Nek family members regulate Rad54 during homologous recombination in developing mice

Abstract

Homologous recombination (HR) represents an important pathway for repairing DNA double-strand breaks (DSBs) but HR factors, including RAD51, also serve to protect and restart stalled replication forks. RAD54 functions during DSB repair where it removes RAD51 from duplex DNA including heteroduplex DNA which is formed during D-loop formation. This allows subsequent DNA repair synthesis and completion of the HR process. We have previously suggested that RAD54s activity is regulated by never-in-mitosis-gene A (NIMA) related kinase 1 (NEK1) in a cell cycle-specific manner to promote RAD51 removal and HR in G2 phase without interfering with RAD51s fork stabilization role during S phase. Here, we establish that Nek1 regulates the phosphorylation of Rad54 at Ser572 (S572) to promote HR in vivo in adult mice and in vitro in fibroblasts derived from such mice. In contrast, embryonic mice and fibroblasts derived from them do not require Nek1 for HR. We further show that HR requires Rad54 phosphorylation at S572 both in embryonic and adult fibroblasts and that this is mediated in embryonic fibroblasts by Nek3 and Nek5 instead of Nek1. Thus, our work identifies a developmental change in the regulation of HR and uncovers two new factors involved in this process.