Regulation of human polλ by ATM-mediated phosphorylation during non-homologous end joining

•Human Polλ is efficiently phosphorylated by DNA-PKcs in vitro and predominantly by ATM after IR-mediated DSBs in vivo.•Polλ Thr204 is the main target for ATM- and DNA-PKcs-mediated phosphorylation.•Phosphorylation of Polλ at Thr204 after IR is required for efficient Polλ-mediated gap-filling NHEJ repair of a subset of DSBs in vivo.•Polλ phosphorylation might favor Polλ interaction with the DNA-PK complex at DSBs. DNA double strand breaks (DSBs) trigger a variety of cellular signaling processes, collectively termed the DNA-damage response (DDR), that are primarily regulated by protein kinase ataxia-telangiectasia mutated (ATM). Among DDR activated processes, the repair of DSBs by non-homologous end joining (NHEJ) is essential. The proper coordination of NHEJ factors is mainly achieved through phosphorylation by an ATM-related kinase, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the molecular basis for this regulation has yet to be fully elucidated. In this study we identify the major NHEJ DNA polymerase, DNA polymerase lambda (Polλ), as a target for both ATM and DNA-PKcs in human cells. We show that Polλ is efficiently phosphorylated by DNA-PKcs in vitro and predominantly by ATM after DSB induction with ionizing radiation (IR) in vivo. We identify threonine 204 (T204) as a main target for ATM/DNA-PKcs phosphorylation on human Polλ, and establish that its phosphorylation may facilitate the repair of a subset of IR-induced DSBs and the efficient Polλ-mediated gap-filling during NHEJ. Molecular evidence suggests that Polλ phosphorylation might favor Polλ interaction with the DNA-PK complex at DSBs. Altogether, our work provides the first demonstration of how Polλ is regulated by phosphorylation to connect with the NHEJ core machinery during DSB repair in human cells.