부산대학교 도서관

The Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to initiate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs), and confirmed these results by a Cas9-mediated DSBs in combination with a bacterial retron system that produces ssDNA templates in vivo. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but unlike other Rad51-independent recombination events, independent of Rdh54. We show that Rad59 acts to alleviate the inhibition of Rad51 on Rad52's strand annealing activity both in SSTR and in single strand annealing (SSA). Gene editing is Rad51-dependent when double-stranded oligonucleotides of the same size and sequence are introduced as templates. The assimilation of mismatches during gene editing is dependent on the activity of Msh2, which acts very differently on the 3' side of the ssODN which can anneal directly to the resected DSB end compared to the 5' end. In addition DNA polymerase Polδ's 3' to 5' proofreading activity frequently excises a mismatch very close to the 3' end of the template. We further report that SSTR is accompanied by as much as a 600-fold increase in mutations in regions adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing. Author summary: DNA double strand breaks (DSBs) are one of the most lethal types of damage that can be inflicted on a chromosome and failure to repair such lesions can result in chromosome instability, commonly associated with human cancer. A knowledge of DNA repair mechanisms is also critical in the exploitation of gene therapy, a process that includes intentionally breaking the DNA to modify the genetic sequence. Here we compared two site-specific methods to create DSBs (HO endonuclease and CRISPR/Cas9) in budding yeast, to modify several DNA targets by single-strand DNA template repair (SSTR). We show that gene editing uses a DSB repair pathway that is independent of the canonical repair protein Rad51 and distinct from previously studied Rad51-independent pathways in its requirements for several other known recombination proteins. We show both in gene editing and in single-strand annealing that Rad59 acts to suppress the modulation of Rad52's strand annealing activity by Rad51. We also determined how mismatches in the template are incorporated into the genome, and that this assimilation reflects different aspects of Msh2-mediated mismatch repair as well as Polδ-mediated proofreading. These insights provide insight into the mechanisms of DSB repair by this important gene-editing pathway. [ABSTRACT FROM AUTHOR]