Two Distinct Pathways Support Gene Correction by Single-Stranded Donors at DNA Nicks.
Nicks are the most common form of DNA damage. The mechanisms of their repair are fundamental to genomic stability and of practical importance for genome engineering. We define two pathways that support homology-directed repair by single-stranded DNA donors. One depends upon annealing-driven strand synthesis and acts at both nicks and double-strand breaks. The other depends upon annealing-driven heteroduplex correction and acts at nicks. Homology-directed repair via these pathways, as well as mutagenic end joining, are inhibited by RAD51 at nicks but largely independent of RAD51 at double-strand breaks. Guidelines for coordinated design of targets and donors for gene correction emerge from definition of these pathways. This analysis further suggests that naturally occurring nicks may have significant recombinogenic and mutagenic potential that is normally inhibited by RAD51 loading onto DNA, thereby identifying a function for RAD51 in maintenance of genomic stability.
Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195, USA.
Luther
L
Davis
Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195, USA; Department of Biochemistry, University of Washington School of Medicine, Seattle, WA 98195, USA; Department of Pathology, University of Washington School of Medicine, Seattle, WA 98195, USA. Electronic address: maizels@u.washington.edu.
Nancy
N
Maizels
D015415:Biomarkers / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D053904:DNA Breaks, Single-Stranded; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000378:metabolism*; D000072669:Gene Editing; D057809:HEK293 Cells; D006801:Humans; D051135:Rad51 Recombinase / Q000378:metabolism; D059767:Recombinational DNA Repair; D051716:Replication Protein A / Q000378:metabolism
D015415:Biomarkers; D004277:DNA, Single-Stranded; D051716:Replication Protein A; D051135:Rad51 Recombinase
DNA double-strand break; DNA single-strand break; breast cancer; deletion; gene correction; gene therapy; genome engineering; mutagenesis; recombination; replication