Interaction between the Msh2 and Msh6 Nucleotide-binding Sites in the Saccharomyces cerevisiae Msh2-Msh6 Complex

Indirect evidence has suggested that the Msh2-Msh6 mispair-binding complex undergoes conformational changes upon binding of ATP and mispairs, resulting in the formation of Msh2-Msh6 sliding clamps and licensing the formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes. Here, we have studied eight mutant Msh2-Msh6 complexes with defective responses to nucleotide binding and/or mispair binding and used them to study the conformational changes required for sliding clamp formation and ternary complex assembly. ATP binding to the Msh6 nucleotide-binding site results in a conformational change that allows binding of ATP to the Msh2 nucleotide-binding site, although ATP binding to the two nucleotide-binding sites appears to be uncoupled in some mutant complexes. The formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes requires ATP binding to only the Msh6 nucleotide-binding site, whereas the formation of Msh2-Msh6 sliding clamps requires ATP binding to both the Msh2 and Msh6 nucleotide-binding sites. In addition, the properties of the different mutant complexes suggest that distinct conformational states mediated by communication between the Msh2 and Msh6 nucleotide-binding sites are required for the formation of ternary complexes and sliding clamps.     

Ataxia telangiectasia: the effects of chemical mutagens and x-rays on sister chromatid exchanges in blood lymphocytes

It is now possible to examine in detail exchanges between sister chromatids (SCEs) and to attempt to investigate the relationships of such exchanges to aberration formation and DNA-repair mechanisms. The frequency of SCEs is dramatically increased by chemical mutagens and may reflect the level of DNA damage. Lymphocytes from patients with ataxia telangiectasis (AT) show high levels of spontaneous chromosome damage and are hypersentive to ionising radiations and it was of interest to examine the levels of SCE induced in these cells by various mutagens. The frequencies of SCE after treatment with X=rays or three chemical mutagens were equivalent to those in normal cells. The effects of fluorodeoxyuridine and deoxycytidine on SCE frequencies were also tested.     

Somatic variations on a yellow mutant in Nicotiana tabacum L. (a1+/a1a2+/a2) I. Non-reciprocal genetic events occurring in leaf cells

A greenish-yellow mutant was obtained after treatment of seeds of Nicotiana tabacum L. var. Xanthi n.c. with ethyl methanesulfonate (EMS). Two genetically independent mutations (a₁ and a₂) were isolated. The first mutation (a₁) antagonizes the function of its partially dominant a₁⁺ allele. The second mutation (a₂) is amorphous but strongly interacts with a₁.Among the nine possible genotypes at the two loci, five varied in somatic cells. The heterozygous state a₁⁺/a₁ strongly increased the frequency of both spontaneous and induced variations. However, two homozygotes also showed variations.Variants were isolated from induced and spontaneous non-reciprocal and reciprocal variations within paliside tissues by bud induction in vitro. They were genetically tested. In this first paper, only non-reciprocal variations are reported.Green variants from the greenish-yellow (J¹) dihybrid a₁⁺/a₁a₂⁺/a₂ clone had two genotypes: the first was due to true reversions of a₁ to a₁⁺, whereas the second was due to amorphous a₁⁰ mutations from a₁. These a₁⁰ mutations may well be deletions.The lightest yellow variants from J¹ were due to mutations either from a₁⁺ into a₁ or from a₂⁺ into a₂.Deletions at the a₁⁺?a₁ locus led to either yellow variations when a₁⁺ was lost, or to false reversions when the antagonistic allele a₁ was lost.Amorphous alleles at the a₁⁺?a₁ locus were also isolated from tissues other than J⁺. They gave zygotic lethality (s) that probably varied with the size of the deletions. Thus, true reversions and deletions at the a₁⁺?a₁ locus could be distinguished from one another by progeny tests.Other variants showed higher frequencies of spontaneous variations (instability). Somatic changes observed in these unstable systems were due to modifications at the marker loci. The genetic nature of this instability is not yet known.There is strong evidence that the genetic events involved in these non-reciprocal variations were deletions, conversions and point mutations. True reversions from a₁ into a₁⁺ and new mutations from a₁⁺ into a₁ were obtained only from a₁⁺/a₁. It was therefore supposed that the changes observed took place only in heterozygotes, and the conversion hypothesis was made. Attempts are being made to prove that conversions do exist in higher plants, and to find out if this process, as deletions, is induced by radiation.     

Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

The bacterial H⁺-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (_KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (_KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted _KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of _KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (_KArg-25, _KArg-26, and _KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.     

Ultraviolet photoproducts at the ochre suppressor mutation site in the glnU gene of Escherichia coli: relevance to "mutation frequency decline"

Summary Ochre suppressor mutations induced by UV in the Escherichia coli glnU tRNA gene are CG to TA transitions at the first letter of the anticodon-encoding triplet, CAA. Premutational UV photoproducts at this site have long been known to exhibit an excision repair anomaly (mutation frequency decline or MFD), whereby post-irradiation inhibition of protein synthesis enhances their excision and reduces suppressor mutation yields ten-fold. We sought to clarify the basis of this unique repair response by determining the spectrum of UV photoproducts on both strands of a 36 by region of glnU which includes the anticodon-encoding triplet. We found that four different photolesions are produced within the 3 by sequence corresponding to the tRNA anticodon: (i) on the transcribed strand, TC (6–4) photoproducts and TC cyclobutane dimers are formed in equal numbers at the site of the C to T transition, indicating that this site is a hotspot for the usually less frequent (6–4) photoproduct; (ii) on the nontranscribed strand, TT dimers are found opposite the second and third letters of the anticodon-encoding triplet, adjacent to the mutation site; and (iii) on the nontranscribed strand, an alkali-sensitive lesion other than a (6–4) photoproduct is formed, apparently at the G in the mutation site. We suggest that mutation frequency decline may reflect excision repair activity at closely spaced UV lesions on opposite strands, resulting in double-strand breaks and the death of potential mutants.     

