DNA Damage Response Factors from Diverse Pathways Mediate Alternative End Joining.

摘要

Alternative end joining (Alt-EJ) chromosomal break repair involves bypassing classical nonhomologous end joining (c-NHEJ), and such repair causes mutations often with microhomology at the repair junction. Since the mediators of Alt-EJ are not well understood, I have sought to identify DNA damage response (DDR) factors important for this repair event. Using chromosomal break reporter assays, I surveyed an RNAi library targeting known DDR factors for siRNAs that cause a specific decrease in Alt-EJ, relative to an EJ event that is a composite of Alt-EJ and c-NHEJ (Distal-EJ between two tandem breaks). From this analysis, I identified several DDR factors that are specifically important for Alt-EJ relative to Distal-EJ.;These factors are from diverse pathways, including DNA crosslink repair factors such as the Fanconi Anemia factor, FANCA. Also, I found that most of these factors also promote homologous recombination (HR). Since bypass of c-NHEJ is likely important for both Alt-EJ and HR, I disrupted the c-NHEJ factor Ku70 in Fanca-deficient mouse cells and found that Ku70 loss significantly diminishes the influence of Fanca on Alt-EJ. In contrast, an inhibitor of poly ADP-ribose polymerase (PARP) causes a decrease in Alt-EJ that is enhanced by Ku70 loss. Additionally, the helicase/nuclease DNA2 appears to have distinct effects from FANCA and PARP on both Alt-EJ, as well as end resection. I suggest that several distinct DDR functions are important for Alt-EJ, which include promoting bypass of c-NHEJ and end resection.;Finally, I examined the influence of the tetratricopeptide repeat factor XAB2 on chromosomal break repair, and found that XAB2 promotes end resection that generates the 3' ssDNA intermediate for HR. XAB2 also forms a complex with ISY1 and PRP19, which show a similar influence as XAB2 on HR and end resection.;In summary, I screened a library of siRNAs targeting DDR factors and found factors from multiple pathways, including DNA crosslink repair, are important for mediating Alt-EJ. In a related study, I found that the XAB2 complex mediates DNA damage response events important for the end resection step of HR.