Are adaptive mutations due to a decline in mismatch repair? The evidence is lacking

The levels of proteins required for methyl-directed mismatch repair appear to decline in stationary-phase and nutritionally-deprived cells of Escherichia coli. It has been hypothesized that error-correction by the system also declines, and this decline is responsible for adaptive or stationary-phase mutations. However, evidence in support of this hypothesis is lacking. The mismatch repair system is no less effective in correcting errors during prolonged selection than it is during growth. Furthermore, mismatch repair proteins supplied in excess reduce both growth-dependent and adaptive mutation.     

Adaptive mutations in Salmonella typhimurium phenotypic of purR super-repression

Under non-lethal selective conditions, a non-dividing or very slowly dividing microbial population gives rise to mutations that relieve selective pressures. This process is described as adaptive mutation. Salmonella typhimurium strain 5-28 has been used as a system for studying adaptive mutations in the chromosomal regulatory gene purR and its target, the purD operator. When this strain is plated on a minimal lactose medium, no apparent growth of parent lawn is observed, yet the revertant colonies accumulate over a period of time. Analysis of the purR mutational spectra showed that the frequencies of transitions and transversions were not significantly different among the growth-dependent and adaptive mutations. But the frequencies for five kinds of -1 frameshifts were significantly different between the growth-dependent and adaptive types. Among the growth-dependent mutations, most one-base deletions occurred in non-iterated bases and were distributed randomly. Among adaptive mutations, the frequency of one-base deletions in small mononucleotide repeats was higher and mutations were concentrated at three hotspots. One-base deletion in small mononucleotide repeats are generally believed to result from DNA polymerase slippage errors, which are not corrected by DNA repair machinery. We further investigated the role of DNA repair on adaptive mutation. Our results showed that the mismatch repair (MMR) might function less efficiently during adaptive mutation. However, DNA oxidative damage repair seemed no less effective in correcting errors under selective pressures than during non-selective growth.     

Adaptation of Mycobacterium smegmatis to Stationary Phase

Mycobacterium tuberculosis can persist for many years within host lung tissue without causing clinical disease. Little is known about the state in which the bacilli survive, although it is frequently referred to as dormancy. Some evidence suggests that cells survive in nutrient-deprived stationary phase. Therefore, we are studying stationary-phase survival of Mycobacterium smegmatis as a model for mycobacterial persistence. M. smegmatis cultures could survive 650 days of either carbon, nitrogen, or phosphorus starvation. In carbon-limited medium, cells entered stationary phase before the carbon source (glycerol) had been completely depleted and glycerol uptake from the medium continued during the early stages of stationary phase. These results suggest that the cells are able to sense when the glycerol is approaching limiting concentrations and initiate a shutdown into stationary phase, which involves the uptake of the remaining glycerol from the medium. During early stationary phase, cells underwent reductive cell division and became more resistant to osmotic and acid stress and pool mRNA stabilized. Stationary-phase cells were also more resistant to oxidative stress, but this resistance was induced during late exponential phase in a cell-density-dependent manner. Upon recovery in fresh medium, stationary-phase cultures showed an immediate increase in protein synthesis irrespective of culture age. Colony morphology variants accumulated in stationary-phase cultures. A flat colony variant was seen in 75% of all long-term-stationary-phase cultures and frequently took over the whole population. Cryo scanning electron microscopy showed that the colony organization was different in flat colony strains, flat colonies appearing less well organized than wild-type colonies. Competition experiments with an exponential-phase-adapted wild-type strain showed that the flat strain had a competitive advantage in stationary phase, as well a providing evidence that growth and cell division occur in stationary-phase cultures of M. smegmatis. These results argue against stationary-phase M. smegmatis cultures entering a quiescent state akin to dormancy but support the idea that they are a dynamic population of cells.     

Hypermutation in stationary-phaseE. coli: tales from thelac operon

Escherichia coli cells are capable of complex regulatory responses to environmental conditions and stresses. In some circumstances, the response includes an increase in the mutation rate, effectively mutagenizing the genome. The classic example is the SOS response to DNA damage. Recent work indicates that other environmental stresses can also result in mutation of the genome. Modulation of mutation rate may be a more prevalent stress response than previously thought. In this review we focus on genome-wide mutation inE. coli cells subjected to a nonlethal genetic selection for reversion of alac frameshift allele. Reversion of thelac frameshift allele occurs via a novel mechanism that requires homologous recombination functions, and is enhanced by transiently diminished postsynthesis mismatch repair. A model for recombination-dependent stationary-phase mutation will be presented and its relevance for genome-wide mutation discussed.     

Population Genetic Study of Mitochondrial DNA in Roseate Spoonbill (Aves; Platalea ajaja) Breeding Colonies from the Pantanal Wetlands, Brazil

Five breeding colonies of the Roseate spoonbill (Aves: Platalea ajaja) from two Brazilian wetland areas (Pantanal and Taim marshes) were sampled, and domain I of the mitochondrial DNA control region (483 bp) was sequenced in 50 birds. The average haplotype diversity (h = 0.75, s = 0.071) and average nucleotide diversity (pi = 0.004, s = 0.003) were evaluated, and nonsignificant differences were found among the colonies studied. The lack of differentiation among breeding colonies revealed by AMOVA analysis was explained either as a consequence of high gene flow or recent expansion. The significantly negative results of the neutrality tests (Fu's F ( s ) = -23.271, P < 0.01; Tajima's D = -1.941, P < 0.01) associated with the star shape of the haplotype tree and mismatch distribution data are evidence supporting the idea that these populations underwent a recent demographic expansion in the Pantanal region. The average time since the expansion is estimated to be 25,773 years, and this agrees with a period of increased moisture that occurred during the last glacial period.     

Evidence for independent mismatch repair processing on opposite sides of a double-strand break in Saccharomyces cerevisiae.

Double-strand break (DSB) induced gene conversion in Saccharomyces cerevisiae during meiosis and MAT switching is mediated primarily by mismatch repair of heteroduplex DNA (hDNA). We used nontandem ura3 duplications containing palindromic frameshift insertion mutations near an HO nuclease recognition site to test whether mismatch repair also mediates DSB-induced mitotic gene conversion at a non-MAT locus. Palindromic insertions included in hDNA are expected to produce a stem-loop mismatch, escape repair, and segregate to produce a sectored (Ura+/-) colony. If conversion occurs by gap repair, the insertion should be removed on both strands, and converted colonies will not be sectored. For both a 14-bp palindrome, and a 37-bp near-palindrome, approximately 75% of recombinant colonies were sectored, indicating that most DSB-induced mitotic gene conversion involves mismatch repair of hDNA. We also investigated mismatch repair of well-repaired markers flanking an unrepaired palindrome. As seen in previous studies, these additional markers increased loop repair (likely reflecting corepair). Among sectored products, few had additional segregating markers, indicating that the lack of repair at one marker is not associated with inefficient repair at nearby markers. Clear evidence was obtained for low levels of short tract mismatch repair. As seen with full gene conversions, donor alleles in sectored products were not altered. Markers on the same side of the DSB as the palindrome were involved in hDNA less often among sectored products than nonsectored products, but markers on the opposite side of the DSB showed similar hDNA involvement among both product classes. These results can be explained in terms of corepair, and they suggest that mismatch repair on opposite sides of a DSB involves distinct repair tracts.     

The conversion gradient at HIS4 of Saccharomyces cerevisiae. I. Heteroduplex rejection and restoration of Mendelian segregation.

In Saccharomyces cerevisiae, some gene loci manifest gradients in the frequency of aberrant segregation in meiosis, with the high end of each gradient corresponding to a hotspot for DNA double-strand breaks (DSBs). The slope of a gradient is reduced when mismatch repair functions fail to act upon heteroduplex DNA-aberrant segregation frequencies at the low end of the gradient are higher in the absence of mismatch repair. Two models for the role of mismatch repair functions in the generation of meiotic "conversion gradients" have been proposed. The heteroduplex rejection model suggests that recognition of mismatches by mismatch repair enzymes limits hybrid DNA flanking the site of a DSB. The restoration-conversion model proposes that mismatch repair does not affect the length of hybrid DNA, but instead increasingly favors restoration of Mendelian segregation over full conversion with increasing distance from the DSB site. In our experiment designed to distinguish between these two models, data for one subset of well repairable mismatches in the HIS4 gene failed to show restoration-type repair but did indicate reduction in the length of hybrid DNA, supporting the heteroduplex rejection model. However, another subset of data manifested restoration-type repair, indicating a relationship between Holliday junction resolution and mismatch repair. We also present evidence for the infrequent formation of symmetric hybrid DNA during meiotic DSB repair.     

