The effect of repeat length on Marcal1-dependent single-strand annealing in Drosophila.

Autor:	Dewey EB; Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA., Korda Holsclaw J; Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA., Saghaey K; Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA., Wittmer ME; Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA., Sekelsky J; Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.; Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Jazyk:	angličtina
Zdroj:	Genetics [Genetics] 2023 Jan 12; Vol. 223 (1).
DOI:	10.1093/genetics/iyac164
Abstrakt:	Proper repair of DNA double-strand breaks is essential to the maintenance of genomic stability and avoidance of genetic disease. Organisms have many ways of repairing double-strand breaks, including the use of homologous sequences through homology-directed repair. While homology-directed repair is often error free, in single-strand annealing homologous repeats flanking a double-strand break are annealed to one another, leading to the deletion of one repeat and the intervening sequences. Studies in yeast have shown a relationship between the length of the repeat and single-strand annealing efficacy. We sought to determine the effects of homology length on single-strand annealing in Drosophila, as Drosophila uses a different annealing enzyme (Marcal1) than yeast. Using an in vivo single-strand annealing assay, we show that 50 base pairs are insufficient to promote single-strand annealing and that 500-2,000 base pairs are required for maximum efficiency. Loss of Marcal1 generally followed the same homology length trend as wild-type flies, with single-strand annealing frequencies reduced to about a third of wild-type frequencies regardless of homology length. Interestingly, we find a difference in single-strand annealing rates between 500-base pair homologies that align to the annealing target either nearer or further from the double-strand break, a phenomenon that may be explained by Marcal1 dynamics. This study gives insights into Marcal1 function and provides important information to guide the design of genome engineering strategies that use single-strand annealing to integrate linear DNA constructs into a chromosomal double-strand break. Competing Interests: Conflicts of interest: None declared. (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)
Databáze:	MEDLINE
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