Initiation.

Genomic DNA is replicated every cell cycle by the programmed activation of replication origins at specific times and chromosomal locations. The factors that define the locations of replication origins and their typical activation times in eukaryotic cells are poorly understood. Previous studies highlighted the role of activating factors and epigenetic modifications in regulating replication initiation. Here, we review the role that repressive pathways - and their alleviation - play in establishing the genomic landscape of replication initiation. Several factors mediate this repression, in particular, factors associated with inactive chromatin. Repression can support organized, yet stochastic, replication initiation, and its absence could explain instances of rapid and random replication or re-replication.

Mutation Rate Variability across Human Y-Chromosome Haplogroups.

A common assumption in dating patrilineal events using Y-chromosome sequencing data is that the Y-chromosome mutation rate is invariant across haplogroups. Previous studies revealed interhaplogroup heterogeneity in phylogenetic branch length. Whether this heterogeneity is caused by interhaplogroup mutation rate variation or nongenetic confounders remains unknown. Here, we analyzed whole-genome sequences from cultured cells derived from >1,700 males. We confirmed the presence of branch length heterogeneity. We demonstrate that sex-chromosome mutations that appear within cell lines, which likely occurred somatically or in vitro (and are thus not influenced by nongenetic confounders) are informative for germline mutational processes. Using within-cell-line mutations, we computed a relative Y-chromosome somatic mutation rate, and uncovered substantial variation (up to 83.3%) in this proxy for germline mutation rate among haplogroups. This rate positively correlates with phylogenetic branch length, indicating that interhaplogroup mutation rate variation is a likely cause of branch length heterogeneity.

TIGER: inferring DNA replication timing from whole-genome sequence data.

MOTIVATION: Genomic DNA replicates according to a reproducible spatiotemporal program, with some loci replicating early in S phase while others replicate late. Despite being a central cellular process, DNA replication timing studies have been limited in scale due to technical challenges. RESULTS: We present TIGER (Timing Inferred from Genome Replication), a computational approach for extracting DNA replication timing information from whole genome sequence data obtained from proliferating cell samples. The presence of replicating cells in a biological specimen leads to non-uniform representation of genomic DNA that depends on the timing of replication of different genomic loci. Replication dynamics can hence be observed in genome sequence data by analyzing DNA copy number along chromosomes while accounting for other sources of sequence coverage variation. TIGER is applicable to any species with a contiguous genome assembly and rivals the quality of experimental measurements of DNA replication timing. It provides a straightforward approach for measuring replication timing and can readily be applied at scale. AVAILABILITY AND IMPLEMENTATION: TIGER is available at https://github.com/TheKorenLab/TIGER. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Quantification of somatic mutation flow across individual cell division events by lineage sequencing.

Mutation data reveal the dynamic equilibrium between DNA damage and repair processes in cells and are indispensable to the understanding of age-related diseases, tumor evolution, and the acquisition of drug resistance. However, available genome-wide methods have a limited ability to resolve rare somatic variants and the relationships between these variants. Here, we present lineage sequencing, a new genome sequencing approach that enables somatic event reconstruction by providing quality somatic mutation call sets with resolution as high as the single-cell level in subject lineages. Lineage sequencing entails sampling single cells from a population and sequencing subclonal sample sets derived from these cells such that knowledge of relationships among the cells can be used to jointly call variants across the sample set. This approach integrates data from multiple sequence libraries to support each variant and precisely assigns mutations to lineage segments. We applied lineage sequencing to a human colon cancer cell line with a DNA polymerase epsilon (POLE) proofreading deficiency (HT115) and a human retinal epithelial cell line immortalized by constitutive telomerase expression (RPE1). Cells were cultured under continuous observation to link observed single-cell phenotypes with single-cell mutation data. The high sensitivity, specificity, and resolution of the data provide a unique opportunity for quantitative analysis of variation in mutation rate, spectrum, and correlations among variants. Our data show that mutations arrive with nonuniform probability across sublineages and that DNA lesion dynamics may cause strong correlations between certain mutations.

Genomic methods for measuring DNA replication dynamics.

