Modulation of error-prone double-strand break repair in mammalian chromosomes by DNA mismatch repair protein Mlh1.
We assayed error-prone double-strand break (DSB) repair in wild-type and isogenic Mlh1-null mouse embryonic fibroblasts containing a stably integrated DSB repair substrate. The substrate contained a thymidine kinase (tk) gene fused to a neomycin-resistance (neo) gene; the tk-neo fusion gene was disrupted in the tk portion by a 22bp oligonucleotide containing the 18 bp recognition site for endonuclease I-SceI. Following DSB-induction by transient expression of I-SceI endonuclease, cells that repaired the DSB by error-prone nonhomologous end-joining (NHEJ) and restored the correct reading frame to the tk-neo fusion gene were recovered by selecting for G418-resistant clones. The number of G418-resistant clones induced by I-SceI expression did not differ significantly between wild-type and Mlh1-deficient cells. While most DSB repair events were consistent with simple NHEJ in both wild-type and Mlh1-deficient cells, complex repair events were more common in wild-type cells. Furthermore, genomic deletions associated with NHEJ events were strikingly larger in wild-type versus Mlh1-deficient cells. Additional experiments revealed that the stable transfection efficiency of Mlh1-null cells is higher than that of wild-type cells. Collectively, our results suggest that Mlh1 modulates error-prone NHEJ by inhibiting the annealing of DNA ends containing noncomplementary base pairs or by promoting the annealing of microhomologies.
Bannister
Laura A
LA
Department of Biological Sciences, University of South Carolina, 700 Sumter Street, Columbia, SC 29208, USA.
Waldman
Barbara Criscuolo
BC
Waldman
Alan S
AS
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D020137:Base Pair Mismatch / Q000235:genetics*; D001483:Base Sequence; D002352:Carrier Proteins; D002460:Cell Line; D019457:Chromosome Breakage / Q000235:genetics*; D033481:Chromosomes, Mammalian / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D017353:Gene Deletion; D051379:Mice; D018345:Mice, Knockout; D000070957:MutL Protein Homolog 1; D009363:Neoplasm Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C501485:Mlh1 protein, mouse; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000070957:MutL Protein Homolog 1