Nucleotide sequence of the Salmonella typhimurium mutL gene required for mismatch repair: homology of MutL to HexB of Streptococcus pneumoniae and to PMS1 of the yeast Saccharomyces cerevisiae.

The mutL gene of Salmonella typhimurium LT2 is required for dam-dependent methyl-directed DNA mismatch repair. We have cloned and sequenced the mutL gene of S. typhimurium LT2 and compared its sequence with those of the hexB gene product of the gram-positive bacterium Streptococcus pneumoniae and the PMS1 gene product of the yeast Saccharomyces cerevisiae. MutL was found to be quite similar to the HexB mismatch repair protein of S. pneumoniae and to the mismatch repair protein PMS1 of the yeast S. cerevisiae. The significant similarities among these proteins were confined to their amino-terminal regions and suggest common evolution of the mismatch repair machinery in those organisms. The DNA sequence for mutL predicted a gene encoding a protein of 618 amino acid residues with a molecular weight of 67,761. The assignment of reading frame was confirmed by the construction of a chimeric protein consisting of the first 30 amino acids of LacZ fused to residues 53 through 618 of MutL. Interestingly, the presence of excess amounts of this fusion protein in wild-type mutL+ cells resulted in a trans-dominant effect causing the cell to exhibit a high spontaneous mutation frequency.     

Mismatch repair genes of Streptococcus pneumoniae: HexA confers a mutator phenotype in Escherichia coli by negative complementation.

DNA repair systems able to correct base pair mismatches within newly replicated DNA or within heteroduplex molecules produced during recombination are widespread among living organisms. Evidence that such generalized mismatch repair systems evolved from a common ancestor is particularly strong for two of them, the Hex system of the gram-positive Streptococcus pneumoniae and the Mut system of the gram-negative Escherichia coli and Salmonella typhimurium. The homology existing between HexA and MutS and between HexB and MutL prompted us to investigate the effect of expressing hex genes in E. coli. Complementation of mutS or mutL mutations, which confer a mutator phenotype, was assayed by introducing on a multicopy plasmid the hexA and hexB genes, under the control of an inducible promoter, either individually or together in E. coli strains. No decrease in mutation rate was conferred by either hexA or hexB gene expression. However, a negative complementation effect was observed in wild-type E. coli cells: expression of hexA resulted in a typical Mut- mutator phenotype. hexB gene expression did not increase the mutation rate either individually or in conjunction with hexA. Since expression of hexA did not affect the mutation rate in mutS mutant cells and the hexA-induced mutator effect was recA independent, it is concluded that this effect results from inhibition of the Mut system. We suggest that HexA, like its homolog MutS, binds to mismatches resulting from replication errors, but in doing so it protects them from repair by the Mut system. In agreement with this hypothesis, an increase in mutS gene copy number abolished the hexA-induced mutator phenotype. HexA protein could prevent repair either by being unable to interact with Mut proteins or by producing nonfunctional repair complexes.     

Cloning and nucleotide sequence of DNA mismatch repair gene PMS1 from Saccharomyces cerevisiae: homology of PMS1 to procaryotic MutL and HexB.

The PMS1 gene from Saccharomyces cerevisiae, implicated in DNA mismatch repair in yeast cells (M. S. Williamson, J. C. Game, and S. Fogel, Genetics 110:609-646, 1985), was cloned, and the nucleotide sequence was determined. The nucleotide sequence showed a 2,712-base-pair open reading frame; the predicted molecular mass of the deduced protein is 103 kilodaltons. Deletion mutants of the open reading frame were constructed and genetically characterized. The deduced amino acid sequence of the PMS1 gene exhibited homology to those of the mutL gene from Salmonella typhimurium and the hexB gene from Streptococcus pneumoniae, genes required for DNA mismatch repair in these organisms. The homology suggests an evolutionary relationship of DNA mismatch repair in procaryotes and eucaryotes.     

Heteroduplex DNA mismatch repair system of Streptococcus pneumoniae: cloning and expression of the hexA gene.

