title: Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair.
abstract: DNA nicks are the most common form of DNA damage, and if unrepaired can give rise to genomic instability. In human cells, nicks are efficiently repaired via the single-strand break repair pathway, but relatively little is known about the fate of nicks not processed by that pathway. Here we show that homology-directed repair (HDR) at nicks occurs via a mechanism distinct from HDR at double-strand breaks (DSBs). HDR at nicks, but not DSBs, is associated with transcription and is eightfold more efficient at a nick on the transcribed strand than at a nick on the nontranscribed strand. HDR at nicks can proceed by a pathway dependent upon canonical HDR factors RAD51 and BRCA2; or by an efficient alternative pathway that uses either ssDNA or nicked dsDNA donors and that is strongly inhibited by RAD51 and BRCA2. Nicks generated by either I-AniI or the CRISPR/Cas9(D10A) nickase are repaired by the alternative HDR pathway with little accompanying mutagenic end-joining, so this pathway may be usefully applied to genome engineering. These results suggest that alternative HDR at nicks may be stimulated in physiological contexts in which canonical RAD51/BRCA2-dependent HDR is compromised or down-regulated, which occurs frequently in tumors.
authors.affiliation: Departments of Immunology, Biochemistry, and Pathology, University of Washington School of Medicine, Seattle, WA 98195.
authors.forename: Luther
authors.initials: L
authors.lastname: Davis
authors.forename: Nancy
authors.initials: N
authors.lastname: Maizels
mesh_terms: D024682:BRCA2 Protein / Q000235:genetics; D002460:Cell Line; D053904:DNA Breaks, Single-Stranded; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000502:physiology; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D008957:Models, Genetic; D034741:RNA, Small Interfering / Q000235:genetics; D051135:Rad51 Recombinase / Q000235:genetics; D059767:Recombinational DNA Repair / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D024682:BRCA2 Protein; C551750:BRCA2 protein, human; D034741:RNA, Small Interfering; C495153:RAD51 protein, human; D051135:Rad51 Recombinase
keywords: cancer; gene conversion; loss of heterozygosity; recombination; targeted gene correction