Non-homologous end joining of resected double-strand breaks in human cells involves a DNA fill-in process

Each cell in the human body is exposed to endogenous and exogenous damaging agents, leading to tens of thousands of DNA lesions daily. If these lesions are not repaired or are repaired incorrectly, they lead to mutations and genomic aberrations that can threaten the cell or organism viability. DNA double-strand breaks (DSBs) represent the most deleterious type of lesions since both strands of the DNA backbone are broken and disrupted, increasing the risk of genomic rearrangements. In order to cope with DNA damage and prevent genomic alterations, cells have developed a complex network of signaling pathways and repair mechanisms. DNA DSBs are generally repaired by two main pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is particularly important as it is the only available repair pathway in G0/G1 phases, where the majority of cells in the human body reside. In contrast, HR repairs DSBs only during the late S and G2 phases when the sister chromatid is available to serve as a template for error-free repair. Previous research revealed that NHEJ repairs IR-induced DSBs in G1 with biphasic kinetics, involving a fast, resection-independent component and a slow, resection-dependent component. The fast component is error-prone, as minimal processing and fast ligation of the broken ends can result in deletions of genetic material. The slow component involves DNA end resection and the function of several nucleases known for their involvement in HR repair. However, the mechanism underlying resection-dependent NHEJ has not yet been entirely uncovered. In this project, the human breast epithelial cell line MCF10A was used as a model system to study the mechanism of resection-dependent NHEJ. Inhibition of resection in G1-MCF10A resulted in repair deficiency, highlighting that certain DSBs must undergo resection before repair can occur. A growing number of studies revealed that transcription plays an important role during DNA DSB repair, impacting genome stability. The cell cycle-specific analyses presented in this thesis support previous findings, demonstrating that transcription is also required for the repair of G1-MCF10A cells. In particular, the inhibition of transcription occurring after damage induction led to a significant reduction of repair in G1-MCF10A and revealed that resected breaks are dependent on transcription processes. Furthermore, results in this thesis suggest that G1-phase MCF10A cells require resection-dependent NHEJ for the repair of DSBs that arise in highly transcriptionally active genomic locations. This is consistent with previous findings showing that DSBs arising in genes in G1-mESCs are repaired via resection dependent NHEJ. Since cells that must preserve genomic integrity throughout generations rely on resection-dependent NHEJ to repair DSBs in coding regions, it is believed this repair process has a high fidelity. Recent research has revealed that a fill-in mechanism similar to that occurring during the replication of telomeres could restore resected nucleotides at DSBs and convert ssDNA into dsDNA. Results in this work demonstrate that a fill-in mechanism dependent on the Pol α/primase complex synthesizes new DNA at resected breaks in G1 phase, allowing for the repair to be finalized. In this thesis, it was shown that a deficiency in resection, transcription, or fill-in synthesis results in the inability to complete the repair at certain DSBs in G1. This provides new insights into the repair processes during G1 phase, including a potential explanation of how cells maintain genomic integrity and stability.