Characterization of functional interactions among the Escherichia coli mismatch repair proteins using a bacterial two-hybrid assay.

Vsr mediates very short patch repair in Escherichia coli, correcting T/G mismatches caused by deamination of 5-methylcytosine to thymine. MutS and MutL, part of the post-replication mismatch repair system, stimulate VSP repair. In this study, we use a bacterial two-hybrid assay to show that MutL interacts with Vsr. We also show that interaction between Vsr and MutL inhibits the ability of MutL to dimerize, to interact with MutS and MutH and to mediate a previously unknown interaction between MutS and MutH. This inhibition may explain why high levels of Vsr are mutagenic in vivo. In addition, we show that the Mut fusion proteins are repair proficient in the bacterial two-hybrid assay, making it possible to study their interactions in various genetic backgrounds, or in the presence of DNA damaging agents.

Very-short-patch repair in Escherichia coli requires the dam adenine methylase.

Strains of Escherichia coli which lack the dam-encoded adenine methylase are mutators due to a reduction in the efficiency of postreplication mismatch repair. In this study, we show that Dam(-) strains are also defective in very-short-patch repair, the system which corrects T/G mismatches arising from the deamination of 5-methylcytosine. This defect is associated with decreased levels of Vsr, the endonuclease which initiates short-patch repair. We also show that production of the dcm-encoded cytosine methylase is unaffected in Dam(-) strains. Since the dcm and vsr genes are cotranscribed, the regulation of Vsr by Dam is probably posttranscriptional.

Lowering S-adenosylmethionine levels in Escherichia coli modulates C-to-T transition mutations.

Deoxycytosine methylase (Dcm) enzyme activity causes mutagenesis in vitro either directly by enzyme-induced deamination of cytosine to uracil in the absence of the methyl donor, S-adenosylmethionine (SAM), or indirectly through spontaneous deamination of [5-methyl]cytosine to thymine. Using a Lac reversion assay, we investigated the contribution of the first mechanism to Dcm mutagenesis in vivo by lowering the levels of SAM. Escherichia coli SAM levels were lowered by reducing SAM synthetase activity via the introduction of a metK84 allele or by hydrolyzing SAM using the bacteriophage T3 SAM hydrolase. The metK84 strains exhibited increased C-to-T mutagenesis. Expression of the T3 SAM hydrolase gene, under the control of the arabinose-inducible P(BAD) promoter, effectively reduced Dcm-mediated genomic DNA methylation. However, increased mutagenesis was not observed until extremely high arabinose concentrations were used, and genome methylation at Dcm sites was negligible.

Effect of interaction between 5-azacytidine and DNA (cytosine-5) methyltransferase on C-to-G and C-to-T mutations in Escherichia coli.

The purpose of this study was to determine the effect of the Dcm cytosine methyltransferase on 5-azacytidine (5-azaC) mutagenesis in Escherichia coli. We used a Lac reversion assay to measure C-to-G and C-to-T mutations at a single, methylatable cytosine in the lacZ gene, in the presence and absence of Dcm. C-to-G mutations are stimulated by 5-azaC but are largely independent of Dcm. In contrast, C-to-T mutations are not stimulated by 5-azaC in either wild type or dcm cells. However, in cells which contain Dcm but are defective in very short patch repair, the normally high frequency of spontaneous C-to-T mutations is decreased by the analog in a dose-dependent manner.

Growth phase-dependent regulation of Vsr endonuclease may contribute to 5-methylcytosine mutational hot spots in Escherichia coli.

Using rabbit polyclonal antibodies, we have shown that the Dcm cytosine methylase of Escherichia coli is maintained at a constant level during cell growth, while Vsr endonuclease levels are growth phase dependent. Decreased production of Vsr relative to Dcm during the log phase may contribute substantially to the mutability of 5-methylcytosine.

The Vsr endonuclease of Escherichia coli: an efficient DNA repair enzyme and a potent mutagen.

The Vsr endonuclease of Escherichia coli initiates the repair of T/G mismatches caused by deamination of 5-methylcytosine to thymine. In this paper, we examine the capacity of Vsr to prevent CG-to-TA mutations in cells with increased transcription of the cytosine methylase gene (dcm). We find that sufficient Vsr is produced by a single chromosomal copy of vsr to prevent mutagenesis. We also investigate the cause of the transition and frameshift mutations in cells overproducing Vsr. Neither the absence of the dcm methylase nor its overproduction affects Vsr-stimulated mutagenesis. However, addition of mutS, mutL, or mutH on multicopy plasmids has a significant effect: mutL or mutH decreases the number of mutations, while mutS stimulates mutagenesis. The mut-containing plasmids have the same effect in cells treated with 2-aminopurine and in cells made defective in DNA proofreading, two experimental situations known to cause transition and frameshift mutations by saturating mismatch repair.

Overexpression of vsr in Escherichia coli is mutagenic.

Overexpression of vsr in Escherichia coli stimulates transition and frameshift mutations. The pattern of mutations suggests that mutagenesis is due to saturation or inactivation of dam-directed mismatch repair.

