title: Requirements for 5'dRP/AP lyase activity in Ku.
abstract: The non-homologous end joining (NHEJ) pathway is used in diverse species to repair chromosome breaks, and is defined in part by a requirement for Ku. We previously demonstrated mammalian Ku has intrinsic 5' deoxyribosephosphate (5'dRP) and apurinic/apyrimidinic (AP) lyase activity, and showed this activity is important for excising abasic site damage from ends. Here we employ systematic mutagenesis to clarify the protein requirements for this activity. We identify lysine 31 in the 70 kD subunit (Ku70 K31) as the primary candidate nucleophile required for catalysis, but additional mutation of Ku70 K160 and six other lysines within Ku80 were required to eliminate all activity. Ku from Saccharomyces cerevisiae also possesses 5'dRP/AP lyase activity, and robust activity was also reliant on lysines in Ku70 analogous to K31 and K160. By comparison, these lysines are not conserved in Xenopus laevis Ku, and Ku from this species has negligible activity. A role for residues flanking Ku70 K31 in expanding the range of abasic site contexts that can be used as substrate was also identified. Our results suggest an active site well located to provide the substrate specificity required for its biological role.
authors.affiliation: Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27514, USA.
authors.forename: Natasha T
authors.initials: NT
authors.lastname: Strande
authors.affiliation: Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27514, USA.
authors.forename: Juan
authors.initials: J
authors.lastname: Carvajal-Garcia
authors.affiliation: Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27514, USA.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Hallett
authors.affiliation: Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27514, USA Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Crystal A
authors.initials: CA
authors.lastname: Waters
authors.affiliation: Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27514, USA.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Roberts
authors.affiliation: Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27514, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Strom
authors.affiliation: Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27514, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Kuhlman
authors.affiliation: Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27514, USA Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599, USA dale_ramsden@med.unc.edu.
authors.forename: Dale A
authors.initials: DA
authors.lastname: Ramsden
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry* / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000072200:Ku Autoantigen; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D012267:Ribosemonophosphates / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D029867:Xenopus Proteins / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: C502791:5'-deoxyribose-5-phosphate; D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D012267:Ribosemonophosphates; D029701:Saccharomyces cerevisiae Proteins; D029867:Xenopus Proteins; D051747:DNA-Activated Protein Kinase; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D008239:Lysine