| Résumé: | <p><b>Background and aims:</b> ERCC6L2 is a member of the Snf2 ATPase superfamily that has been linked to the DNA damage response (DDR). Past work from our group demonstrated that ERCC6L2KO cells are sensitive to the double-strand break (DSB) inducers etoposide and phleomycin. However, the precise nature of the role that ERCC6L2 plays in the DDR and how it functions to preserve genome stability, remain unclear. Several mutations have been identified in ERCC6L2 which result in a distinct subtype of inherited bone marrow failure (IBMF). The aim of my project has been to improve understanding of the physiological function of ERCC6L2 using a combination of cellular and biochemical approaches. <b>Results:</b> ERCC6L2<sup>KO</sup> cells accumulate higher levels of DNA damage, compared to wildtype cells, under treatment with both etoposide and phleomycin. Cell cycle analysis revealed that this causes a higher proportion of ERCC6L2KO cells to accumulate in G2 phase. Assessment of the ability of ERCC6L2<sup>KO</sup> cells to recover from acute phleomycin treatment showed that cells lacking ERCC6L2 have significantly higher levels of DNA damage after a short release but return to levels comparable to untreated cells after a longer recovery period. Following release from phleomycin treatment, ERCC6L2<sup>KO</sup> cells progressively accumulate higher levels of single-stranded DNA (ssDNA) than wildtype cells. We hypothesised that this is due to a DNA end processing defect and that ERCC6L2 functions as an anti-resection factor. Loss or inactivation of anti-resection factors has been shown to suppress PARP inhibitor sensitivity in BRCA1-deficient cells. However, I found that loss of ERCC6L2 does not mediate this effect. Furthermore, I found that ERCC6L2<sup>KO</sup> cells are mildly sensitive to the replication inhibitor hydroxyurea (HU). Under HU-induced replication stress, ERCC6L2<sup>KO</sup> cells show significantly lower levels of ssDNA than wildtype cells and have increased chromatin bound levels of the mismatch repair factors MSH2/6. Finally, I show that a fragment of ERCC6L2 containing the uncharacterised C-terminal VIGSSK domain, which localises to sites of DNA damage, can bind DNA. A BioID screen with the VIGSSK fragment identified several exciting interaction candidates which may indicate that ERCC6L2 is important for maintaining genetic stability at specific regions of the genome. Conclusions: Loss of ERCC6L2 leads to an increase in DNA end resection following treatment with DSB inducing drugs and therefore likely functions in an anti-resection capacity and in effect, as a non-homologous end joining promoting factor. ERCC6L2 may be involved in DNA replication though it is unclear whether this role is distinct from its function in DSB repair. Collectively, these findings offer useful insights into the physiological function of ERCC6L2 in a cellular and biochemical context and provide a strong foundation for future studies. It is hoped that these results will help to elucidate the pathological basis of ERCC6L2-assoicated IBMF. </p>
|
|---|