A simple assay for monitoring the mutagenic effects of 5-methylcytosine deamination in Escherichia coli.

We have developed and tested a simple phenotypic assay which monitors C to T transition mutations at the second C of a CCAGG sequence in the lacZ gene of Escherichia coli. The assay is based on new data concerning amino acid requirements on either side of a crucial active site residue in beta-galactosidase, glutamic acid 461. We show that the frequency of occurrence of the mutation is influenced by two genes: dcm, the cytosine methylase gene, and vsr, one of the genes involved in very short patch repair. The assay has been used to evaluate the function of vsr cloned from a potential very short patch repair mutant.

MutY repair is mutagenic in mutT- strains of Escherichia coli.

We have determined the numbers and types of mutations that occur in strains of Escherichia coli defective in mutT and (or) mutY repair. High rates of C.G to A.T mutations in mutY- cells are unaffected by the status of mutT. However, mutT-/mutY+ strains have higher rates of A.T to C.G mutations than mutT-/mutY- strains. This result indicates that the high rates of A.T to C.G mutations seen in mutT- strains of E. coli are due in part to the activity of the mutY repair system. We conclude that mutY repair is mutagenic in a mutT- background.

Isolation and characterization of an Escherichia coli strain with a high frequency of C-to-T mutations at 5-methylcytosines.

We used a genetic selection system to isolate a strain of Escherichia coli with a high frequency of C-to-T transition mutations at the second C of the sequence CCAGG. Cytosines in other sequences do not mutate to thymine at a high frequency in this strain, and the frequencies of other base substitution mutations are not increased to the same extent. The gene responsible for the mutator phenotype has been mapped to 43 min on the E. coli chromosome. Several lines of evidence indicate that this gene is distinct from the very short patch repair gene vsr.

Site specific mutants of beta-galactosidase show that Tyr-503 is unimportant in Mg2+ binding but that Glu-461 is very important and may be a ligand to Mg2+.

The Mg2+ concentrations required for half maximal activity, the dissociation constants, and the free energies of binding for Mg2+ bound to wild type beta-galactosidase and several site specific mutants are reported. The mutants have one of the following substitutions: Glu-461 substituted with Asp, Gln, Gly, His, or Lys; or Tyr-503 substituted with Phe, His or Cys. Substitutions for Tyr-503 had little effect on the affinity of the enzyme for Mg2+, implying that Tyr-503 is not involved in Mg2+ binding. Neutrally charged amino acids substituted for the negatively charged Glu-461 significantly decreased the affinity of the enzyme for Mg2+ and substitution of positively charged amino acids at this position further decreased the affinity. On the other hand, substitution by Asp (negative charge) at position 461 had no effect on the binding. Thus, the negatively charged side chain of Glu-461 is important for divalent cation binding to beta-galactosidase.

A set of lacZ mutations in Escherichia coli that allow rapid detection of specific frameshift mutations.

We have used site-directed mutagenesis to alter bases in lacZ near the region encoding essential residues in the active site of beta-galactosidase. The altered sequences generate runs of six or seven identical base pairs which create a frameshift, resulting in a Lac- phenotype. Reversion to Lac+ in each strain can occur only by a specific frameshift at these sequences. Monotonous runs of A's (or of T's on the opposite strand) and G's (or C's) have been constructed, as has an alternating -C-G- sequence. These specific frameshift indicator strains complement a set of six previously described strains which detect each of the base substitutions. We have examined a variety of mutagens and mutators for their ability to cause reversion to Lac+. Surprisingly, frameshifts are well stimulated at many of these runs by ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-amino-purine, mutagens not widely known to induce frameshifts. A comparison of ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-aminopurine frameshift specificity with that found with a mutH strain suggests that these mutagens partially or fully saturate or inactivate the methylation-directed mismatch repair system and allow replication errors leading to frameshifts to escape repair. This results in a form of indirect mutagenesis, which can be detected at certain sites.

Determination of the roles of Glu-461 in beta-galactosidase (Escherichia coli) using site-specific mutagenesis.

Site-directed substitutions (Asp, Gly, Gln, His, and Lys) were made for Glu-461 of beta-galactosidase (Escherichia coli). All substitutions resulted in loss of most activity. Substrates and a substrate analog inhibitor were bound better by the Asp-substituted enzyme than by the normal enzyme, about the same for enzyme substituted with Gly, but only poorly when Gln, His, or Lys was substituted. This shows that Glu-461 is involved in substrate binding. Binding of the positively charged transition state analog 2-aminogalactose was very much reduced with Gly, Gln, His, and Lys, whereas the Asp-substituted enzyme bound this inhibitor even better than did the wild-type enzyme. Since Asp, like Glu, is negatively charged, this strongly supports the proposal that one role of Glu-461 is to electrostatically interact with a positively charged galactosyl transition state intermediate. The substitutions also affected the ability of the enzyme to bind L-ribose, a planar analog of D-galactose that strongly inhibits beta-galactosidase activity. This indicates that the binding of a planar "galactose-like" compound is somehow mediated through Glu-461. The data indicated that the presence of Glu-461 is highly important for the acid catalytic component of kappa 2 (glycosylic bond cleavage or "galactosylation"), and therefore Glu-461 must be involved in a concerted acid catalytic reaction, presumably by stabilizing a developing carbonium ion. The kappa 2 values with o- and p-nitrophenyl-beta-D-galactopyranoside as substrates varied more or less as did the K8 values, indicating that most of the glycolytic bond breaking activity found for the enzymes from the mutants with these substrates was probably a result of strain or other such effects. The kappa 3 values (hydrolysis or "degalactosylation") of the substituted enzymes were also low, indicating that Glu-461 is important for that part of the catalysis. The enzyme with His substituted for Glu-461 had the highest kappa 3 value. This is probably a result of the formation of a covalent bond between His and the galactosyl part of the substrate.

