title: Regulation of human polλ by ATM-mediated phosphorylation during non-homologous end joining.
abstract: 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.
authors.affiliation: Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid/CSIC, Madrid 28049, Spain.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Sastre-Moreno
authors.affiliation: Department of Biochemistry and Biophysics and Curriculum in Genetics and Molecular Biology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Pryor
authors.affiliation: Departamento Bioquímica Vegetal y Biología Molecular, Universidad de Sevilla, Sevilla 41092, Spain; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla/CSIC, Sevilla 41092, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Moreno-Oñate
authors.affiliation: Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla/CSIC, Sevilla 41092, Spain.
authors.forename: Andrés M
authors.initials: AM
authors.lastname: Herrero-Ruiz
authors.affiliation: Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla/CSIC, Sevilla 41092, Spain.
authors.forename: Felipe
authors.initials: F
authors.lastname: Cortés-Ledesma
authors.affiliation: Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid/CSIC, Madrid 28049, Spain.
authors.forename: Luis
authors.initials: L
authors.lastname: Blanco
authors.affiliation: Department of Biochemistry and Biophysics and Curriculum in Genetics and Molecular Biology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Dale A
authors.initials: DA
authors.lastname: Ramsden
authors.affiliation: Departamento Bioquímica Vegetal y Biología Molecular, Universidad de Sevilla, Sevilla 41092, Spain; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla/CSIC, Sevilla 41092, Spain. Electronic address: jose.ruiz@cabimer.es.
authors.forename: Jose F
authors.initials: JF
authors.lastname: Ruiz
mesh_terms: D000595:Amino Acid Sequence; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D019951:DNA Polymerase beta / Q000737:chemistry / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D016415:Sequence Alignment
chemical_list: D009687:Nuclear Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; C415367:DNA polymerase beta2; D019951:DNA Polymerase beta