Fidelity of replication of repetitive DNA in mutS and repair proficient Escherichia coli

Replication fidelity is not constant among strains within a species or at a ll genetic loci within a genome. Altered fidelity of replication may affect patterns of pathogenesis and the evolution of these strains. We have been studying replication fidelity in Escherichia coli, both in laboratory atten uated strains and in food-borne pathogens. To understand the altered patter ns of mutagenesis at the molecular level, we used a shuttle vector plasmid with a tRNA mutational marker gene which had been altered to include homopo lymeric runs of five, seven and nine [G:C] pairs, as well as non-repetitive DNA. Replication of the plasmid in mutS strains resulted in a 20-fold incr ease in mutant progeny plasmids. The mutations were almost all (>90%) frame shift mutations, while base substitution mutations were rare. Most mutation s were insertions or deletions of one or two [G:C] pairs in the longest hom opolymeric runs. Larger deletions (5 to >70 bp), also targeted to the repet itive sequence, were likewise common. Mutations increased exponentially wit h the length of the homopolymeric run. These patterns of mutation, includin g unexpectedly high levels in repair proficient strains, led to an examinat ion of the E. coli K-12 genome for homopolymeric DNA. This sequence motif w as found to be rare, particularly in genes and open reading frames. Amino a cid homotrimers were found to avoid usage of homopolymeric codons, even whe n they are preferred among synonymous codons in E. coli. There appears to b e active selection against tandem direct nucleotide repeats in the E. coli genome, correlated with the inability of the organism to accurately replica te such sequence. Published by Elsevier Science B.V.