RIF1-ASF1-mediated high-order chromatin structure safeguards genome integrity.

The 53BP1-RIF1 pathway antagonizes resection of DNA broken ends and confers PARP inhibitor sensitivity on BRCA1-mutated tumors. However, it is unclear how this pathway suppresses initiation of resection. Here, we identify ASF1 as a partner of RIF1 via an interacting manner similar to its interactions with histone chaperones CAF-1 and HIRA. ASF1 is recruited to distal chromatin flanking DNA breaks by 53BP1-RIF1 and promotes non-homologous end joining (NHEJ) using its histone chaperone activity. Epistasis analysis shows that ASF1 acts in the same NHEJ pathway as RIF1, but via a parallel pathway with the shieldin complex, which suppresses resection after initiation. Moreover, defects in end resection and homologous recombination (HR) in BRCA1-deficient cells are largely suppressed by ASF1 deficiency. Mechanistically, ASF1 compacts adjacent chromatin by heterochromatinization to protect broken DNA ends from BRCA1-mediated resection. Taken together, our findings identify a RIF1-ASF1 histone chaperone complex that promotes changes in high-order chromatin structure to stimulate the NHEJ pathway for DSB repair.

Cancer Mutational Processes Vary in Their Association with Replication Timing and Chromatin Accessibility.

Cancer somatic mutations are the product of multiple mutational and repair processes, both of which are tightly associated with DNA replication. Distinctive patterns of somatic mutation accumulation, termed mutational signatures, are indicative of processes sustained within tumors. However, the association of various mutational processes with replication timing (RT) remains an open question. In this study, we systematically analyzed the mutational landscape of 2,787 tumors from 32 tumor types separately for early and late replicating regions using sequence context normalization and chromatin data to account for sequence and chromatin accessibility differences. To account for sequence differences between various genomic regions, an artificial genome-based approach was developed to expand the signature analyses to doublet base substitutions and small insertions and deletions. The association of mutational processes and RT was signature specific: Some signatures were associated with early or late replication (such as SBS7b and SBS7a, respectively), and others had no association. Most associations existed even after normalizing for genome accessibility. A focused mutational signature identification approach was also developed that uses RT information to improve signature identification; this approach found that SBS16, which is biased toward early replication, is strongly associated with better survival rates in liver cancer. Overall, this novel and comprehensive approach provides a better understanding of the etiology of mutational signatures, which may lead to improved cancer prevention, diagnosis, and treatment. SIGNIFICANCE: Many mutational processes associate with early or late replication timing regions independently of chromatin accessibility, enabling development of a focused identification approach to improve mutational signature detection.

Chromosomal coordination and differential structure of asynchronous replicating regions.

Stochastic asynchronous replication timing (AS-RT) is a phenomenon in which the time of replication of each allele is different, and the identity of the early allele varies between cells. By taking advantage of stable clonal pre-B cell populations derived from C57BL6/Castaneous mice, we have mapped the genome-wide AS-RT loci, independently of genetic differences. These regions are characterized by differential chromatin accessibility, mono-allelic expression and include new gene families involved in specifying cell identity. By combining population level mapping with single cell FISH, our data reveal the existence of a novel regulatory program that coordinates a fixed relationship between AS-RT regions on any given chromosome, with some loci set to replicate in a parallel and others set in the anti-parallel orientation. Our results show that AS-RT is a highly regulated epigenetic mark established during early embryogenesis that may be used for facilitating the programming of mono-allelic choice throughout development.

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.

Perturbations in the Replication Program Contribute to Genomic Instability in Cancer.

Cancer and genomic instability are highly impacted by the deoxyribonucleic acid (DNA) replication program. Inaccuracies in DNA replication lead to the increased acquisition of mutations and structural variations. These inaccuracies mainly stem from loss of DNA fidelity due to replication stress or due to aberrations in the temporal organization of the replication process. Here we review the mechanisms and impact of these major sources of error to the replication program.

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement.

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge. This article describes in detail the "Copy Number Ratio of S/G1 for mapping genomic Time of Replication" method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.