title: Ku is a 5'-dRP/AP lyase that excises nucleotide damage near broken ends.
abstract: Mammalian cells require non-homologous end joining (NHEJ) for the efficient repair of chromosomal DNA double-strand breaks. A key feature of biological sources of strand breaks is associated nucleotide damage, including base loss (abasic or apurinic/apyrimidinic (AP) sites). At single-strand breaks, 5'-terminal abasic sites are excised by the 5'-deoxyribose-5-phosphate (5'-dRP) lyase activity of DNA polymerase beta (pol beta): here we show, in vitro and in cells, that accurate and efficient repair by NHEJ of double-strand breaks with such damage similarly requires 5'-dRP/AP lyase activity. Classically defined NHEJ is moreover uniquely effective at coupling this end-cleaning step to joining in cells, helping to distinguish this pathway from otherwise robust alternative NHEJ pathways. The NHEJ factor Ku can be identified as an effective 5'-dRP/AP lyase. In a similar manner to other lyases, Ku nicks DNA 3' of an abasic site by a mechanism involving a Schiff-base covalent intermediate with the abasic site. We show by using cell extracts that Ku is essential for the efficient removal of AP sites near double-strand breaks and, consistent with this result, that joining of such breaks is specifically decreased in cells complemented with a lyase-attenuated Ku mutant. Ku had previously been presumed only to recognize ends and recruit other factors that process ends; our data support an unexpected direct role for Ku in end-processing steps as well.
authors.affiliation: Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, and Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Roberts
authors.forename: Natasha
authors.initials: N
authors.lastname: Strande
authors.forename: Martin D
authors.initials: MD
authors.lastname: Burkhalter
authors.forename: Christina
authors.initials: C
authors.lastname: Strom
authors.forename: Jody M
authors.initials: JM
authors.lastname: Havener
authors.forename: Paul
authors.initials: P
authors.lastname: Hasty
authors.forename: Dale A
authors.initials: DA
authors.lastname: Ramsden
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002457:Cell Extracts; D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D051379:Mice; D012267:Ribosemonophosphates / Q000378:metabolism*; D012545:Schiff Bases / Q000737:chemistry
chemical_list: C502791:5'-deoxyribose-5-phosphate; D034961:Antigens, Nuclear; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D012267:Ribosemonophosphates; D012545:Schiff Bases; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase
references.citation: DNA Repair (Amst). 2004 Oct 5;3(10):1323-34
references.pmid: 15336627
references.citation: Biochim Biophys Acta. 2008 Nov;1784(11):1777-85
references.pmid: 18757043
references.citation: J Biol Chem. 1998 Jun 12;273(24):15263-70
references.pmid: 9614142
references.citation: Mol Cell. 2004 Oct 22;16(2):163-71
references.pmid: 15494304
references.citation: Crit Rev Eukaryot Gene Expr. 1992;2(4):283-314
references.pmid: 1486241
references.citation: Nature. 2001 Aug 9;412(6847):607-14
references.pmid: 11493912
references.citation: DNA Repair (Amst). 2008 Jan 1;7(1):67-76
references.pmid: 17881298
references.citation: Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12244-8
references.pmid: 9770471
references.citation: Mutat Res. 1990 Jul;239(1):1-16
references.pmid: 2195330
references.citation: Radiat Res. 2001 Oct;156(4):365-78
references.pmid: 11554848
references.citation: J Exp Med. 2007 Dec 24;204(13):3209-19
references.pmid: 18070939
references.citation: Nucleic Acids Res. 2008 May;36(8):2561-9
references.pmid: 18332040
references.citation: J Biol Chem. 1998 May 1;273(18):11121-6
references.pmid: 9556598
references.citation: J Biol Chem. 2005 May 20;280(20):20051-8
references.pmid: 15778218
references.citation: Oncogene. 2009 Apr 23;28(16):1875-8
references.pmid: 19330025
references.citation: Int J Radiat Biol. 1994 Nov;66(5):427-32
references.pmid: 7983426
references.citation: Int J Radiat Biol Relat Stud Phys Chem Med. 1982 Jun;41(6):671-6
references.pmid: 6288603
references.citation: Science. 1995 Aug 4;269(5224):699-702
references.pmid: 7624801
references.citation: Nature. 2000 Jun 15;405(6788):807-10
references.pmid: 10866204
references.citation: Nucleic Acids Res. 2009 Apr;37(6):1868-77
references.pmid: 19188258
references.citation: Chem Rev. 1998 May 7;98(3):1089-1108
references.pmid: 11848926
references.citation: Trends Genet. 2008 Nov;24(11):529-38
references.pmid: 18809224
references.citation: J Biol Chem. 1996 Jul 26;271(30):17811-5
references.pmid: 8663612
references.citation: Proc Natl Acad Sci U S A. 2007 Dec 26;104(52):20902-7
references.pmid: 18093953
references.citation: Nucleic Acids Res. 1998 Dec 1;26(23):5333-42
references.pmid: 9826756
references.citation: EMBO J. 2001 Dec 3;20(23):6919-26
references.pmid: 11726527
references.citation: J Biol Chem. 2001 Sep 14;276(37):34659-63
references.pmid: 11457865