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.