Mutations affecting heteroduplex DNA mismatch repair in Streptococcus pneumoniae were localized in two genes, hexA and hexB, by fractionation of restriction fragments carrying mutant alleles. A fragment containing the hexA4 allele was cloned in the S. pneumoniae cloning system, and the hexA+ allele was introduced into the recombinant plasmid by chromosomal facilitation of plasmid transfer. Subcloning localized the functional hexA gene to a 3.5-kilobase segment of the cloned pneumococcal DNA. The product of this gene was shown in Bacillus subtilis minicells to be a polypeptide with an Mr of 86,000. Two mutant alleles of hexA showed partial expression of the repair system when present in multicopy plasmids. A model for mismatch repair, which depends on the interaction of two protein components to recognize the mismatched base pair and excise a segment of DNA between strand breaks surrounding the mismatch, is proposed.     

Molecular cloning, characterization, and nucleotide sequence of an extracellular amylase gene from Aeromonas hydrophila.

The structural gene for excreted amylase from Aeromonas hydrophila JMP636 has been cloned within a 2.1-kilobase SmaI fragment of DNA. The amylase gene is transcribed from its own promoter in Escherichia coli, producing a gene product of Mr 49,000. The amylase gene product is secreted to the periplasm of E. coli; however, it is not excreted. Nucleotide sequencing revealed an open reading frame of 1,392 base pairs corresponding to a protein of 464 amino acid residues. A potential signal peptide of 21 amino acid residues is present at the NH2 terminal of the predicted protein. Three regions of homology with other procaryotic and eucaryotic alpha-amylases were detected within the predicted amino acid sequence.     

Dual requirement in yeast DNA mismatch repair for MLH1 and PMS1, two homologs of the bacterial mutL gene.

We have identified a new Saccharomyces cerevisiae gene, MLH1 (mutL homolog), that encodes a predicted protein product with sequence similarity to DNA mismatch repair proteins of bacteria (MutL and HexB) and S. cerevisiae yeast (PMS1). Disruption of the MLH1 gene results in elevated spontaneous mutation rates during vegetative growth as measured by forward mutation to canavanine resistance and reversion of the hom3-10 allele. Additionally, the mlh1 delta mutant displays a dramatic increase in the instability of simple sequence repeats, i.e., (GT)n (M. Strand, T. A. Prolla, R. M. Liskay, and T. D. Petes, Nature [London] 365:274-276, 1993). Meiotic studies indicate that disruption of the MLH1 gene in diploid strains causes increased spore lethality, presumably due to the accumulation of recessive lethal mutations, and increased postmeiotic segregation at each of four loci, the latter being indicative of inefficient repair of heteroduplex DNA generated during genetic recombination. mlh1 delta mutants, which should represent the null phenotype, show the same mutator and meiotic phenotypes as isogenic pms1 delta mutants. Interestingly, mutator and meiotic phenotypes of the mlh1 delta pms1 delta double mutant are indistinguishable from those of the mlh1 delta and pms1 delta single mutants. On the basis of our data, we suggest that in contrast to Escherichia coli, there are two MutL/HexB-like proteins in S. cerevisiae and that each is a required component of the same DNA mismatch repair pathway.     

Uracil-DNA glycosylase affects mismatch repair efficiency in transformation and bisulfite-induced mutagenesis in Streptococcus pneumoniae.

The generalized mismatch repair system of Streptococcus pneumoniae (the Hex system) can eliminate base pair mismatches arising in heteroduplex DNA during transformation or by DNA polymerase errors during replication. Mismatch repair is most likely initiated at nicks or gaps. The present work was started to examine the hypothesis that strand discontinuities arising after removal of uracil by uracil DNA-glycosylase (Ung) can be utilised as strand discrimination signals. We show that mismatch repair efficiency is enhanced 3- to 6-fold when using uracil-containing DNA as donor in transformation. In order to assess the contribution of Ung to nascent strand discrimination for postreplication mismatch repair, we developed a positive selection procedure to isolate S. pneumoniae Ung- mutants. We succeeded in isolating Ung- mutants using this procedure based on chromosomal integration of uracil-containing hybrid DNA molecules. Cloning and characterization of the ung gene was achieved. Comparison of spontaneous mutation rates in strains either proficient or deficient in mismatch and/or uracil repair gave no support to the hypothesis that Ung plays a major role in targeting the Hex system to neosynthesized DNA strands. However Ung activity is responsible for the increased efficiency of mismatch repair observed in transformation with uracil-containing DNA. In addition Ung is involved in repair of bisulfite-treated transforming DNA.     

Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.     

Hex1: a new human Rad2 nuclease family member with homology to yeast exonuclease 1.

Nucleolytic processing of chromosomal DNA is required in operations such as DNA repair, recombination and replication. We have identified a human gene, named HEX1 forhumanexonuclease 1, by searching the EST database for cDNAs that encode a homolog to the Saccharomyces cerevisiae EXO1 gene product. Based on its homology to this and other DNA repair proteins of the Rad2 family, most notably Schizosaccharomyces pombe exonuclease 1 (Exo1), Hex1 presumably functions as a nuclease in aspects of recombination or mismatch repair. Similar to the yeast proteins, recombinant Hex1 exhibits a 5'-->3' exonuclease activity. Northern blot analysis revealed that HEX1 expression is highest in fetal liver and adult bone marrow, suggesting that the encoded protein may operate prominently in processes specific to hemopoietic stem cell development. HEX1 gene equivalents were found in all vertebrates examined. The human gene includes 14 exons and 13 introns that span approximately 42 kb of genomic DNA and maps to the chromosomal position 1q42-43, a region lost in some cases of acute leukemia and in several solid tumors.     

Endonucleolytic function of MutLalpha in human mismatch repair

Half of hereditary nonpolyposis colon cancer kindreds harbor mutations that inactivate MutLalpha (MLH1*PMS2 heterodimer). MutLalpha is required for mismatch repair, but its function in this process is unclear. We show that human MutLalpha is a latent endonuclease that is activated in a mismatch-, MutSalpha-, RFC-, PCNA-, and ATP-dependent manner. Incision of a nicked mismatch-containing DNA heteroduplex by this four-protein system is strongly biased to the nicked strand. A mismatch-containing DNA segment spanned by two strand breaks is removed by the 5'-to-3' activity of MutSalpha-activated exonuclease I. The probable endonuclease active site has been localized to a PMS2 DQHA(X)(2)E(X)(4)E motif. This motif is conserved in eukaryotic PMS2 homologs and in MutL proteins from a number of bacterial species but is lacking in MutL proteins from bacteria that rely on d(GATC) methylation for strand discrimination in mismatch repair. Therefore, the mode of excision initiation may differ in these organisms.     

Nucleotide sequence of the Salmonella typhimurium mutS gene required for mismatch repair: homology of MutS and HexA of Streptococcus pneumoniae.

The mutS gene product of Escherichia coli and Salmonella typhimurium is one of at least four proteins required for methyl-directed mismatch repair in these organisms. A functionally similar repair system in Streptococcus pneumoniae requires the hex genes. We have sequenced the S. typhimurium mutS gene, showing that it encodes a 96-kilodalton protein. Amino-terminal amino acid sequencing of purified S. typhimurium MutS protein confirmed the initial portion of the deduced amino acid sequence. The S. typhimurium MutS protein is homologous to the S. pneumoniae HexA protein, suggesting that they arose from a common ancestor before the gram-negative and gram-positive bacteria diverged. Overall, approximately 36% of the amino acids of the two proteins are identical when the sequences are optimally aligned, including regions of stronger homology which are of particular interest. One such region is close to the amino terminus. Another, located closer to the carboxy terminus, includes homology to a consensus sequence thought to be diagnostic of nucleotide-binding sites. A third one, adjacent to the second, is homologous to the consensus sequence for the helix-turn-helix motif found in many DNA-binding proteins. We found that the S. typhimurium MutS protein can substitute for the E. coli MutS protein in vitro as it can in vivo, but we have not yet been able to demonstrate a similar in vitro complementation by the S. pneumoniae HexA protein.     

