title: A role for DNA mismatch repair protein Msh2 in error-prone double-strand-break repair in mammalian chromosomes.
abstract: We examined error-prone nonhomologous end joining (NHEJ) in Msh2-deficient and wild-type Chinese hamster ovary cell lines. A DNA substrate containing a thymidine kinase (tk) gene fused to a neomycin-resistance (neo) gene was stably integrated into cells. The fusion gene was rendered nonfunctional due to a 22-bp oligonucleotide insertion, which included the 18-bp I-SceI endonuclease recognition site, within the tk portion of the fusion gene. A double-strand break (DSB) was induced by transiently expressing the I-SceI endonuclease, and deletions or insertions that restored the tk-neo fusion gene's reading frame were recovered by selecting for G418-resistant colonies. Overall, neither the frequency of recovery of G418-resistant colonies nor the sizes of NHEJ-associated deletions were substantially different for the mutant vs. wild-type cell lines. However, we did observe greater usage of terminal microhomology among NHEJ events recovered from wild-type cells as compared to Msh2 mutants. Our results suggest that Msh2 influences error-prone NHEJ repair at the step of pairing of terminal DNA tails. We also report the recovery from both wild-type and Msh2-deficient cells of an unusual class of NHEJ events associated with multiple deletion intervals, and we discuss a possible mechanism for the generation of these "discontinuous deletions."
authors.affiliation: Department of Biological Sciences, University of South Carolina, Columbia, 29208, USA.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Smith
authors.forename: Barbara Criscuolo
authors.initials: BC
authors.lastname: Waldman
authors.forename: Alan S
authors.initials: AS
authors.lastname: Waldman
mesh_terms: D000818:Animals; D015139:Blotting, Southern; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004260:DNA Repair / Q000502:physiology*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA