Repair of hydrolytic DNA deamination damage in thermophilic bacteria: cloning and characterization of a Vsr endonuclease homolog from Bacillus stearothermophilus.
Hydrolytic deamination of 5-methyl cytosine in double stranded DNA results in formation of a T/G mismatch that-if left unrepaired-leads to a C-->T transition mutation in half of the progeny. In addition to several mismatch-specific glycosylases that have been found in both pro- and eukaryotes to channel this lesion into base excision repair by removing the T from the mismatch, Vsr endonuclease from Escherichia coli has been described which initiates repair by an endonucleolytic strand incision 5' to the mismatched T. We have isolated a gene coding for a homolog of E.coli Vsr endonuclease from the thermophilic bacterium Bacillus stearothermophilus H3 (Vsr.Bst) using a method that allows PCR amplification with degenerated primers of gene segments which code for only one highly conserved amino acid region. Vsr.Bst was produced heterologously in E.coli and purified to apparent homogeneity. Vsr.Bst specifically incises heteroduplex DNA with a preference for T/G mismatches. The selectivity of Vsr.Bst for the sequence context of the T/G mismatch appears less pronounced than for Vsr.Eco.
Laging
Martin
M
Abteilung Molekulare Genetik und Präparative Molekularbiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstrasse 8, D-37077 Göttingen, Germany.
Lindner
Eric
E
Fritz
Hans-Joachim
HJ
Kramer
Wilfried
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004249:DNA Damage; D004260:DNA Repair; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003641:Deamination; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004269:DNA, Bacterial; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D004706:Endodeoxyribonucleases; C071421:vsr endonuclease