"Plasmid mutability/stability in E. coli dam cells is affected by MutS and/or MutL levels".

MARTINA, M.A; JACQUELÍN, D. K; ARGARAÑA, C. E; BARRA , J. L.

Rosario,Santa Fe,Argentina.

Congreso; XLII Reunión anual de SAIB.; 2006

SAIB

In Escherichia coli MutS, MutL and MutH/Dam proteins are the principal components of the mismatch repair system (MMRS), which repairs DNA replication errors. We have previously observed that overproduction of MutS or MutL in E. coli improve the functioning of the MMRS. As the MMRS present in E. coli dam cells is active but non-directed the action of these proteins can result in a double-strand break, and consequently in DNA degradation. Here we analyzed the mutability/stability of plasmids in dam and dam cells overproducing MutS and/or MutL. We first analyzed the effect of the endogenous MutS/L/H proteins on plasmid mutability, using a plasmid carrying a mutated copy of a gene that confer resistance to tetracycline (tr). E. coli dam cells generated only 4% of the tr resistant colonies that can be obtained with E. coli MMRS-deficient strains. This result shows that the endogenous MutS/L/H proteins are able to act on plasmid DNA eliminating most of the copies in which a mutation occurred (96% of the putative revertants). We then analyzed the effect of MutS and/or MutL overproduction on plasmid stability after 5 days of successive culture in reach media without antibiotic. Two different plasmids were analyzed. While empty plasmids are stable maintained in E. coli dam cells, one of the plasmid (P1) can be lost from cells overproducing MutS or -L (40% of the cells), and both plasmids can be eliminated when both proteins are simultaneously overproduced, however in different proportion (80% for P1, 15% for P2).