The DNA damage tolerance system in health and disease

DNA damage tolerance (DDT) is an evolutionarily ancient system that safeguards genome integrity during replication when DNA lesions are encountered. Multiple partially redundant DDT pathways exist, each with distinct advantages, allowing cells to deploy specific DDT proteins across a wide range of genetic contexts. While the importance of DNA damage repair disorders has been well characterized, the precise contribution of DDT to organismal wellbeing — particularly in mammals — remained less clear. This thesis set out to address that question. We examined mice deficient for two central DDT activation pathways: PCNA ubiquitination, which is required for recruitment of translesion synthesis (TLS) polymerases and enables template switching and fork reversal, and REV1, which independently facilitates TLS polymerase recruitment. Combined inactivation of both pathways proved embryonic lethal. Double mutant embryos were smaller than controls, suffered from severe anemia, and showed a near-complete collapse of hematopoietic stem and progenitor cells (HSPCs). Development of both T and B cells was blocked at early stages. Single-cell RNA sequencing revealed transcriptional signatures consistent with replication stress, DNA damage, checkpoint activation, and apoptosis. These findings were recapitulated in adult mice following conditional induction of the double mutant genotype, with hematopoietic collapse and HSPC depletion again being the dominant phenotype. Outside of the hematopoietic compartment and germ cell niche, no gross pathologies were observed in unperturbed conditions, underscoring the particular vulnerability of rapidly dividing stem cell populations to DDT loss. Further analysis clarified the distinct contributions of each pathway to interstrand crosslink (ICL) repair. PCNA ubiquitination was found to be essential for cellular resistance to ICL-inducing agents such as cisplatin and mitomycin-C, while REV1 deficiency conferred no such sensitivity. Instead, REV1-deficient cells showed selective sensitivity to alkylating agents and, intriguingly, to steroid hormones — consistent with REV1's role in bypassing guanine adducts. Investigating the epistatic relationship between PCNA ubiquitination and the Fanconi anemia (FA) pathway revealed near-complete synthetic lethality in double mutant mice, and in p53-immortalized cells, PCNA ubiquitination and FA-mediated ICL repair appeared epistatic. Mass spectrometry of the FA core complex interactome in PcnaK164R cells revealed enrichment of MMR proteins MSH2 and MSH6, which were found to constitute an alternative ICL repair pathway in the absence of PCNA ubiquitination. Whole-genome sequencing of DDT-deficient lymphoma cells uncovered a striking mutational signature: deletions of 400 bp to 4 kb, a class of structural variant frequently observed in human cancers. These deletions were absent in DDT-proficient cells, suggesting DDT suppresses a specific class of genomic instability. Whether these arise from aberrant double-strand break processing or represent loss of unreplicated DNA — potentially at common fragile sites — remains to be determined. TLS polymerase κ (Pol κ) emerged as a clinically relevant factor. Pol κ-deficient tumor-bearing mice showed significantly improved survival upon cisplatin treatment compared to Pol κ-proficient controls, and TCGA data indicate that Pol κ-deficient tumors constitute a meaningful subset of several cancer types. These findings suggest that Pol κ status may guide treatment decisions involving ICL-inducing chemotherapy. Finally, an unbiased whole-genome CRISPR dropout screen in DDT-deficient cells identified novel synthetic lethal interactions, including with components of the CST complex involved in telomere maintenance and replication of common fragile sites. Additional hits — including TRIM33, DCAF15, SERBP1, and KIFC1 — represent candidate regulators of genome maintenance whose roles in the DNA damage response remain incompletely understood. Together, these findings reveal that DDT is indispensable for mammalian hematopoiesis and germ cell maintenance even under physiological conditions, establish distinct and complementary roles for PCNA ubiquitination and REV1, and identify Pol κ as a potential biomarker for cisplatin sensitivity in cancer.