Separation-of-Function Mutations in Saccharomyces cerevisiae MSH2 That Confer Mismatch Repair Defects but Do Not Affect Nonhomologous-Tail Removal during Recombination

Yeast Msh2p forms complexes with Msh3p and Msh6p to repair DNA mispairs that arise during DNA replication. In addition to their role in mismatch repair (MMR), the MSH2 and MSH3 gene products are required to remove 3' nonhomologous DNA tails during genetic recombination. The mismatch repair genes MSH6, MLH1, and PMS1, whose products interact with Msh2p, are not required in this process. We have identified mutations in MSH2 that do not disrupt genetic recombination but confer a strong defect in mismatch repair. Twenty-four msh2 mutations that conferred a dominant negative phenotype for mismatch repair were isolated. A subset of these mutations mapped to residues in Msh2p that were analogous to mutations identified in human nonpolyposis colorectal cancer msh2 kindreds. Approximately half of the these MMR-defective mutations retained wild-type or nearly wild-type activity for the removal of nonhomologous DNA tails during genetic recombination. The identification of mutations in MSH2 that disrupt mismatch repair without affecting recombination provides a first step in dissecting the Msh-effector protein complexes that are thought to play different roles during DNA repair and genetic recombination.     

Stochastic Processes and Component Plasticity Governing DNA Mismatch Repair

DNA mismatch repair (MMR) is a DNA excision–resynthesis process that principally enhances replication fidelity. Highly conserved MutS (MSH) and MutL (MLH/PMS) homologs initiate MMR and in higher eukaryotes act as DNA damage sensors that can trigger apoptosis. MSH proteins recognize mismatched nucleotides, whereas the MLH/PMS proteins mediate multiple interactions associated with downstream MMR events including strand discrimination and strand-specific excision that are initiated at a significant distance from the mismatch. Remarkably, the biophysical functions of the MLH/PMS proteins have been elusive for decades. Here we consider recent observations that have helped to define the mechanics of MLH/PMS proteins and their role in choreographing MMR. We highlight the stochastic nature of DNA interactions that have been visualized by single-molecule analysis and the plasticity of protein complexes that employ thermal diffusion to complete the progressions of MMR.     

Plant histones. Relevance to the evolution of prokaryotes to eukaryotes

There are many procaryotic and eucaryotic organisms in plant kingdom. It is hoped that the study of plant histones will be useful in evolutionary studies. The histones of great variety of animal species have been studied and well characterized. Less information is available concerning plant histones. The general conclusion drawn from these investigations is that most organisms of eucaryotic plant and animal species contain the same five major histone fractions. Recently the histone-like proteins were found in some primitive eucaryotes and procaryotes. Data on histones from higher and lower eucaryotes and histone-like proteins of procaryotes are reviewed. Evolution of histones and their appearance prior to that of eucaryotic cell is postulated. The role of histones in evolution of nucleosomes is discussed.     

A novel interaction between human DNA polymerase η and MutLα

Human DNA polymerase η (Polη) is the gene product underlying xeroderma pigmentosum variant, and plays principal roles in translesion DNA synthesis. Here, we identified human MLH1, an essential component of mismatch repair (MMR), as a Polη-interacting protein. The middle area residues, which include the little finger domain, of Polη are important for the interaction with MLH1. Polη also interacts with the MLH1/PMS2 heterodimer (MutLα). Co-immunoprecipitation analyses revealed that MutLα, and also MSH2 and MSH6, components of the MutSα heterodimer, form complexes with Polη in human cells. Although MutSα had been reported to interact with C-terminal residues of Polη, MutLα and MutSα co-precipitated with C-terminally truncated Polη, suggesting that MutSα can interact with Polη through MutLα. MMR proteins were more abundant in the Polη complex on the chromatin of S phase-synchronized cells than of asynchronous cells, suggesting that the interaction between Polη and MLH1 is involved in DNA replication.     

The predicted protein product of a pathogenicity locus from Pseudomonas syringae pv. phaseolicola is homologous to a highly conserved domain of several procaryotic regulatory proteins.