Isolation of the RAD18 gene of Saccharomyces cerevisiae and construction of rad18 deletion mutants

Summary The RAD18 gene of Saccharomyces cerevisiae is involved in mutagenic DNA repair. We describe its isolation from a yeast library introduced into the centromeric YCp50 vector, a low copy number plasmid. The insert was sublconed into YCp50 and into the multicopy YRp7 plasmid. RAD18 is not toxic when present in multiple copies but the UV survival response indicates an heterogeneity in the cell population, a fraction of it being more sensitive. A DNA segment, close to RAD18, is toxic on the multicopy plasmid and may correspond to the tRAN sup61 known to be tightly linked to RAD18. Chromosomal deletions of RAD18 were constructed. The gene is not essential and the deleted strains have the properties of single site mutants. Thus, RAD18 appears to be essentially involved in DNA repair metabolism.     

Mechanism of comutagenesis of sodium arsenite withn-methyl-n-nitrosourea

Arsenic compounds are known carcinogens. Although many carcinogens are also mutagens, we have previously shown that sodium arsenite is not mutagenic at either the Na⁺/K⁺ ATPase orhprt locus in Chinese hamster V79 cells. It can, however, enhance UV-mutagenesis. We now confirm the nonmutagenicity of sodium arsenite in line G12, a pSV2gpt-transformed V79 (hprt ⁻) cell line, which is able to detect multilocus deletions in addition to point mutations and small deletions. The lack of arsenic mutagenicity has led to studies emphasizing its comutagenicity. Sodium arsenite at relatively nontoxic concentrations (5 μM for 24 h or 10 μM for 3 h) is comutagenic withN-methyl-N-nitrosourea (MMU) at thehprt locus in V79 cells. Using a nick translation assay, which measures DNA strand breaks by incorporating radioactive deoxyribonucleoside monophosphate at their 3′OH ends in permeabilized cells, we found that much more incorporation was seen in cells treated with MNU (4 mM, 15 min) followed by 3-h incubation with 10 μM sodium arsenite compared with cells exposed to the same MNU treatment followed by 3-h incubation without sodium arsenite. This result shows that in the presence of arsenite, strand breaks resulting from MNU or its repair accumulate over a 3-h period. We suggest that the repair of MNU-induced DNA lesions may be inhibited by arsenite either by affecting the incorporation of dNMPs into the MNU-damaged DNA template or by interfering with the ligation step.     

Genetic,biochemical and immunological evidence for the involvement of two alcohol dehydrogenases in the metabolism of propane by Rhodococcus rhodochrous PNKb1

NAD⁺-dependent propan-1-ol and propan-2-ol dehydrogenase activities were detected in cell-free extracts of Rhodococcus rhodochrous PNKb1 grown on propane and potential intermediates of propane oxidation. However, it was unclear whether this activity was mediated by one or more enzymes. The isolation of mutants unable to utilize propan-1-ol (alcA^-) or propan-2-ol (alcB^-) as sole carbon and energy sources demonstrated that these substrates are metabolized by different alcohol dehydrogenases. These mutants were also unable to utilize propane as a growth substrate indicating that both alcohols are intermediates of propane metabolism. Therefore, propane is metabolized by terminal and sub-terminal oxidation pathways. Westernblot analysis demonstrated that a previously purified NAD⁺-dependent propan-2-ol dehydrogenase (Ashraf and Murrell 1990) was only synthesized after growth on propane and sub-terminal oxidation intermediates (but not acetone), and not propan-1-ol or terminal oxidation intermediates. Therefore, our evidence suggest that another dehydrogenase is involved in the metabolism of propan-1-ol and this agrees with the isolation of the alcA^- and alcB^- phenotypes. The previously characterized NAD⁺-dependent propan-2-ol dehydrogenase from R. rhodochrous PNKb1 is highly conserved amongst members of the propane-utilizing Rhodococcus-Nocardia complex.     

DNA sequence changes in mutations induced by ultraviolet light in the gpt gene on the chromosome of Escherichia coli uvr+ and urvA cells

Summary Sequence changes in mutations induced by ultraviolet light are reported for the chromosomal Escherichia coli gpt gene in almost isogenic E. coli uvr ⁺ and excision-deficient uvrA cells. Differences between the mutagenic spectra are ascribed to preferential removal of photoproducts in the transcribed strand by excision repair in uvr ⁺ cells. This conclusion is confirmed by analysis of published results for genes in both uvr ⁺ and uvr ^– cells, showing a similar selective removal of mutagenic products from the transcribed strand of the E. coli lacI gene and of the lambda phage cl repressor gene. Comparison of these data with published results for ultraviolet mutagenesis of gpt on a chromosome in Chinese hamster ovary cells showed that a mutagenic hot spot in mammalian cells is not present in E. coli; the possibility is suggested that the hot spot might arise from localized lack of excision repair. Otherwise, mutagenesis in hamster cells appeared similar to that in E. coli uvr ⁺ cells, except there appears to be a smaller fraction of single-base additions and deletions (frameshifts) in mammalian than in bacterial cells. Phenotypes of 6-thioguanine-resistant E. coli showed there is a gene (or genes) other than gpt involved in the utilization of thioguanine by bacteria.