ABSTRACT
BACKGROUND: Not all of the enzymatic pathways involved in genetic rearrangements have been elucidated. While some rearrangements occur by recombination at areas of high homology, others are mediated by short, often interrupted homologies. We have previously constructed an Escherichia coli strain that allows us to examine inversions at microhomologies, and have shown that inversions can occur at short inverted repeats in a recB,C-dependent fashion. RESULTS: Here, we report on the use of this strain to define genetic loci involved in limiting rearrangements on an F' plasmid carrying the lac genes. Employing mini-Tn10 derivatives to generate insertions near or into genes of interest, we detected three loci (rmuA,B,C) that, when mutated, increase inversions. We have mapped, cloned and sequenced these mutator loci. In one case, inactivation of the sbcC gene leads to an increase in rearrangements, and in another, insertions near the recE gene lead to an even larger increase. The third gene involved in limiting inversions, rmuC, has been mapped at 86 min on the E. coli chromosome and encodes a protein of unknown function with a limited homology to myosins, and some of the SMC (structural maintenance of chromosomes) proteins. CONCLUSIONS: This work presents the first example of an anti-mutator role of the sbcC,D genes, and defines a new gene (rmuC) involved in DNA recombination.
Subject(s)
Chromosome Inversion , DNA, Bacterial/genetics , Escherichia coli Proteins , Escherichia coli/genetics , Genes, Regulator/genetics , Repetitive Sequences, Nucleic Acid/genetics , Cloning, Molecular , DNA Mutational Analysis , DNA Transposable Elements , Exodeoxyribonucleases/genetics , Genes, Bacterial/genetics , Lac Operon/genetics , Mutagenesis, Insertional , Physical Chromosome Mapping , Plasmids/genetics , Recombination, GeneticABSTRACT
Proteins containing the Nudix box "GX(5)EX(7)REUXEEXGU" (where U is usually Leu, Val, or Ile) are Nudix hydrolases, which catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Here we report cloning and characterization of a human cDNA encoding a novel nudix hydrolase NUDT5 for the hydrolysis of ADP-sugars. The deduced amino acid sequence of NUDT5 contains 219 amino acids, including a conserved Nudix box sequence. The recombinant NUDT5 was expressed in Escherichia coli and purified to near homogeneity. At the optimal pH of 7, the purified recombinant NUDT5 catalyzed hydrolysis of two major substrates ADP-ribose and ADP-mannose with K(m) values of 32 and 83 microM, respectively; the V(max) for ADP-mannose was about 1.5 times that with ADP-ribose. The murine NUDT5 homolog was also cloned and characterized. mNudT5 has 81% amino acid identity to NUDT5 with catalytic activities similar to NUDT5 under the optimal pH of 9. Both NUDT5 and mNudT5 transcripts were ubiquitously expressed in tissues analyzed with preferential abundance in liver. The genomic structures of both NUDT5 and mNudT5 were determined and located on human chromosome 10 and mouse chromosome 2, respectively. The role of NUDT5 in maintaining levels of free ADP-ribose in cells is discussed.
Subject(s)
Amino Acid Motifs , Conserved Sequence , Escherichia coli Proteins , Multigene Family , Pyrophosphatases/genetics , Adenosine Diphosphate Ribose/metabolism , Adenosine Diphosphate Sugars/metabolism , Amino Acid Sequence , Animals , Bacterial Proteins/genetics , Base Sequence , Chromosome Mapping , Chromosomes, Human, Pair 10 , Cloning, Molecular , Escherichia coli/genetics , Genetic Complementation Test , Humans , Mice , Molecular Sequence Data , Phosphoric Monoester Hydrolases/genetics , Phylogeny , Pyrophosphatases/classification , Pyrophosphatases/isolation & purification , Recombinant Proteins/isolation & purification , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
We have previously described the hMYH cDNA and genomic clones (M. M. Slupska et al., J. Bacteriol. 178:3885-3892, 1996). Here, we report that the enzyme expressed from an hMYH cDNA clone in Escherichia coli complements the mutator phenotype in a mutY mutant and can remove A from an A. 8-hydroxydeoxyguanine mismatch and to a lesser extent can remove A from an A. G mismatch in vitro.
Subject(s)
DNA Glycosylases , DNA Repair , Mutagenesis , N-Glycosyl Hydrolases/genetics , 8-Hydroxy-2'-Deoxyguanosine/analogs & derivatives , Base Pair Mismatch , Deoxyadenine Nucleotides/metabolism , Escherichia coli/genetics , Genetic Complementation Test , Guanine/analogs & derivatives , Guanine/metabolism , Humans , N-Glycosyl Hydrolases/biosynthesis , Recombinant Proteins/metabolism , Species SpecificityABSTRACT
We have cloned the human mutY gene (hMYH) from both genomic and cDNA libraries. The human gene contains 15 introns and is 7.1 kb long. The 16 exons encode a protein of 535 amino acids that displays 41% identity to the Escherichia coli protein, which provides an important function in the repair of oxidative damage to DNA and helps to prevent mutations from oxidative lesions. The human mutY gene maps on the short arm of chromosome 1, between p32.1 and p34.3.