Effect of base pair mismatches on recombination via the RecBCD pathway

Summary The effect of base pair mismatches on recombination via the RecBCD pathway was studied in mutS and wild-type Escherichia coli, using substrates that contain single or multiple mismatches. Recombination between homologous DNA inserts in lambda phage and pBR322-derived plasmids forms phage-plasmid cointegrates that result from an odd number of crossovers. In the mutS host, when the sequence homology of a pair of 405 bp substrates decreased from 100% to 89%, the recombinant frequency decreased by about 9-fold, while in the wild-type host the decrease was about 240-fold. These results suggest that multiple mismatches can reduce recombinant frequencies by impeding the mechanism of recombination itself, and by provoking mismatch repair. Single mismatches in 31 bp substrates caused reductions in recombinant frequencies of 2-or 12-fold, depending on the location of the mismatch. However, unlike the reduction by multiple mismatches, the reduction of the recombinant frequencies by single mismatches was the same in both mutS and wild-type hosts. Thus a single match repair seems unable to act on single mismatches in very short homologies during recombination.     

Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene

The mismatch repair (MMR) system is one of the key molecular devices that prokaryotic cells have for ensuring fidelity of DNA replication. While the canonical MMR of E. coli involves 3 proteins (encoded by mutS, mutL and mutH), the soil bacterium Pseudomonads putida has only 2 bona fide homologues (mutS and mutL) and the sensitivity of this abridged system to different types of mismatches is unknown. In this background, sensitivity to MMR of this bacterium was inspected through single stranded (ss) DNA recombineering of the pyrF gene (the prokaryotic equivalent to yeast's URA3) with mutagenic oligos representative of every possible mispairing under either wild-type conditions, permanent deletion of mutS or transient loss of mutL activity (brought about by the thermoinducible dominant negative allele mutL_E36K). Analysis of single nucleotide mutations borne by clones resistant to fluoroorotic acid (5FOA, the target of wild type PyrF) pinpointed prohibited and tolerated single-nucleotide replacements and exposed a clear grading of mismatch recognition. The resulting data unequivocally established the hierarchy A:G < C:C < G:A < C:A, A:A, G:G, T:T, T:G, A:C, C:T < G:T, T:C as the one prevalent in Pseudomonas putida. This information is vital for enabling recombineering strategies aimed at single-nucleotide changes in this biotechnologically important species.     

The yeast gene MSH3 defines a new class of eukaryotic MutS homologues

We have identified a gene in Saccharomyces cerevisiae, MSH3, whose predicted protein product shares extensive sequence similarity with bacterial proteins involved in DNA mismatch repair as well as with the predicted protein product of the Rep-3 gene of mouse. MSH3 was obtained by performing a polymerase chain reaction on yeast genomic DNA using degenerate oligonucleotide primers designed to anneal with the most conserved regions of a gene that would be homologous to Rep-3 and Salmonella typhimurium mutS. MSH3 seems to play some role in DNA mismatch repair, inasmuch as its inactivation results in an increase in reversion rates of two different mutations and also causes an increase in postmeiotic segregation. However, the effect of MSH3 disruption on reversion rates and postmeiotic segregation appears to be much less than that of previously characterized yeast DNA mismatch repair genes. Alignment of the MSH3 sequence with all of the known MutS homologues suggests that its primary function may be different from the role of MutS in repair of replication errors. MSH3 appears to be more closely related to the mouse Rep-3 gene and other similar eukaryotic mutS homologues than to the yeast gene MSH2 and other mutS homologues that are involved in replication repair. We suggest that the primary function of MSH3 may be more closely related to one of the other known functions of mutS, such as its role in preventing recombination between non-identical sequences.     