Escherichia coli frameshift mutation rate depends on the chromosomal context but not on the GATC content near the mutation site

Different studies have suggested that mutation rate varies at different positions in the genome. In this work we analyzed if the chromosomal context and/or the presence of GATC sites can affect the frameshift mutation rate in the Escherichia coli genome. We show that in a mismatch repair deficient background, a condition where the mutation rate reflects the fidelity of the DNA polymerization process, the frameshift mutation rate could vary up to four times among different chromosomal contexts. Furthermore, the mismatch repair efficiency could vary up to eight times when compared at different chromosomal locations, indicating that detection and/or repair of frameshift events also depends on the chromosomal context. Also, GATC sequences have been proved to be essential for the correct functioning of the E. coli mismatch repair system. Using bacteriophage heteroduplexes molecules it has been shown that GATC influence the mismatch repair efficiency in a distance- and number-dependent manner, being almost nonfunctional when GATC sequences are located at 1 kb or more from the mutation site. Interestingly, we found that in E. coli genomic DNA the mismatch repair system can efficiently function even if the nearest GATC sequence is located more than 2 kb away from the mutation site. The results presented in this work show that even though frameshift mutations can be efficiently generated and/or repaired anywhere in the genome, these processes can be modulated by the chromosomal context that surrounds the mutation site.     

The cellular mismatch repair system is able to repair mismatches within MLV retroviral double-stranded DNA at a low frequency

Eukaryotic cells possess several distinct mismatch repair pathways. A mismatch can be introduced in retroviral double-stranded DNA by a pre-existing mutation within the primer binding site (PBS) of the viral RNA genome. In order to evaluate mismatch repair of retroviral double-stranded DNA, Moloney leukemia virus (MLV)-based vectors with a mutation in their PBS were used to infect mismatch repair-competent as well as mismatch repair-deficient cell lines. If the target cells were capable of repairing the mismatch before an infected cell divided, the mismatch within the PBS could be repaired to the wild-type or mutant PBS. If the target cells were unable to repair the mismatch, half the cells in the colony should contain the mutant PBS while the other half should contain the wild-type PBS. To evaluate these predictions, individual colonies were isolated and analyzed by PCR. Almost all mismatch-deficient cell colonies analyzed (cell lines HCT 116 and PMS2–/–) contained both the wild-type and mutated PBS, therefore, mismatches within retroviral double-strand DNA could not be repaired by the mismatch-deficient cells. In contrast, mismatches in ~25% of the mismatch repair-competent cell clones analyzed (cell lines HeLa and PMS2+/+) were repaired, while 75% were not. Therefore, the cellular mismatch repair system is able to repair mismatches within viral double-stranded DNA, but at a low frequency.     

Structural and functional divergence of MutS2 from bacterial MutS1 and eukaryotic MSH4-MSH5 homologs

MutS homologs, identified in nearly all bacteria and eukaryotes, include the bacterial proteins MutS1 and MutS2 and the eukaryotic MutS homologs 1 to 7, and they often are involved in recognition and repair of mismatched bases and small insertion/deletions, thereby limiting illegitimate recombination and spontaneous mutation. To explore the relationship of MutS2 to other MutS homologs, we examined conserved protein domains. Fundamental differences in structure between MutS2 and other MutS homologs suggest that MutS1 and MutS2 diverged early during evolution, with all eukaryotic homologs arising from a MutS1 ancestor. Data from MutS1 crystal structures, biochemical results from MutS2 analyses, and our phylogenetic studies suggest that MutS2 has functions distinct from other members of the MutS family. A mutS2 mutant was constructed in Helicobacter pylori, which lacks mutS1 and mismatch repair genes mutL and mutH. We show that MutS2 plays no role in mismatch or recombinational repair or deletion between direct DNA repeats. In contrast, MutS2 plays a significant role in limiting intergenomic recombination across a range of donor DNA tested. This phenotypic analysis is consistent with the phylogenetic and biochemical data suggesting that MutS1 and MutS2 have divergent functions.     

Defects in the Error Prevention Oxidized Guanine System Potentiate Stationary-Phase Mutagenesis in Bacillus subtilis

Previous studies showed that a Bacillus subtilis strain deficient in mismatch repair (MMR; encoded by the mutSL operon) promoted the production of stationary-phase-induced mutations. However, overexpression of the mutSL operon did not completely suppress this process, suggesting that additional DNA repair mechanisms are involved in the generation of stationary-phase-associated mutants in this bacterium. In agreement with this hypothesis, the results presented in this work revealed that starved B. subtilis cells lacking a functional error prevention GO (8-oxo-G) system (composed of YtkD, MutM, and YfhQ) had a dramatic propensity to increase the number of stationary-phase-induced revertants. These results strongly suggest that the occurrence of mutations is exacerbated by reactive oxygen species in nondividing cells of B. subtilis having an inactive GO system. Interestingly, overexpression of the MMR system significantly diminished the accumulation of mutations in cells deficient in the GO repair system during stationary phase. These results suggest that the MMR system plays a general role in correcting base mispairing induced by oxidative stress during stationary phase. Thus, the absence or depression of both the MMR and GO systems contributes to the production of stationary-phase mutants in B. subtilis. In conclusion, our results support the idea that oxidative stress is a mechanism that generates genetic diversity in starved cells of B. subtilis, promoting stationary-phase-induced mutagenesis in this soil microorganism.     

