The essential roles of DNA resection AND the impact of loss of function NHEJ mutations on DSB repair in human somatic cells

Abstract

DNA resection is a highly conserved biological process that protects the genome. It links DNA damage to checkpoint activation and is required for the homologous recombination (HR) pathway. The relatively recent discovery of CtIP, EXO1, and DNA2 as the nucleases responsible for DNA resection in the HR pathway, has generated significant interest in this field of DNA repair. The majority of studies depend upon "knockdowns" via the use of siRNA to address the function of CtIP, EXO1, and DNA2, and then usually only in the presence of exogenous DNA damage. With our conditionally-null knockout human cell lines, we find that CtIP, EXO1, and DNA2 are essential in human somatic cells and that they are required for normal DNA replication fork progression even in the absence of exogenous DNA damage. Furthermore, we find that CtIP, EXO1, and DNA2 are co-regulated and that they coordinately regulate the stability of FANCD2, BRCA2, Rad51, and CHK1. Current chemotherapeutic drugs work by inhibiting DNA replication. Since HR is required for the repair of damaged replication forks, the process of DNA resection is a promising drug target, either alone or in combination in combination with current chemotherapeutic drugs. Therefore, it is important that we understand the mechanisms and regulation of CtIP, EXO1, and DNA2. The nonhomologous end-joining pathway (NHEJ) is subdivided into a Ku -dependent pathway {Classical NHEJ (C-NHEJ)} and a Ku-independent, {Alternative NHEJ (A-NHEJ)}. C-NHEJ repair normally dominates in human cells but how or why this happens has remained obscure. Using C- NHEJ loss-of-function mutant cell lines for Ku86, LIGIV, DNA-PKcs, and XLF, we found that Ku86 uniquely and strongly represses the A-NHEJ pathway, which suggests that Ku86 regulates DSB repair by controlling pathway choice decisions.