甲基营养菌MB200中mutS的缺失及高浓度甲醇和甲醛诱变

【背景】甲基营养菌(Methylobacterium)是一类能够以单碳或非C-C键低碳化合物(如甲烷、甲醇、甲醛等)为底物生长,并可生产多种代谢产物如氨基酸、工业酶和辅助因子、多羟基烷酸酯(polyhydroxyalkanoates,PHA)、多糖和类胡萝卜素等的革兰氏阴性细菌。【目的】通过突变甲基营养菌MB200的mutS基因,在胁迫条件下定向诱导,以获得可以耐受高浓度甲醇和甲醛的生产菌株。【方法】利用三亲本结合构建mutS基因缺失的高突变菌株MB200sTB,逐步提升培养液中甲醇、甲醛的浓度进行定向诱导突变,对获得的高耐受性突变株进行回补,分析菌株的生长情况。【结果】构建了mutS基因的缺失突变体MB200sTB,并且得到了高耐受甲醇和甲醛的菌株MB200sHBc和MB200sHBq。MB200sHBc与野生株MB200相比其甲醇耐受性得到了极显著的提高,甲醇耐受浓度从8g/L提升到44g/L,但生长量不受影响。MB200sHBq在以甲醛为0.45g/L的碳源条件下,生长量相较于野生型MB200提高了1.69倍。【结论】通过定向诱导缺失mutS基因的突变体,可获得具有生产应用潜力的...     

Separation of mutation avoidance and antirecombination functions in an Escherichia coli mutS mutant

DNA mismatch repair in Escherichia coli has been shown to be involved in two distinct processes: mutation avoidance, which removes potential mutations arising as replication errors, and antirecombination which prevents recombination between related, but not identical (homeologous), DNA sequences. We show that cells with the mutSΔ800 mutation (which removes the C-terminal 53 amino acids of MutS) on a multicopy plasmid are proficient for mutation avoidance. In interspecies genetic crosses, however, recipients with the mutSΔ800 mutation show increased recombination by up to 280-fold relative to mutS⁺. The MutSΔ800 protein binds to O⁶-methylguanine mismatches but not to intrastrand platinated GG cross-links, explaining why dam bacteria with the mutSΔ800 mutation are resistant to cisplatin, but not MNNG, toxicity. The results indicate that the C-terminal end of MutS is necessary for antirecombination and cisplatin sensitization, but less significant for mutation avoidance. The inability of MutSΔ800 to form tetramers may indicate that these are the active form of MutS.     

Mutations in the mutY gene of Escherichia coli enhance the frequency of targeted G:C → T:A transversions induced by a single 8-oxoguanine residue in single-stranded DNA

Oxidative damage to guanine in DNA results in the formation of 8-oxoguanine, which has been shown to induce G → T transversions targeted to this site. The mutagenicity of this lesion was studied in several mutator strains of Escherichia coli, using single-stranded DNA containing a single 8-oxoguanine residue. The frequencies of targeted G → T transversions increased markedly in mutY strains, while this mutagenic event was not affected in mutM or mutS strains. Introdution of a mutM mutation into a mutY strain caused a somewhat higher frequency of G → T transversions than that in the mutY strain and the effect of a mutS mutation was marginal. We conclude that the mutY gene plays a crucial role in preventing targeted G → T mutations derived from misreplication of the 8-oxoguanine-containing template DNA.     

New functional sites in MutS affect DNA mismatch repair

The MutS protein plays an important role in the DNA mismatch repair system. Mutations in the mutS gene can lead to genome instability and ultimately cell malfunction. Here we have established a method for identifying functional defective mutants of MutS by random mutation and rifampicin screening. Some novel functional sites in MutS were identified. The MutS mutant strains were analyzed using surface plasmon resonance, gel filtration and far-western methods to determine the molecular mechanisms behind the DNA mismatch repair function of MutS.     

