Saturation of mismatch repair in the mutD5 mutator strain of Escherichia coli

Abstract

The mutD (dnaQ) gene of Escherichia coli codes for the proofreading activity of DNA polymerase III. The very strong mutator phenotype of mutD5 strains seems to indicate that their postreplicational mismatch repair activity is also impaired. We show that the mismatch repair system of mutDS strains is functional but saturated, presumably by the excess of DNA replication errors, since it is recovered by inhibiting chromosomal DNA replication. This recovery depends on de novo protein synthesis.The mechanisms ensuring accuracy of DNA replication have been elucidated by study of mutator mutants affecting specific accuracy functions (for a review, see reference 4). In Escherichia coli, mutator mutations mapping in the dnaE gene presumably affect nucleotide selection by DNA polymerase III (28). Mutations mapping in the dnaQ gene (also called mutD) affect the proofreading 3'->5' exonuclease function of the DNA polymerase III holoenzyme (6, 8), whereas mutH, mutL, mutS, and mutU mutations cause defects in methyl-directed postreplicative mismatch correction (for reviews, see references 21 and 24). mutD5 and dnaQ49 mutants are the most potent mutator strains known. All classes of transition, transversion, and frameshift mutations are increased up to 105-fold when mutD5 strains are grown in rich medium (5, 10, 11). The mutD5 gene of E. coli encodes the epsilon subunit, which carries out the 3'-*5' proofreading exonuclease function of the DNA polymerase III holoenzyme (6,8). However, the high-mutation-rate effect of mutD5 strains cannot be explained only by the defect in exonuclease activity, as this mutation rate is comparable to the error rate of in vitro replication with a polymerase devoid of proofreading activity (9, 16). The strong mutator phenotype of mutD5 suggested that mismatch repair functions may be impaired as well. This prediction was supported by results from DNA heteroduplex transfection experiments (23,25,26). One hypothesis is that the high error rate in DNA replication saturates mismatch repair in mutD5 mutants (23). Therefore, we have tested the mismatch repair capacity of mutD5 strains under conditions in which chromosomal DNA replication was or was not allowed. Mismatch repair activity was determined by the extent of pure infective centers derived from infection with packaged hemimethylated heteroduplexes of lambda DNA. Under these conditions, bacteria proficient in mismatch repair produced essentially pure infective centers, whereas bacteria deficient in mismatch repair produced mostly mixed infective centers.To evaluate mismatch repair activity, pure hemimethy-* Corresponding author. t Present address: Institute of Molecular Biology, University of Oregon, Eugene, OR 97403. lated heteroduplexes of lambda DNA containing an A. C or a G T mismatch were artificially constructed and introduced into mutD5 and other appropriate strains. Hemimethylated heteroduplexes of lambda DNA with defined mismatches were prepared as previously described (19) by reannealing the separated DNA strands of a lambda mutant carry...

Adaptive mutation sequences reproduced by mismatch repair deficiency.

“…Means by which mismatch repair could be disabled transiently during adaptive mutation include down-regulation of mutS, -L, -H, or -U gene expression (34); depletion of any of those gene products due to starvation (19) or due to such high levels of polymerase error that the repair system is saturated (35); transient undermethylation of the DNA due to diminution of Dam protein before cessation of DNA synthesis during starvation; or transient overmethylation of the unreplicating DNA that blocks action of the MutH endonuclease on DNA (20).…”

Depletion of the cellular amounts of the MutS and MutH methyl-directed mismatch repair proteins in stationary-phase Escherichia coli K-12 cells

“…Thus, it appears that the amount of cellular MutS can be repressed but not increased in response to the stress conditions tested so far. It remains to be determined whether other situations that titrate MDM repair and lead to a strong mutator phenotype, such as the growth of mutD mutants in rich medium (10,53), change the cellular amounts or levels of activity of the MDM repair proteins. In mutD mutants, limitation for MutL and MutH, rather than MutS, may diminish MDM repair capacity (53).…”

“…The cellular amounts of the MDM repair proteins have never been determined directly. Amounts of 10 to 30 molecules of MutL, MutS, and MutH per cell were estimated from ␤-galactosidase activities of gene fusions between the individual mutHLS genes and lacZ (10). The amount of MutH was also estimated at about 20 copies per cell from comparisons of the in vitro repair activity of purified MutH added to extracts of mutH mutants with that of extracts from wild-type bacteria (63).…”

Homeologous recombination and mismatch repair during transformation in Streptococcus pneumoniae: saturation of the Hex mismatch repair system.

“…Saturation has been observed in a mutD, proofreading deficient, strain that accumulates excessive DNA replication errors (34,35), during treatments with mutagens whose mutagenic intermediates are subject to mismatch repair (36), or in the presence of multicopy single-stranded DNAs with mismatches (37). Given the biological consequences of mutational inactivation of mismatch repair genes in humans (8), even transient saturation could be highly detrimental.…”