Mismatch repair at stop codons is directed independent of GATC methylation on the Escherichia coli chromosome

Abstract

The mismatch repair system (MMR) corrects replication errors that escape proofreading. Previous studies on extrachromosomal DNA in Escherichia coli suggested that MMR uses hemimethylated GATC sites to identify the newly synthesized strand. In this work we asked how the distance of GATC sites and their methylation status affect the occurrence of single base substitutions on the E. coli chromosome. As a reporter system we used a lacZ gene containing an early TAA stop codon. We found that occurrence of point mutations at this stop codon is unaffected by GATC sites located more than 115 base pairs away. However, a GATC site located about 50 base pairs away resulted in a decreased mutation rate. This effect was independent of Dam methylation. The reversion rate of the stop codon increased only slightly in dam mutants compared to mutL and mutS mutants. We suggest that unlike on extrachromosomal DNA, GATC methylation is not the only strand discrimination signal for MMR on the E. coli chromosome.

Figure 1. Structure and chromosomal context of the reporter constructs used in this study.

The reporter constructs (A–E) contained the zeocin resistance cassette and the lacZ gene which was inactivated by the C20A substitution resulting in a stop codon (red line). The positions of GATC sequences in the different constructs are indicated by vertical lines. Arrowheads indicate the direction of transcription. The replication fork proceeds from left to right in this region. The local sequence context of the stop codon is shown on the top. Measured mutation rates (M) and 95% confidence intervals (95% CI) are shown on the right. The mutation rates and 95% confidence intervals observed upon mutS deletion were 3.1 (2.2–4.1), 13.3 (11.2–15.6), 3.8 (2.8–4.9), and 13.6 (11.1–16.3) × 10⁻⁹/generation for strains ‘A’–‘D’, respectively.

Figure 2. Efficiency of mismatch repair as function of MutS diffusion rate along the DNA.

At each value of the diffusion rate, MutS located at the mismatch is released to perform one-dimensional random walks along the DNA. The simulation was repeated 10000 times. The efficiency of the mismatch repair is scored as the fraction of the released MutS that reach either a site 2400 bp downstream of the mismatch or a site 5550 bp upstream of the mismatch within 90 seconds. These 90 seconds correspond to the average lifetime of a hemi-methylated GATC. The shown behavior was reproduced (within a factor 2 in diffusion constant) in a more elaborate model where many independently methylated GATC sites (methylated in 90 seconds on average) are placed at 256 bp intervals outside the −2400 to 5550 bp region. In that more complicated model the repair efficiency was scored as the probability that MutS reached any of these sites in a hemimethylated state.