Homology modelling of the DNA 5mC methyltransferase M.BssHII. Is permutation of functional subdomains common to all subfamilies of DNA methyltransferases?

This work presents a full tertiary model of the M.BssHII methyltransferase (MTase) complexed with substrate DNA and cofactor S-adenosyl-L-methionine, built by homology modelling based on previously solved complete structures of DNA MTases M.HaeIII and M. HhaI. M.BssHII and the template proteins show high sequence similarity, which indicates that they are evolutionary related. However, they are topologically different: M.BssHII is a circularly permuted variant of template MTases (Xu et al. Nucleic Acids Res 1997;25:3991). The model developed in this work will be a good starting point and valuable help in designing mutagenesis experiments to better understand the biological function of methyltransferases and the process of domain swapping.     

Native supercoiling of DNA: The effects of DNA gyrase and ω protein in E. coli

Molecular Genetics and Genomics - This study deals with the effects of a temperature-sensitive (ts) mutation at the gene encoding the DNA gyrase B subunit (gyrB ts) and a deletion of the top gene...     

Effects of glutathione on chromium‐induced DNA. Crosslinking and DNA polymerase arrest

Hexavalent chromium (Cr (VI)) is reduced intracellularly to Cr (V), Cr (IV) and Cr (III) by ascorbate (Asc), cysteine and glutathione (GSH). These metabolites induce a spectrum of genomic DNA damage resulting in the inhibition of DNA replication. Our previous studies have shown that treatment of DNA with Cr (III) or Cr (VI) plus Asc results in the formation of DNACrDNA crosslinks (CrDDC) and guaninespecific arrests of both prokaryotic and mammalian DNA polymerases. GSH not only acts as a reductant of Cr (VI) but also becomes crosslinked to DNA by Cr, thus, the focus of the present study was to examine the role of GSH in Crinduced DNA damage and polymerase arrests. Coincubation of Cr (III) with plasmid DNA in the presence of GSH led to the crosslinking of GSH to DNA. GSH cotreatment with Cr (III) also led to a decrease in the degree of Crinduced DNA interstrand crosslinks relative to Cr (III) alone, without affecting total Cr DNA binding. DNA polymerase arrests were observed following treatment of DNA with Cr (III) alone, but were markedly reduced when GSH was added to the reaction mixture. Preformed polymerasearresting lesions (CrDDC) were not removed by subsequent addition of GSH. Treatment of DNA with Cr (VI), in the presence of GSH, resulted in crosslinking of GSH to DNA, but failed to produce detectable DNA interstrand crosslinks or polymerase arrests. The inhibitory effect of GSH on Crinduced polymerase arrest was further confirmed in human genomic DNA using quantitative PCR (QPCR) analysis. Treatment of genomic DNA with Cr (III) resulted in a marked inhibition of the amplification of a 1.6 kb target fragment of the p53 gene by Taq polymerase. This was almost completely prevented by cotreatment with GSH and Cr (III). These results indicate that Crinduced DNA interstrand crosslinks, and not DNACrGSH crosslinks, are the principal lesions responsible for blocking DNA replication. Moreover, the formation of DNACrGSH crosslinks may actually preclude the formation of the polymerase arresting lesions.     

Mobile DNA distributions refine the phylogeny of “matsutake” mushrooms, Tricholoma sect. Caligata

“Matsutake” mushrooms are formed by several species of Tricholoma sect. Caligata distributed across the northern hemisphere. A phylogenetic analysis of matsutake based on virtually neutral mutations in DNA sequences resolved robust relationships among Tricholoma anatolicum, Tricholoma bakamatsutake, Tricholoma magnivelare, Tricholoma matsutake, and Tricholoma sp. from Mexico (=Tricholoma sp. Mex). However, relationships among these matsutake and other species, such as Tricholoma caligatum and Tricholoma fulvocastaneum, were ambiguous. We, therefore, analyzed genomic copy numbers of σ _marY1 , marY1, and marY2N retrotransposons by comparing them with the single-copy mobile DNA megB1 using real-time polymerase chain reaction (PCR) to clarify matsutake phylogeny. We also examined types of megB1-associated domains, composed of a number of poly (A) and poly (T) reminiscent of RNA-derived DNA elements among these species. Both datasets resolved two distinct groups, one composed of T. bakamatsutake, T. fulvocastaneum, and T. caligatum that could have diverged earlier and the other comprising T. magnivelare, Tricholoma sp. Mex, T. anatolicum, and T. matsutake that could have evolved later. In the first group, T. caligatum was the closest to the second group, followed by T. fulvocastaneum and T. bakamatsutake. Within the second group, T. magnivelare was clearly differentiated from the other species. The data suggest that matsutake underwent substantial evolution between the first group, mostly composed of Fagaceae symbionts, and the second group, comprised only of Pinaceae symbionts, but diverged little within each groups. Mobile DNA markers could be useful in resolving difficult phylogenies due to, for example, closely spaced speciation events.     

Phylogeny of Salsoleae s.l. (Chenopodiaceae) based on DNA sequence data from ITS, psbB–psbH, and rbcL, with emphasis on taxa of northwestern China

To reconstruct phylogeny and verify the monophyly of major subgroups, a total of 52 species representing almost all species of Salsoleae s.l. in China were sampled, with analysis based on three molecular markers (nrDNA ITS, cpDNA psbB–psbH and rbcL), using maximum parsimony, maximum likelihood, and Bayesian inference methods. Our molecular evidence provides strong support for the following: (1) Camphorosmeae is nested within Salsoleae s.l. instead of the previously suggested sister relationship. (2) Tribe Salsoleae s.l. is monophyletic and is composed of three monophyletic subunits, Caroxyloneae, the Kali clade, and Salsoleae s.str. (3) Climacoptera is separated from Salsola s.l. It does not form a monophyletic group but is split into two monophyletic parts, Climacoptera I and Climacoptera II. (4) Halogeton is clearly polyphyletic, as are Anabasis and the genus Salsola s.l. (5) Caroxylon, Haloxylon, Kali, and Petrosimonia are well-supported monophyletic genera. Additional evidence is needed regarding the monophyly of Halimocnemis, which remains unclear.     

Cloning and Characterization of the Human Mitochondrial DNA Polymerase, DNA Polymerase γ

The nuclear-encoded DNA polymerase γ (DNA POLγ) is the sole DNA polymerase required for the replication of the mitochondrial DNA. We have cloned the cDNA for human DNA POLγ and have mapped the gene to the chromosomal location 15q24. Additionally, the DNA POLγ gene fromDrosophila melanogasterand a partial cDNA for DNA POLγ fromGallus gallushave been cloned. The predicted human DNA POLγ polypeptide is 1239 amino acids, with a calculated molecular mass of 139.5 kDa. The human amino acid sequence is 41.6, 43.0, 48.7, and 77.6% identical to those ofSchizosaccharomyces pombe, Saccharomyces cerevisiae, Drosophila melanogaster,and the C-terminal half ofG. gallus,respectively. Polyclonal antibodies raised against the polymerase portion of the protein reacted specifically with a 140-kDa protein in mitochondrial extracts and immunoprecipitated a protein with DNA POLγ like activity from mitochondrial extracts. The human DNA POLγ is unique in that the first exon of the gene contains a CAG₁₀trinucleotide repeat.