The yeast gene MSH3 defines a new class of eukaryotic MutS homologues

We have identified a gene in Saccharomyces cerevisiae, MSH3, whose predicted protein product shares extensive sequence similarity with bacterial proteins involved in DNA mismatch repair as well as with the predicted protein product of the Rep-3 gene of mouse. MSH3 was obtained by performing a polymerase chain reaction on yeast genomic DNA using degenerate oligonucleotide primers designed to anneal with the most conserved regions of a gene that would be homologous to Rep-3 and Salmonella typhimurium mutS. MSH3 seems to play some role in DNA mismatch repair, inasmuch as its inactivation results in an increase in reversion rates of two different mutations and also causes an increase in postmeiotic segregation. However, the effect of MSH3 disruption on reversion rates and postmeiotic segregation appears to be much less than that of previously characterized yeast DNA mismatch repair genes. Alignment of the MSH3 sequence with all of the known MutS homologues suggests that its primary function may be different from the role of MutS in repair of replication errors. MSH3 appears to be more closely related to the mouse Rep-3 gene and other similar eukaryotic mutS homologues than to the yeast gene MSH2 and other mutS homologues that are involved in replication repair. We suggest that the primary function of MSH3 may be more closely related to one of the other known functions of mutS, such as its role in preventing recombination between non-identical sequences.     

Frameshift lesions induced by oxazolopyridocarbazoles are recognized by the mismatch repair system in Escherichia coli

The simple reversible intercalating agent isopropyl-OPC (iPr-OPC) induces frameshift-1 mutations in Salmonella typhimurium and Escherichia coli. The mutagenic responses of S. typhimurium and E. coli wild-type strains are not proportional to the amount of drug intercalated into double-stranded nucleic acids in living bacteria; it occurs only above a minimum level of binding. The fact that mismatch-repair-deficient (mutS) as well as adenine-methylation-deficient (dam) E. coli mutants are hypermutable at low concentrations of iPr-OPC suggests that the majority of mutants induced by this intercalating drug occur as mismatch-repairable mutations (or lesions) in the newly synthesized DNA strand close to the replication fork.     

Mismatch repair ensures fidelity of replication and recombination in the radioresistant organism<Emphasis Type="Italic"> Deinococcus radiodurans</Emphasis>

We have characterized the mismatch repair system (MMR) of the highly radiation-resistant type strain of Deinococcus radiodurans, ATCC 13939. We show that the MMR system is functional in this organism, where it participates in ensuring the fidelity of DNA replication and recombination. The system relies on the activity of two key proteins, MutS1 and MutL, which constitute a conserved core involved in mismatch recognition. Inactivation of MutS1 or MutL resulted in a seven-fold increase in the frequency of spontaneous Rif^R mutagenesis and a ten-fold increase in the efficiency of integration of a donor point-mutation marker during bacterial transformation. Inactivation of the mismatch repair-associated UvrD helicase increased the level of spontaneous mutagenesis, but had no effect on marker integration—suggesting that binding of MutS1 and MutL proteins to a mismatched heteroduplex suffices to inhibit recombination between non identical (homeologous) DNAs. In contrast, inactivation of MutS2, encoded by the second mutS -related gene present in D. radiodurans, had no effect on mutagenesis or recombination. Cells devoid of MutS1 or MutL proteins were as resistant to -rays, mitomycin C and UV-irradiation as wild-type bacteria, suggesting that the mismatch repair system is not essential for the reconstitution of a functional genome after DNA damage.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. Baldacci     

Mutator mutations in Escherichia coli induced by the insertionof phage Mu and the transposable resistance elements Tn5 and Tn10

Mutator mutations in the mutS gene induced by the insertion of phage Mu or the transposable resistance elements Tn5 or Tn10 and those in the mutL gene induced by Tn5 and T10 gave mutagenic activities similar to that of the previously described mutS3 and mutL25 mutations. Various combinations of mutS::Tn5,mutL::Tn5, uvrE156, and the deletion mutation δmutH2 did not produce an additive effect. This supports the idea that the products of these genes function in the same pathway of error correction during DNA synthesis.     

