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SummaryAging is characterized by genome instability, which contributes to cancer formation and cell lethality leading to organismal decline. The high levels of DNA double‐strand breaks (DSBs) observed in old cells and premature aging syndromes are likely a primary source of genome instability, but the underlying cause of their formation is still unclear. DSBs might result from higher levels of damage or repair defects emerging with advancing age, but repair pathways in old organisms are still poorly understood. Here, we show that premeiotic germline cells of young and old flies have distinct differences in their ability to repair DSBs by the error‐free pathway homologous recombination (HR). Repair of DSBs induced by either ionizing radiation (IR) or the endonuclease I‐SceI is markedly defective in older flies. This correlates with a remarkable reduction in HR repair measured with the DR ‐white DSB repair reporter assay. Strikingly, most of this repair defect is already present at 8 days of age. Finally, HR defects correlate with increased expression of early HR components and increased recruitment of Rad51 to damage in older organisms. Thus, we propose that the defect in the HR pathway for germ cells in older flies occurs following Rad51 recruitment. These data reveal that DSB repair defects arise early in the aging process and suggest that HR deficiencies are a leading cause of genome instability in germ cells of older animals.

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.

Centromeres serve a critical function in preserving genome integrity across sequential cell divisions, by mediating symmetric chromosome segregation. The repetitive, heterochromatic nature of centromeres is thought to be inhibitory to DNA replication, but has also led to their underrepresentation in human reference genome assemblies. Consequently, centromeres have been excluded from genomic replication timing analyses, leaving their time of replication unresolved. However, the most recent human reference genome, hg38, included models of centromere sequences. To establish the experimental requirements for achieving replication timing profiles for centromeres, we sequenced G1- and S-phase cells from five human cell lines, and aligned the sequence reads to hg38. We were able to infer DNA replication timing profiles for the centromeres in each of the five cell lines, which showed that centromere replication occurs in mid-to-late S phase. Furthermore, we found that replication timing was more variable between cell lines in the centromere regions than expected, given the distribution of variation in replication timing genome-wide. These results suggest the potential of these, and future, sequence models to enable high-resolution studies of replication in centromeres and other heterochromatic regions.