title: Essential role for polymerase specialization in cellular nonhomologous end joining.
abstract: Nonhomologous end joining (NHEJ) repairs chromosome breaks and must remain effective in the face of extensive diversity in broken end structures. We show here that this flexibility is often reliant on the ability to direct DNA synthesis across strand breaks, and that polymerase (Pol) μ and Pol λ are the only mammalian DNA polymerases that have this activity. By systematically varying substrate in cells, we show each polymerase is uniquely proficient in different contexts. The templating nucleotide is also selected differently, with Pol μ using the unpaired base adjacent to the downstream 5' phosphate even when there are available template sites further upstream of this position; this makes Pol μ more flexible but also less accurate than Pol λ. Loss of either polymerase alone consequently has clear and distinguishable effects on the fidelity of repair, but end remodeling by cellular nucleases and the remaining polymerase helps mitigate the effects on overall repair efficiency. Accordingly, when cells are deficient in both polymerases there is synergistic impact on NHEJ efficiency, both in terms of repair of defined substrates and cellular resistance to ionizing radiation. Pol μ and Pol λ thus provide distinct solutions to a problem for DNA synthesis that is unique to this pathway and play a key role in conferring on NHEJ the flexibility required for accurate and efficient repair.
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;
authors.forename: John M
authors.initials: JM
authors.lastname: Pryor
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;
authors.forename: Crystal A
authors.initials: CA
authors.lastname: Waters
authors.affiliation: Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas-Universidad Autonoma de Madrid), 28049 Madrid, Spain;
authors.forename: Ana
authors.initials: A
authors.lastname: Aza
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;
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Asagoshi
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;
authors.forename: Christina
authors.initials: C
authors.lastname: Strom
authors.affiliation: Department of Genetics, University of North Carolina, Chapel Hill, NC 27599.
authors.forename: Piotr A
authors.initials: PA
authors.lastname: Mieczkowski
authors.affiliation: Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas-Universidad Autonoma de Madrid), 28049 Madrid, 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; dale_ramsden@med.unc.edu.
authors.forename: Dale A
authors.initials: DA
authors.lastname: Ramsden
mesh_terms: D000818:Animals; D049109:Cell Proliferation; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D059766:DNA End-Joining Repair; D019951:DNA Polymerase beta / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D004307:Dose-Response Relationship, Radiation; D005347:Fibroblasts / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009711:Nucleotides / Q000737:chemistry; D011839:Radiation, Ionizing
chemical_list: D009711:Nucleotides; D004247:DNA; C415367:DNA polymerase beta2; C407743:DNA polymerase mu; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase
keywords: Pol X; double-strand break repair; nonhomologous end joining; polymerase lambda; polymerase mu