Papillation in Bacillus anthracis colonies: a tool for finding new mutators

Colonies of Bacillus anthracis Sterne allow the growth of papillation after 6 days of incubation at 30°C on Luria–Bertani medium. The papillae are due to mutations that allow the cells to overcome the barriers to continued growth. Cells isolated from papillae display two distinct gross phenotypes (group A and group B). We determined that group A mutants have mutations in the nprR gene including frameshifts, deletions, duplications and base substitutions. We used papillation as a tool for finding new mutators as the mutators generate elevated levels of papillation. We discovered that disruption of yycJ or recJ leads to a spontaneous mutator phenotype. We defined the nprR/papillation system as a new mutational analysis system for B. anthracis. The mutational specificity of the new mutator yycJ is similar to that of mismatch repair‐deficient strains (MMR^‐) such as those with mutations in mutL or mutS. Deficiency in recJ results in a unique specificity, generating only tandem duplications.     

The maxillary palp of Drosophila : ultrastructure and physiology depends on the lozenge gene

The ultrastructure and physiology of the maxillary palp of Drosophila melanogaster have been studied in wild-type and lozenge mutants. Olfactory physiology in the maxillary palp is shown to depend upon the lozenge(lz) gene. Reduced response amplitudes were recorded for all odorants tested, and the physiological defect was shown to map to the lz locus. The structure of the maxillary palp sensilla is described by scanning electron microscopy (SEM) at high magnification, initially in the wild-type. A linear arrangement of pores, connected by furrows, was found in one class of sensilla, the basiconic sensilla. In the lz ³ mutant, morphological alterations in the basiconic sensilla and duplications of sensilla are documented by SEM. The correlation of structural abnormalities in the lz sensilla and physiological abnormalities in odorant response are consistent with an olfactory role for the basiconic sensilla of the maxillary palp. Accepted: 10 September 1996     

Inefficient mismatch repair: genetic defects and down regulation

The mismatch repair system is involved in the maintenance of genomic integrity by editing DNA replication and recombination. However, although most mutations are neutral or deleterious, a mutator phenotype due to an inefficient mismatch repair may generate advantageous variants and may therefore be selected for. We review the evidence for inefficient mismatch repair due either to genetic defects in mismatch repair genes or to physiological conditions. Among natural isolates ofEscherichia coli andSalmonella enterica, about 1% are mutator bacteria, mostly deficient in mismatch repair (most of them defective in themutS gene). Characterization of mutators derived from laboratory strains led also to the isolation of mismatch repair mutants in which the most frequently found defects are inmutL andmutS. The correlation of the size of the antimutator genes with the frequency of their defective alleles amongE. coli andSalmonella strains reveals thatmutU mutants are underrepresented. Analysis of the progeny of a defined M13 phage heteroduplex DNA transfected intoE. coli cells shows that mismatch repair efficiency progressively decreases from the end of the exponential growth in K-12 and is variable among natural isolates. Implications of this defective mismatch repair activity for evolution and tumorigenesis will be discussed.     

Mutational analysis of the arginine repressor of Escherichia coli

Arginine biosynthesis in Escherichia coli is negatively regulated by a hexameric repressor protein, encoded by the gene argR and the corepressor arginine. By hydroxylamine mutagenesis two types of argR mutants were isolated and mapped. The first type is transdominant. In heterodiploids, these mutant polypeptides reduce the activity of the wild-type repressor, presumably by forming heteropolymers. Four mutant repressor proteins were purified. Two of these map in the N-terminal half of the protein. Gel retardation experiments showed that they bind poorly to DNA, but they could be precipitated by l -arginine at the same concentration as the wild-type repressor. The other two mutant repressors map in the C-terminal half of the protein. They are poorly precipitated by L-arginine and they bind poorly to DNA. In addition, one of these mutants appears to exist as a dimer. The second type of argR mutant repressor consists of super-repressors. Such mutants behave as arginine auxotrophs as a result of hyper-repression of arginine biosynthetic enzymes. They map at many locations throughout the argR gene. Three arginine super-repressor proteins were purified, in comparison with the wild-type repressor, two of them were shown to have a higher DNA-binding affinity in the absence of bound arginine, while the third was shown to have a higher DNA-binding affinity when bound to arginine.     