Dimerization of MLH1 and PMS2 limits nuclear localization of MutLalpha

DNA mismatch repair maintains genomic stability by detecting and correcting mispaired DNA sequences and by signaling cell death when DNA repair fails. The mechanism by which mismatch repair coordinates DNA damage and repair with cell survival or death is not understood, but it suggests the need for regulation. Since the functions of mismatch repair are initiated in the nucleus, we asked whether nuclear transport of MLH1 and PMS2 is limiting for the nuclear localization of MutLalpha (the MLH1-PMS2 dimer). We found that MLH1 and PMS2 have functional nuclear localization signals (NLS) and nuclear export sequences, yet nuclear import depended on their C-terminal dimerization to form MutLalpha. Our studies are consistent with the idea that dimerization of MLH1 and PMS2 regulates nuclear import by unmasking the NLS. Limited nuclear localization of MutLalpha may thus represent a novel mechanism by which cells fine-tune mismatch repair functions. This mechanism may have implications in the pathogenesis of hereditary non-polyposis colon cancer.     

A Genetic Strategy to Demonstrate the Occurrence of Spontaneous Mutations in Nondividing Cells within Colonies of Escherichia Coli

A genetic strategy was designed to examine the occurrence of mutations in stationary-phase populations. In this strategy, a parental population of cells is able to survive under both permissive and restrictive conditions whereas mutants at a particular target locus exhibit a conditional-lethal phenotype. Thus, by growing the population to stationary phase under restrictive conditions and then shifting it to permissive conditions, mutations that had arisen in stationary phase can be studied without confounding effects caused by the occurrence of similar mutations during growth of the population. In two different applications of this strategy, we have studied the reversion to Lac(+) in stationary phase of several Lac(-) mutations in Escherichia coli. Our results indicate that a variety of spontaneous point mutations and deletions, particularly those that are sensitive to the mechanisms of replication slippage (for their generation) and methyl-directed mismatch repair (for their correction), can arise in nondividing populations of cells within a colony. The frequency of their occurrence was also elevated in mutS strains, which are defective in such mismatch repair. These data have relevance to the ongoing debate on adaptive or directed mutations in bacteria.     

Spontaneous mutagenesis in exponentially growing and stationary-phase, umuDC-proficient and -deficient, Escherichia coli dnaQ49

Spontaneous mutations arise not only in exponentially growing bacteria but also in non-dividing or slowly dividing stationary-phase cells. In the latter case mutations are called adaptive or stationary-phase mutations. High spontaneous mutability has been observed in temperature sensitive Escherichia coli dnaQ49 strain deficient in 3'-->5' proofreading activity assured by the e subunit of the main replicative polymerase, Pol III. The aim of this study was to evaluate the effects of the dnaQ49 mutation and deletion of the umuDC operon encoding polymerase V (Pol V) on spontaneous mutagenesis in growing and stationary-phase E. coli cells. Using the argE3(OC) -->Arg+ reversion system in the AB1157 strain, we found that the level of growth-dependent and stationary-phase Arg+ revertants was significantly increased in the dnaQ49 mutant at the non-permissive temperature of 37 degrees C. At this temperature, in contrast to cultures grown at 28 degrees C, SOS functions were dramatically increased. Deletion of the umuDC operon in the dnaQ49 strain led to a 10-fold decrease in the level of Arg+ revertants in cultures grown at 37 degrees C and only to a 2-fold decrease in cultures grown at 28 degrees C. Furthermore, in stationary-phase cultures Pol V influenced spontaneous mutagenesis to a much lesser extent than in growing cultures. Our results indicate that the level of Pol III desintegration, dependent on the temperature of incubation, is more critical for spontaneous mutagenesis in stationary-phase dnaQ49 cells than the presence or absence of Pol V.     

Levels of the Vsr Endonuclease Do Not Regulate Stationary-Phase Reversion of a Lac− Frameshift Allele in Escherichia coli

Vsr endonuclease, which initiates very short patch repair, has been hypothesized to regulate mutation in stationary-phase cells. Overexpression of Vsr does dramatically increase the stationary-phase reversion of a Lac⁻ frameshift allele, but the absence of Vsr has no effect. Thus, at least in this case, Vsr has no regulatory role in stationary-phase mutation, and the effects of Vsr overproduction are likely to be artifactual.     

