DNA double-strand breaks (DSBs) represent the most deleterious type of DNA damage as they pose a serious threat to genome integrity. Two major pathways are available for the repair of DSBs: canonical non-homologous end joining (c-NHEJ) and homologous recombination (HR). During c-NHEJ, the DSB ends are re-ligated after minimal end processing steps. The HR pathway is more complex and is initiated by CtIP-dependent DSB end resection to form 3’ ssDNA overhangs for subsequent homology search in the sister chromatid. In wild type human G1-phase cells only c-NHEJ is available for DSB repair, as in this cell cycle phase the homologous sister chromatid required for HR is missing. DSB repair in G1, as well as in G2, shows biphasic kinetics consisting of a fast component that repairs the majority of breaks within the first few hours after damage induction, followed by a slow component that repairs the remaining breaks. The fast component in both G1 and G2 phase is well characterized and represents c-NHEJ, while the slow component in G2 represents repair by HR. Previous work has suggested that the slow repair component in G1 represents a sub-pathway of NHEJ that requires the activities of Artemis and ATM. However, the mechanism underlying the slow repair component in G1 is not fully understood and its characterization was the focus of this work.
To specifically study slow repair in G1, high LET α-particle radiation was used to induce complex DNA damages that are repaired with slow kinetics. RPA rapidly binds ssDNA in the cell to protect it from nucleolytic degradation and is phosphorylated in response to DNA damage. Exploiting the qualities of α–particle radiation, an assay was developed to monitor pRPA-foci formation in G1 and used as a tool to measure DSB end resection in this cell-cycle phase. Another approach to study the slow repair component was the quantification of γH2AX foci, a histone modification in response to DSBs, at late time points post IR.
Collectively, it was shown that slowly repairing DSBs in G1 undergo resection and subsequent repair by c-NHEJ. This pathway is regulated by Plk3, which after DNA damage phosphorylates CtIP on amino acids Ser327 and Thr847 in G1. Using the pRPA assay, it was demonstrated that Plk3 phosphorylates CtIP on these amino acid residues to promote resection. CtIP phosphorylation on Ser327 also mediates its interaction with Brca1 in G1, which antagonizes 53BP1 to allow resection. The results indicate that the interaction of CtIP and Brca1 is required to promote resection in G1, while depletion of 53BP1 causes hyper-resection of DSBs in G1. The primary function of Brca1 in G1 appears to be the displacement of 53BP1, similar to the mechanism in G2.
Furthermore, a number of nucleases required for G1 resection were identified. Similar to the process in G2, G1 resection requires the exonuclease activities of Exo1, EXD2 and Mre11. Contrary to G2, the endonuclease activity of Mre11 is dispensable in G1, as are the activities of BLM/DNA2. Thus, it is proposed that resection in G1 might be initiated from the break end and therefore differs from the mechanism in G2 where Mre11 endonuclease function initiates bi-directional resection several hundred nucleotides away from the break end. γH2AX studies indicated that Artemis, an endonuclease which is specifically required for DBS repair during the slow component, functions downstream of the aforementioned factors. Thus, it is proposed that once resection is initiated in G1, resection intermediates have to be resolved by Artemis to complete repair.
Finally, the results indicate that break ends are rejoined via a c-NHEJ process, therefore it was hypothesized that the Ku70/80 heterodimer stays bound to the DSB ends throughout the entire repair time and translocates inwards to expose DNA ends for resection while at the same time limiting the process. Immunofluorescence data support this notion by providing evidence that Ku80 co-localizes with pRPA in G1. Compared to resection in G2, which is always followed up by error-free repair via HR, resection in G1 needs to be much more limited in length. Future work will focus on the elucidation of the mechanisms restricting the extent of resection in G1
