Molecular characterization of the Helicobacter pylori uvr B gene.
Helicobacter pylori persists in the human stomach where it may encounter a variety of DNA-damaging conditions, including gastric acidity. To determine whether the nucleotide excision repair (NER) pathway contributes to the repair of acid-induced DNA damage, we have cloned the putative H. pylori NER gene, uvrB. Degenerate oligonucleotide primers based on conserved amino acid residues of bacterial UvrB proteins were used in PCR with genomic DNA from H. pylori strain 84-183, and the 1.3-kb PCR product from this reaction was used as a probe to clone uvrB from an H. pylori genomic library. This plasmid clone had a 5.5-kb insert containing a 2.0-kb ORF whose predicted product (658 amino acids; 75.9 kDa) exhibited 69.5% similarity to E. coli UvrB. We constructed an isogenic H. pylori uvrB mutant by inserting a kanamycin-resistance cassette into uvrB and verified its proper placement by Southern hybridization. As with uvrB mutants of other bacteria, the H. pylori uvrB mutant showed a greatly increased sensitivity to the DNA-damaging agents methylmethane sulfonate and ultraviolet radiation. The uvrB mutant also was significantly more sensitive than the wild-type strain to killing by low pH, suggesting that the H. pylori nucleotide excision repair (NER) pathway is involved in the repair of acid-induced DNA damage.
Thompson
S A
SA
Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-2605, USA. thompssa@ctrvax.vanderbilt.edu
Latch
R L
RL
Blaser
J M
JM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004249:DNA Damage; D004265:DNA Helicases; D017931:DNA Primers; D004260:DNA Repair / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D019143:Evolution, Molecular; D015698:Genomic Library; D016480:Helicobacter pylori / Q000235:genetics* / Q000254:growth & development / Q000528:radiation effects; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; C058093:UvrB protein, E coli; D004265:DNA Helicases