A mutation with pleiotropic effects on macronuclearly differentiated functions in Paramecium tetraurelia

The mtF^E mutation isolated in Paramecium tetraurelia affects mating type differentiation, trichocyst excretion, and viability. Its effect on mating type has already been shown to correspond to a restriction to the E mating type interpreted by an inefficiency of nuclear O-determining factors. In this paper we study the other two phenotypic characteristics whose hereditary transmission displays two unusual features. (1) In crosses between a wild-type strain and the mutant strain, the mutant characteristics do not reappear in F2 in the wild-type cytoplasmic lineage but only in F3 after the homozygous clones have undergone an additional nuclear reorganization. (2) Some F2 wild-type clones, in the mutant cytoplasmic lineage, retain some of the phenotypic characteristics of the mutant. We propose that the mtF gene product plays a role in the control of several macronuclearly differentiated functions.     

Inactivation of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) improves its virulence towards its insect host

Some baculovirus have been genetically modified for the inactivation of their ecdysteroid glucosyltransferase (egt) gene, and these viruses were shown to kill infected larvae more rapidly when compared to wild-type virus infections. We have previously identified, cloned, and sequenced the egt gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV). Here we present data regarding the construction of an egt minus (egt−) AgMNPV and its virulence towards its insect host. We have inserted an hsp70-lacZ (3.7 kb) gene cassette into the egt gene open reading frame (ORF) and purified a recombinant AgMNPV (vAgEGTΔ-lacZ). Bioassays with third-instar A. gemmatalis larvae showed that viral occlusion body (OB) production were consistently lower from infections with vAgEGTΔ-lacZ compared to the wild-type virus. A mean of 20.4×10⁸ OBs/g/larva and 40.7×10⁸ OBs/g/larva was produced from vAgEGTΔ-lacZ and AgMNPV infections, respectively. The mean lethal concentration which killed 50% of insects in a treatment group (LC₅₀) for the 10th day after virus treatment (DAT) was 3.9-fold higher for the wild-type virus compared to vAgEGTΔ-lacZ. The recombinant virus killed A. gemmatalis larvae significantly faster (ca. 1–2.8 days), than the wild-type AgMNPV. Therefore, the vAgEGTΔ-lacZ was more efficacious for the control of A. gemmatalis larvae (in bioassays) compared to wild-type AgMNPV.     

Isolation and Characterization of Carotenosomes from a Bacteriochlorophyll c-less Mutant ofChlorobium tepidum

Chlorosomes are the light-harvesting organelles in photosynthetic green bacteria and typically contain large amounts of bacteriochlorophyll (BChl) c in addition to smaller amounts of BChl a, carotenoids, and several protein species. We have isolated vestigial chlorosomes, denoted carotenosomes, from a BChl c-less, bchK mutant of the green sulfur bacterium Chlorobium tepidum. The physical shape of the carotenosomes (86 ± 17 nm × 66 ± 13 nm × 4.3 ± 0.8 nm on average) was reminiscent of a flattened chlorosome. The carotenosomes contained carotenoids, BChl a, and the proteins CsmA and CsmD in ratios to each other comparable to their ratios in wild-type chlorosomes, but all other chlorosome proteins normally found in wild-type chlorosomes were found only in trace amounts or were not detected. Similar to wild-type chlorosomes, the CsmA protein in the carotenosomes formed oligomers at least up to homo-octamers as shown by chemical cross-linking and immunoblotting. The absorption spectrum of BChl a in the carotenosomes was also indistinguishable from that in wild-type chlorosomes. Energy transfer from the bulk carotenoids to BChl a in carotenosomes was poor. The results indicate that the carotenosomes have an intact baseplate made of remarkably stable oligomeric CsmA–BChl a complexes but are flattened in structure due to the absence of BChl c. Carotenosomes thus provide a valuable material for studying the biogenesis, structure, and function of the photosynthetic antennae in green bacteria.