Functional analysis of Arabidopsis thaliana MutS and MutL complexes in yeast

GÓMEZ, R.; GALLES, C.; CIRIBILLI, Y.; INGA, A.; CLAUDIA PATRICIA SPAMPINATO

Rosario

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica (SAIB); 2006

Highly conserved mismatch repair (MMR) systems promote genomic stability by correcting DNA replication errors, antagonizing homeologous recombination, and responding to various DNA lesions. Arabidopsis and other plants encode a suite of MMR protein orthologs: the recognition heterodimers MSH2-MSH6 and MSH2-MSH7, and the recognition-excision coupling component MLH1-PMS1. To analyze the function of these complexes in yeast we constructed vectors for expression of AtMSH2, AtMSH7, AtMLH1 and AtPMS1. The vectors utilize a relaxed 2µ origin, the inducible GAL1-10 promoter to achieve high level of protein expression and different selection markers. The effects of AtMMR protein expression on mutation rates were assessed in strains carrying a sensitive reporter based on a long homonucleotide run of 14 A-T base pairs within the LYS2 locus resulting in a +1 reading frame. Hence, reversion to LYS+ occurs by deletion of a single A-T base pair within the homonucleotide run. Our results suggest that expression of AtMSH2 and/or AtMSH7 has not impact on the mutation rate of the msh6 mutant strain, and expression of the individual proteins does not increase the low mutation rate of a wild-type strain. However, co-expression of AtMSH2 and AtMSH7 at high levels in wild-type yeast reduced reversion events compared to the empty vector.