Mismatch repair modulation of MutY activity drives Bacillus subtilis stationary-phase mutagenesis

Stress-promoted mutations that occur in nondividing cells (adaptive mutations) have been implicated strongly in causing genetic variability as well as in species survival and evolutionary processes. Oxidative stress-induced DNA damage has been associated with generation of adaptive His(+) and Met(+) but not Leu(+) revertants in strain Bacillus subtilis YB955 (hisC952 metB5 leuC427). Here we report that an interplay between MutY and MutSL (mismatch repair system [MMR]) plays a pivotal role in the production of adaptive Leu(+) revertants. Essentially, the genetic disruption of MutY dramatically reduced the reversion frequency to the leu allele in this model system. Moreover, the increased rate of adaptive Leu(+) revertants produced by a MutSL knockout strain was significantly diminished following mutY disruption. Interestingly, although the expression of mutY took place during growth and stationary phase and was not under the control of RecA, PerR, or σ(B), a null mutation in the mutSL operon increased the expression of mutY several times. Thus, in starved cells, saturation of the MMR system may induce the expression of mutY, disturbing the balance between MutY and MMR proteins and aiding in the production of types of mutations detected by reversion to leucine prototrophy. In conclusion, our results support the idea that MMR regulation of the mutagenic/antimutagenic properties of MutY promotes stationary-phase mutagenesis in B. subtilis cells.     

Single-amino-acid substitutions in open reading frame (ORF) 1b-nsp14 and ORF 2a proteins of the coronavirus mouse hepatitis virus are attenuating in mice

A reverse genetic system was recently established for the coronavirus mouse hepatitis virus strain A59 (MHV-A59), in which cDNA fragments of the RNA genome are assembled in vitro into a full-length genome cDNA, followed by electroporation of in vitro-transcribed genome RNA into cells with recovery of viable virus. The "in vitro-assembled" wild-type MHV-A59 virus (icMHV-A59) demonstrated replication identical to laboratory strains of MHV-A59 in tissue culture; however, icMHV-A59 was avirulent following intracranial inoculation of C57BL/6 mice. Sequencing of the cloned genome cDNA fragments identified two single-nucleotide mutations in cloned genome fragment F, encoding a Tyr6398His substitution in open reading frame (ORF) 1b p59-nsp14 and a Leu94Pro substitution in the ORF 2a 30-kDa protein. The mutations were repaired individually and together in recombinant viruses, all of which demonstrated wild-type replication in tissue culture. Following intracranial inoculation of mice, the viruses encoding Tyr6398His/Leu94Pro substitutions and the Tyr6398His substitution alone demonstrated log10 50% lethal dose (LD50) values too great to be measured. The Leu94Pro mutant virus had reduced but measurable log10 LD5), and the "corrected" Tyr6398/Leu94 virus had a log10 LD50 identical to wild-type MHV-A59. The experiments have defined residues in ORF 1b and ORF 2a that attenuate virus replication and virulence in mice but do not affect in vitro replication. The results suggest that these proteins serve roles in pathogenesis or virus survival in vivo distinct from functions in virus replication. The study also demonstrates the usefulness of the reverse genetic system to confirm the role of residues or proteins in coronavirus replication and pathogenesis.     

Evidence that stationary-phase hypermutation in the Escherichia coli chromosome is promoted by recombination

Adaptive (or stationary-phase) mutation is a group of phenomena in which mutations appear to occur more often when selected than when not. They may represent cellular responses to the environment in which the genome is altered to allow survival. The best-characterized assay system and mechanism is reversion of a lac allele on an F' sex plasmid in Escherichia coli, in which the stationary-phase mutability requires homologous recombination functions. A key issue has concerned whether the recombination-dependent mutation mechanism is F' specific or is general. Hypermutation of chromosomal genes occurs in association with adaptive Lac(+) mutation. Here we present evidence that the chromosomal hypermutation is promoted by recombination. Hyperrecombinagenic recD cells show elevated chromosomal hypermutation. Further, recG mutation, which promotes accumulation of recombination intermediates proposed to prime replication and mutation, also stimulates chromosomal hypermutation. The coincident mutations at lac (on the F') and chromosomal genes behave as independent events, whereas coincident mutations at lac and other F-linked sites do not. This implies that transient covalent linkage of F' and chromosomal DNA (Hfr formation) does not underlie chromosomal mutation. The data suggest that recombinational stationary-phase mutation occurs in the bacterial chromosome and thus can be a general strategy for programmed genetic change.     

How carcinogens (or telomere dysfunction) induce genetic instability: associated-selection model

Carcinogens induce carcinogen-specific genetic instability (defects in DNA repair). According to the 'direct-selection' model, defects in DNA repair per se provide an immediate growth advantage. According to the 'associated-selection' model, carcinogens merely select for cells with adaptive mutations. Like any mutations, adaptive mutations occur predominantly in genetically unstable cells. The 'associated-selection' model predicts that carcinogen-driven selection minimizes cytotoxic but maximizes mutagenic effects of carcinogens. A purely mutagenic (neither cytotoxic, nor cytostatic) environment will favor effective DNA repair, whereas any growth-limiting conditions (telomerase deficiency, anticancer drugs) will select for genetically unstable cells. Genetic instability is a postmark of selective pressure rather than a hallmark of cancer per se. Once selected, genetic instability facilitates the development of resistance to any other growth-limiting conditions. As an example, a putative link between prior exposure to carcinogens and the ability to develop a telomerase-independent growth is discussed.