A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions.

We describe the construction of six strains of Escherichia coli with different mutations at the same coding position in the lacZ gene, which specifies the active site glutamic acid residue at position 461 in beta'-galactosidase. Each strain is Lac- and reverts to Lac+ only by restoring the glutamic acid codon. The strains have been designed so that each reverts via one of the six base substitutions. The set of strains allows detection of each transition and transversion simply by monitoring the Lac- to Lac+ frequency, as demonstrated here with characterized mutagens and mutator alleles. These strains are useful for rapidly determining the mutagenic specificity of mutagens at a single site, for detecting low levels of stimulation of certain base substitutions, for monitoring specific base changes in response to various experimental conditions or strain backgrounds, and for isolating new mutator strains.

Effects of amino acid substitutions at the active site in Escherichia coli beta-galactosidase.

Forty-nine amino acid substitutions were made at four positions in the Escherichia coli enzyme beta-galactosidase; three of the four targeted amino acids are thought to be part of the active site. Many of the substitutions were made by converting the appropriate codon in lacZ to an amber codon, and using one of 12 suppressor strains to introduce the replacement amino acid. Glu-461 and Tyr-503 were replaced, independently, with 13 amino acids. All 26 of the strains containing mutant enzymes are Lac-. Enzyme activity is reduced to less than 10% of wild type by substitutions at Glu-461 and to less than 1% of wild type by substitutions at Tyr-503. Many of the mutant enzymes have less than 0.1% wild-type activity. His-464 and Met-3 were replaced with 11 and 12 amino acids, respectively. Strains containing any one of these mutant proteins are Lac+. The results support previous evidence that Glu-461 and Tyr-503 are essential for catalysis, and suggest that His-464 is not part of the active site. Site-directed mutagenesis was facilitated by construction of an f1 bacteriophage containing the complete lacZ gene on a single EcoRI fragment.

The mutY gene: a mutator locus in Escherichia coli that generates G.C----T.A transversions.

We have used a strain with an altered lacZ gene, which reverts to wild type via only certain transversions, to detect transversion-specific mutators in Escherichia coli. Detection relied on a papillation technique that uses a combination of beta-galactosides to reveal blue Lac+ papillae. One class of mutators is specific for the G.C----T.A transversion as determined by the reversion pattern of a set of lacZ mutations and by the distribution of forward nonsense mutations in the lacI gene. The locus responsible for the mutator phenotype is designated mutY and maps near 64 min on the genetic map of E. coli. The mutY locus may act in a similar but reciprocal fashion to the previously characterized mutT locus, which results in A.T----C.G transversions.

Isolation and characterization of the actin gene from Tetrahymena thermophila.

The macronucleus of Tetrahymena thermophila contains a single actin gene. We have isolated this gene from a partial plasmid library by using the yeast actin gene as a probe. The nucleotide sequence of the gene has been determined and the amino acid sequence of the potential protein deduced. The encoded protein is 375 amino acids long, one amino acid longer than the yeast actin. It is one of the most divergent actins sequenced to date, being only 75% homologous to yeast actin. Unlike the actin genes from most other organisms, it does not contain introns. The coding region contains TAA and TAG codons; the translation termination codon is TGA. Comparison of the amino acid sequence of the Tetrahymena actin with that of actins from other organisms suggests that TAG may code for glutamic acid. The gene is transcribed from multiple initiation sites between 57 and 98 nucleotides upstream of the translation start codon. The 5' flanking region is very A+T-rich and contains numerous "TATA-like" sequences upstream of the transcription start sites.

Effect of human interferon preparations on lymphoblastogenesis in Down's syndrome.

The best characterised properties of human interferon, its antiviral (AV) and cell multiplication inhibitory (CMI) activities, are controlled, in an unexplained manner, by genes on chromosomes 21 (refs 1-4; 14-16). Human and animal interferons have various immunosuppressive effects, among them the inhibition in vitro of DNA synthesis in activated lymphocytes. Using mitogen- and antigen-stimulated lymphocytes from normal subjects (disomic 21) and others with Down's syndrome (trisomic 21), we have found that DNA synthesis is inhibited to a greater degree in the latter by both fibroblastoid and leukocyte interferons. We suggest that this property is also regulated by genes on chromosome 21.