Inactivation of yeast DNA mismatch repair by expression of plant PMS1 protein

GALLES, C.; SPAMPINATO, C.

Puerto Madryn

Congreso; XLVI Reunión Anual de la Sociedad Argentina de Investigación Bioquímica; 2010

SAIB

Highly conserved post-replicative mismatch repair system is crucial for maintaining genomic stability in all organisms. The main proteins involved in this DNA repair pathway include MutS and MutL homologues. Among the last complexes, MutLã , a heterodimer of MLH1 and PMS1, represents the major MutL activity in Arabidopsis thaliana. To analyze the in vivo function of AtPMS1, we cloned its cDNA in the expression vector YEP112SPGAL, under the control of galactose-inducible promoter GAL1, to carry out the expression of this plant protein in Saccharomyces cereviciae strain E134. This strain allows the assessment of spontaneous mutation rates in specific loci (lys2::InsEA14 and his7-2). Galactose induced expression was corroborated by Western Blot analysis, using previously generated rabbit anti-AtPMS1 antibodies. Following this, rates of His+ and Lys+ reversion were measured by fluctuation analysis revealing that expression of plant PMS1 dramatically increases yeast mutation rates at both loci studied (approximately 13 times at his7-2 locus and close to 2,000 times at locus lys2::InsEA14). These results suggest the inactivation of yeast MMR by heterologous protein expression, probably due to the formation of non-functional complexes, either AtPMS1 homodimers or yMLH1-AtPMS1 heterodimers.