A ca. 20-kilobase (kb) region (hrp) that controls the interaction of Pseudomonas syringae pv. phaseolicola with its host (pathogenicity) and nonhost plants (hypersensitive reaction) was previously cloned and partially characterized. In this study we defined the limits and determined the nucleotide sequence of a hrp locus (hrpS), located near the right end of the hrp cluster. The largest open reading frame (ORF302) in hrpS has a coding capacity for a 302-amino-acid polypeptide. The predicted amino acid sequence of the translation product of ORF302 (HrpS) shows significant similarity to several procaryotic regulatory proteins, including the NtrC, NifA, and DctD proteins of Rhizobium spp., the NtrC and NifA proteins of Klebsiella pneumoniae, and the TyrR protein of Escherichia coli. These proteins regulate diverse operons involved in nitrogen fixation, transport and metabolism of amino acids, and transport of C-4 dicarboxylic acids. The HrpS protein appears to be the shortest naturally occurring member of this family of proteins, corresponding for the most part to the highly conserved central domain of these proteins, which contains a putative ATP-binding site. A C-terminal segment analogous to the less-well-conserved domain, involved in DNA binding of NtrC and NifA, is also present in HrpS. These similarities suggest that HrpS is a regulatory protein. In line with this prediction is the finding that a functional hrpS gene is necessary for the activation of another hrp locus during the plant-bacterium interaction.     

Purification, gene cloning, and sequence analysis of an L-isoaspartyl protein carboxyl methyltransferase from Escherichia coli

Mammalian tissues contain protein carboxyl methyltransferases that catalyze the transfer of methyl groups from S-adenosylmethionine to the free carboxyl groups of D-aspartyl or L-isoaspartyl residues (EC 2.1.1.77). These enzymes have been postulated to play a role in the repair and/or degradation of spontaneously damaged proteins. We have now characterized a similar activity from Escherichia coli that recognizes L-isoaspartyl-containing peptides as well as protein substrates such as ovalbumin. The enzyme was purified by DEAE-cellulose, hydroxylapatite, Sephadex G-100, polyaspartate, and reversed-phase chromatography and was shown to consist of a single 24-kDa polypeptide chain. The sequence determined for the N-terminal 39 residues was used to design an oligonucleotide probe that allowed the precise localization of its structural gene (pcm) on the physical map of the E. coli chromosome at 59 min. Transformation of E. coli cells with a plasmid containing DNA from this region results in a 3-4-fold overproduction of enzyme activity. The nucleotide sequence determined for the pcm gene and its flanking regions was used to deduce a mature amino acid sequence of 207 residues with a calculated molecular weight of 23,128. This sequence shows 30.8% sequence identity with the human L-isoaspartyl/D-aspartyl methyltransferase and suggests that this enzyme catalyzes a fundamental reaction in both procaryotic and eucaryotic cells.     

Conservation of structure and location of Rhizobium meliloti and Klebsiella pneumoniae nifB genes.

Using transposon Tn5-mediated mutagenesis, an essential Rhizobium meliloti nitrogen fixation (nif) gene was identified and located directly downstream of the regulatory gene nifA. Maxicell and DNA sequence analysis demonstrated that the new gene is transcribed in the same direction as nifA and codes for a 54-kilodalton protein. In Klebsiella pneumoniae, the nifBQ operon is located directly downstream of a gene which is structurally and functionally homologous to the R. meliloti nifA gene. The DNA sequences of the K. pneumoniae nifB and nifQ genes (which code for 51- and 20-kilodalton proteins, respectively) were determined. The DNA sequence of the newly identified R. meliloti gene was approximately 50% homologous to the K. pneumoniae nifB gene. R. meliloti does not contain a gene homologous to nifQ directly downstream of nifB. The R. meliloti nifB product shares approximately 40% amino acid homology with the K. pneumoniae nifB product, and 10 of the 12 cysteine residues of the R. meliloti nifB product are conserved with 10 of the 17 cysteine residues of the K. pneumoniae nifB product.