title: Specificity of the dRP/AP lyase of Ku promotes nonhomologous end joining (NHEJ) fidelity at damaged ends.
abstract: Nonhomologous end joining (NHEJ) is essential for efficient repair of chromosome breaks. However, the NHEJ ligation step is often obstructed by break-associated nucleotide damage, including base loss (abasic site or 5'-dRP/AP sites). Ku, a 5'-dRP/AP lyase, can excise such damage at ends in preparation for the ligation step. We show here that this activity is greatest if the abasic site is within a short 5' overhang, when this activity is necessary and sufficient to prepare such termini for ligation. In contrast, Ku is less active near 3' strand termini, where excision would leave a ligation-blocking α,β-unsaturated aldehyde. The Ku AP lyase activity is also strongly suppressed by as little as two paired bases 5' of the abasic site. Importantly, in vitro end joining experiments show that abasic sites significantly embedded in double-stranded DNA do not block the NHEJ ligation step. Suppression of the excision activity of Ku in this context therefore is not essential for ligation and further helps NHEJ retain terminal sequence in junctions. We show that the DNA between the 5' terminus and the abasic site can also be retained in junctions formed by cellular NHEJ, indicating that these sites are at least partly resistant to other abasic site-cleaving activities as well. High levels of the 5'-dRP/AP lyase activity of Ku are thus restricted to substrates where excision of an abasic site is required for ligation, a degree of specificity that promotes more accurate joining.
authors.affiliation: Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics and Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: Natasha
authors.initials: N
authors.lastname: Strande
authors.forename: Steven A
authors.initials: SA
authors.lastname: Roberts
authors.forename: Sehyun
authors.initials: S
authors.lastname: Oh
authors.forename: Eric A
authors.initials: EA
authors.lastname: Hendrickson
authors.forename: Dale A
authors.initials: DA
authors.lastname: Ramsden
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004249:DNA Damage; D059766:DNA End-Joining Repair; D004265:DNA Helicases / Q000378:metabolism; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics*; D006801:Humans; D000072200:Ku Autoantigen; D009841:Oligonucleotides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D011995:Recombination, Genetic; D013379:Substrate Specificity
chemical_list: D009841:Oligonucleotides; D011506:Proteins; D011994:Recombinant Proteins; D004247:DNA; D004265:DNA Helicases; C106893:XRCC5 protein, human; D000072200:Ku Autoantigen; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase