Enhanced Use of Backup Pathways of NHEJ in G₂in Chinese Hamster Mutant Cells with Defects in the Classical Pathway of NHEJ

Abstract

In higher eukaryotes DNA double-strand breaks (DSBs) are repaired by homologous recombination repair (HRR) or nonhomologous end joining (NHEJ). In addition to the DNA-PK dependent pathway of NHEJ (D-NHEJ), cells employ a backup pathway (B-NHEJ) using DNA ligase III and PARP1. We have reported previously that mouse embryo fibroblasts (MEFs) defective in D-NHEJ show enhanced repair of DSBs in G2 not reflecting a contribution of HRR. Here we extend these studies to Chinese hamster mutant cells with defects in the DNA-PKcs, Ku80 or XRCC4 components of D-NHEJ or in the XRCC2 and XRCC3 components of HRR. Using cell sorting to separate cells at defined times after irradiation, we measure repair of DSBs with pulsed-field gel electrophoresis in unperturbed G1- and G2-phase cells. Wild-type cells and mutants of XRCC2 and XRCC3 repair DSBs with similar efficiency in G1 and G2. Mutants of DNA-PKcs, Ku80 and XRCC4 show more pronounced repair in G2 than in G1. These and previously published results provide support for the notion that the increased efficacy of DSB repair in G2 reflects the enhanced function of B-NHEJ, which may be a general feature of rodent cells that also holds for human cells.