ThemutTDefect Does Not Elevate Chromosomal Fragmentation inEscherichia coliBecause of the Surprisingly Low Levels of MutM/MutY-Recognized DNA Modifications

Abstract

Nucleotide pool sanitizing enzymes Dut (dUTPase), RdgB (dITPase), and MutT (8-oxo-dGTPase) of Escherichia coli hydrolyze noncanonical DNA precursors to prevent incorporation of base analogs into DNA. Previous studies reported dramatic AT3CG mutagenesis in mutT mutants, suggesting a considerable density of 8-oxo-G in DNA that should cause frequent excision and chromosomal fragmentation, irreparable in the absence of RecBCD-catalyzed repair and similar to the lethality of dut recBC and rdgB recBC double mutants. In contrast, we found mutT recBC double mutants viable with no signs of chromosomal fragmentation. Overproduction of the MutM and MutY DNA glycosylases, both acting on DNA containing 8-oxo-G, still yields no lethality in mutT recBC double mutants. Plasmid DNA, extracted from mutT mutM double mutant cells and treated with MutM in vitro, shows no increased relaxation, indicating no additional 8-oxo-G modifications. Our ⌬mutT allele elevates the AT3CG transversion rate 27,000-fold, consistent with published reports. However, the rate of AT3CG transversions in our mutT ؉ progenitor strain is some two orders of magnitude lower than in previous studies, which lowers the absolute rate of mutagenesis in ⌬mutT derivatives, translating into less than four 8-oxo-G modifications per genome equivalent, which is too low to cause the expected effects. Introduction of various additional mutations in the ⌬mutT strain or treatment with oxidative agents failed to increase the mutagenesis even twofold. We conclude that, in contrast to the previous studies, there is not enough 8-oxo-G in the DNA of mutT mutants to cause elevated excision repair that would trigger chromosomal fragmentation.

Mutation rate dynamics in a bacterial population reflect tension between adaptation and genetic load

“…However, mutY mutations have an antimutator effect in the context of a MutT defect because MutY mis-repairs 8-oxoG:A base pairs in DNA. The 60% reduction in overall mutation rates reported in mutT mutY double mutants compared with mutT single mutants (29) is similar to the rate changes we observed in both the phylogenomic analysis (Fig. 1B) and fluctuation tests (Fig.…”

“…The MutT protein is a hydrolase that purges the cellular nucleotide pool of oxidized guanine nucleotides (8-oxo-dGTP), which can mis-pair with adenine and lead to A:T→C:G (adenine or thymine to cytosine or guanine) transversions after DNA replication. Loss-of-function mutations in mutY, which encodes a DNA repair glycosylase that excises mis-paired bases from DNA helices, also lead to elevated mutation rates on their own (29). However, mutY mutations have an antimutator effect in the context of a MutT defect because MutY mis-repairs 8-oxoG:A base pairs in DNA.…”

“…Mutations after the rise of the mutT hypermutator were highly skewed toward the A: T→C:G transversions typical of this defect (29). This same overall bias dominated along all of the later branches in the tree (Table 1), but the proportion of C:G→A:T substitutions increased from only 2/414 in the initial mutT genetic background to 38/274 in the mutT mutY-E lineage (one-tailed Fisher's exact test, P = 4 × 10…”

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

“…The following additional deletions of the AB1157 strain were applied: Dgpt-proA, DlacA-lacY, DuxaB-yneF, DgadAgadB (coordinates: 255,800,361,100,1,610,611,000,2,233,239,200) and the region gadB (1,570,700-1,571,700), which give abnormal high reads in all strains. The DtnaA-tnaB deletion with coordinates 3,888,800-3,890,600 was removed from all strains, except the SRK strains.…”

“…The initial objectives of this study were, by pushing E. coli cells to maximize chromosomal replication complexity (in the rep and seqA mutants or in HU-treated cells): (1) to confirm the maximal chromosomal replication complexity that E. coli can routinely achieve without major adverse consequences-the natural CRC limit; (2) to determine the maximal chromosomal replication complexity at which E. coli can still slowly grow-the functional CRC limit; (3) to push E. coli to develop the maximal chromosomal replication complexity that the cells can survive-the tolerance CRC limit; (4) to understand the nature of growth inhibition due to the increased CRC. In our efforts to maximize CRC, we specifically avoided the subject of regulation of the replication initiation at the chromosomal origin and, in fact, removed this consideration altogether in subsequent experimentation by initiating chromosome replication from an inducible plasmid replication origin.…”