Genomic DNA replicates according to a defined temporal program in which early-replicating loci are associated with open chromatin, higher gene density, and increased gene expression levels, while late-replicating loci tend to be heterochromatic and show higher rates of genomic instability. The ability to measure DNA replication dynamics at genome scale has proven crucial for understanding the mechanisms and cellular consequences of DNA replication timing. Several methods, such as quantification of nucleotide analog incorporation and DNA copy number analyses, can accurately reconstruct the genomic replication timing profiles of various species and cell types. More recent developments have expanded the DNA replication genomic toolkit to assays that directly measure the activity of replication origins, while single-cell replication timing assays are beginning to reveal a new level of replication timing regulation. The combination of these methods, applied on a genomic scale and in multiple biological systems, promises to resolve many open questions and lead to a holistic understanding of how eukaryotic cells replicate their genomes accurately and efficiently.

Cell-of-origin chromatin organization shapes the mutational landscape of cancer.

Cancer is a disease potentiated by mutations in somatic cells. Cancer mutations are not distributed uniformly along the human genome. Instead, different human genomic regions vary by up to fivefold in the local density of cancer somatic mutations, posing a fundamental problem for statistical methods used in cancer genomics. Epigenomic organization has been proposed as a major determinant of the cancer mutational landscape. However, both somatic mutagenesis and epigenomic features are highly cell-type-specific. We investigated the distribution of mutations in multiple independent samples of diverse cancer types and compared them to cell-type-specific epigenomic features. Here we show that chromatin accessibility and modification, together with replication timing, explain up to 86% of the variance in mutation rates along cancer genomes. The best predictors of local somatic mutation density are epigenomic features derived from the most likely cell type of origin of the corresponding malignancy. Moreover, we find that cell-of-origin chromatin features are much stronger determinants of cancer mutation profiles than chromatin features of matched cancer cell lines. Furthermore, we show that the cell type of origin of a cancer can be accurately determined based on the distribution of mutations along its genome. Thus, the DNA sequence of a cancer genome encompasses a wealth of information about the identity and epigenomic features of its cell of origin.

DNA replication timing during development anticipates transcriptional programs and parallels enhancer activation.

In dividing cells, DNA replication occurs in a precise order, but many questions remain regarding the mechanisms of replication timing establishment and regulation. We now have generated genome-wide, high-resolution replication timing maps throughout zebrafish development. Unexpectedly, in the rapid cell cycles preceding the midblastula transition, a defined timing program was present that predicted the initial wave of zygotic transcription. Replication timing was thereafter progressively and continuously remodeled across the majority of the genome, and epigenetic changes involved in enhancer activation frequently paralleled developmental changes in replication timing. The long arm of Chromosome 4 underwent a dramatic developmentally regulated switch to late replication during gastrulation, reminiscent of mammalian X Chromosome inactivation. This study reveals that replication timing is dynamic and tightly linked to epigenetic and transcriptional changes throughout early zebrafish development. These data provide insight into the regulation and functions of replication timing and will enable further mechanistic studies.

Germline DNA replication timing shapes mammalian genome composition.

Mammalian DNA replication is a highly organized and regulated process. Large, Mb-sized regions are replicated at defined times along S-phase. Replication Timing (RT) is thought to play a role in shaping the mammalian genome by affecting mutation rates. Previous analyses relied on somatic RT profiles. However, only germline mutations are passed on to offspring and affect genomic composition. Therefore, germ cell RT information is necessary to evaluate the influences of RT on the mammalian genome. We adapted the RT mapping technique for limited amounts of cells, and measured RT from two stages in the mouse germline - primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). RT in germline cells exhibited stronger correlations to both mutation rate and recombination hotspots density than those of RT in somatic tissues, emphasizing the importance of using correct tissues-of-origin for RT profiling. Germline RT maps exhibited stronger correlations to additional genetic features including GC-content, transposable elements (SINEs and LINEs), and gene density. GC content stratification and multiple regression analysis revealed independent contributions of RT to SINE, gene, mutation, and recombination hotspot densities. Together, our results establish a central role for RT in shaping multiple levels of mammalian genome composition.