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.

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Cellular DNA is under constant attack by a wide variety of agents, both endogenous and exogenous. To counteract DNA damage, human cells have a large collection of DNA repair factors. Among them, DNA polymerase lambda (Polλ) stands out for its versatility, as it participates in different DNA repair and damage tolerance pathways in which gap-filling DNA synthesis is required. In this work we show that human Polλ is conjugated with Small Ubiquitin-like MOdifier (SUMO) proteins both in vitro and in vivo, with Lys27 being the main target of this covalent modification. Polλ SUMOylation takes place in the nuclear pore complex and is mediated by the E3 ligase RanBP2. This post-translational modification promotes Polλ entry into the nucleus, which is required for its recruitment to DNA lesions and stimulated by DNA damage induction. Our work represents an advance in the knowledge of molecular pathways that regulate cellular localization of human Polλ, which are essential to be able to perform its functions during repair of nuclear DNA, and that might constitute an important point for the modulation of its activity in human cells.

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