The frequency and structure of recombinant products is determined by the cellular level of MutL

Abstract

The presence of repeated DNA sequences is a genomic liability, because interrepeat recombination can result in chromosomal rearrangements. The mismatch repair system prevents recombination between nonidentical repeats, but the mechanism of antirecombination has not been established. Although the MutS protein binds to base pair mismatches in heteroduplex DNA, the role of the MutL protein in preventing recombination is unknown. In a screen designed to identify new cellular functions that suppress deletion formation involving nonidentical DNA repeats, we isolated a mutL mutant having a separation-of-function phenotype. The mutant showed an increased frequency of deletions but not of mutations. The split phenotype is due to a decreased MutL level, indicating that recombination, but not replication editing, is highly sensitive to MutL level. By altering the MutL level, we found that the frequency of deletion-generating recombination is inversely related to the amount of cellular MutL. DNA sequence analysis of the recombined repeats shows that the tolerance of base pair mismatches in heteroduplex DNA is also inversely correlated with MutL level. Unlike recombination, correction of misincorporation errors by mismatch repair is insensitive to fluctuations in MutL level. Overproduction of MutS does not affect either of these phenotypes, suggesting that, unlike MutL, MutS is not limiting for mismatch repair activities. These results indicate that MutL (i) determines effective DNA homology in recombination processes and (ii) fine tunes the process of deletion formation involving repeated, diverged DNA sequences.

Fig. 1.

Chromosomal construct used to measure deletion formation through recombination between identical or diverged repeats. Two nonfunctional lacZ genes are cloned in close proximity (0.5 kb) on the E. coli chromosome. The first lacZ gene contains a C-terminal deletion (lacZ′ΔC), whereas the second contains an N-terminal deletion (′lacZΔN). The two gene fragments share an overlapping region of 1.3 kb (filled boxes) that is 100% identical or 4% diverged at the DNA sequence level. Nonoverlapping lacZ gene C-terminal and N-terminal regions are presented as hatched boxes. A single recombination event between two gene fragments reconstitutes a functional lacZ gene and deletes the intervening region. plac, lactose promoter; lacZ, gene coding for β-galactosidase; lacY, gene coding for lactose permease; yfp, gene coding for yellow fluorescent protein.

Fig. 2.

Quantification of MutL protein level in different strains. (A) Position of the miniTn10 insertion within the amiB-mutL–miaA-hfq-hflX superoperon. pmutL and pmutLHS, mutL gene promoters; miniTn10 (hatched box), transposon; ptetA and ptetR, Tn10 tetracycline-inducible promoters. (B) Cellular level of MutL protein determined by immunoblotting. Values represent fold difference over the control calculated from the average of three independent experiments. wt, wild-type strain; mutL_down, mutant strain with miniTn10 insertion in the amiB gene; pMutL and pMutS, wild-type strains carrying plasmids that overexpress MutL or MutS proteins, respectively; pVector, wild-type strain carrying control plasmid; tc, tetracycline; nd, not detected.

Fig. 3.

Correlation between varying the level of MutL protein and the efficacy of recombination between diverged DNA sequences. (A) RecA-dependent recombination between 4% diverged lacZ repeats (R² = 0.79). (B) RecA-dependent conjugational recombination between Salmonella enterica serovar Typhimurium Hfr and Escherichia coli F⁻ recipients, which are 20% diverged between homologous genes (R² = 0.94). Wild-type strain (filled squares), strain with 33-fold less MutL (filled circles), strain with 4-fold less MutL (open circles), and wild-type strain overexpressing MutL (open squares) are shown. The dotted curves show the 95% confidence interval around the regression. For MutL protein quantification see Fig. 2B.

Fig. 4.

Structure of lacZ⁺ recombinants as a function of cellular MutL and MutS level. For each recombinant, the junction is assigned to a region delimited by mismatches in aligned lacZ repeats. The cumulative distribution of junction positions for the strains examined is shown. Mismatches that are well (red filled circles; G–T and A–C), less efficiently (blue filled circles; A–G and C–T), or not at all (black filled circles; C–C) recognized by the MMR system are shown on the x axes. (A) RecA-dependent lacZ⁺ recombinants. Wild-type strain (pink open squares and lines), mutL strain (green open circles and lines), strain with 4-fold less MutL (red open diamonds and lines), wild-type strains overexpressing MutL (purple open triangles and lines), and MutS proteins (gray open circles and lines) are shown. (B) RecA-independent lacZ⁺ recombinants. recA strain (brown open diamonds and lines) and recA mutL strain (blue open squares and lines) are shown. For comparison wild-type strain (pink open squares and lines) and mutL strain (green open circles and lines) are given. For sequencing protocol see SI Text.