Interaction between the mismatch repair and nucleotide excision repair pathways in the prevention of 5-azacytidine-induced CG-to-GC mutations in Escherichia coli

5-Azacytidine induces CG-to-GC transversion mutations in Escherichia coli. The results presented in this paper provide evidence that repair of the drug-induced lesions that produce these mutations involves components of both the mismatch repair and nucleotide excision repair systems. Strains deficient in mutL, mutS, uvrA, uvrB or uvrC all showed an increase in mutation in response to 5-azacytidine. Using a bacterial two-hybrid assay, we showed that UvrB interacts with MutL and MutS in a drug-dependent manner, while UvrC interacts with MutL independent of drug. We suggest that 5-azacytidine-induced mismatches recruit MutS and MutL, but are poorly processed by mismatch repair. Instead, the stalled MutS–MutL complex recruits the Uvr proteins to complete repair.     

The Escherichia coli histone-like protein HU has a role in stationary phase adaptive mutation

Stationary phase adaptive mutation in Escherichia coli is thought to be a mechanism by which mutation rates are increased during stressful conditions, increasing the possibility that fitness-enhancing mutations arise. Here we present data showing that the histone-like protein, HU, has a role in the molecular pathway by which adaptive Lac(+) mutants arise in E. coli strain FC40. Adaptive Lac(+) mutations are largely but not entirely due to error-prone DNA polymerase IV (Pol IV). Mutations in either of the HU subunits, HUalpha or HUbeta, decrease adaptive mutation to Lac(+) by both Pol IV-dependent and Pol IV-independent pathways. Additionally, HU mutations inhibit growth-dependent mutations without a reduction in the level of Pol IV. These effects of HU mutations on adaptive mutation and on growth-dependent mutations reveal novel functions for HU in mutagenesis.     

The endonuclease domain of MutL interacts with the β sliding clamp

Mismatch repair corrects errors that have escaped polymerase proofreading enhancing replication fidelity by at least two orders of magnitude. The β and PCNA sliding clamps increase the polymerase processivity during DNA replication and are important at several stages of mismatch repair. Both MutS and MutL, the two proteins that initiate the mismatch repair response, interact with β. Binding of MutS to β is important to recruit MutS and MutL to foci. Moreover, the endonuclease activity of human and yeast MutLα is stimulated by PCNA. However, the concrete functions of the processivity clamp in the repair steps preceding DNA resynthesis remain obscure. Here, we demonstrate that the C-terminal domain of MutL encompasses a bona fide β-binding motif that mediates a weak, yet specific, interaction between the two proteins. Mutation of this conserved motif correlates with defects in mismatch repair, demonstrating that the direct interaction with β is important for MutL function. The interaction between the C-terminal domain of MutL and β is conserved in both Bacillus subtilis and Escherichia coli, but the repair defects associated with mutation of this β-binding motif are more severe in the former, suggesting that this interaction may have a more prominent role in methyl-independent than methyl-directed mismatch repair systems. Together with previously published data, our work strongly suggests that β may stimulate the endonuclease activity of MutL through its direct interaction with the C-terminal domain of MutL.     

Conditional DNA repair mutants enable highly precise genome engineering

Oligonucleotide-mediated multiplex genome engineering is an important tool for bacterial genome editing. The efficient application of this technique requires the inactivation of the endogenous methyl-directed mismatch repair system that in turn leads to a drastically elevated genomic mutation rate and the consequent accumulation of undesired off-target mutations. Here, we present a novel strategy for mismatch repair evasion using temperature-sensitive DNA repair mutants and temporal inactivation of the mismatch repair protein complex in Escherichia coli. Our method relies on the transient suppression of DNA repair during mismatch carrying oligonucleotide integration. Using temperature-sensitive control of methyl-directed mismatch repair protein activity during multiplex genome engineering, we reduced the number of off-target mutations by 85%, concurrently maintaining highly efficient and unbiased allelic replacement.     

The effect of mutation him1 characterized by enhanced induced mutagenesis on the genetic effects of 6-N-hydroxylaminopurine in Saccharomyces cerevisiae

The him1 mutation has been shown to influence the genetic effects of the mutagenic purine base analog 6-hydroxylaminopurine, i. e. inactivation of haploid cells, mutation induction, and inhibition of DNA synthesis in vivo. The influence observed is well consistent with the idea that the him1 mutation affects mismatch correction. We present evidence that during in vivo DNA replication 6-hydroxylaminopurine incorporates into the yeast DNA.