DNA repair of 8-oxo-7,8-dihydroguanine lesions in Porphyromonas gingivalis.
The persistence of Porphyromonas gingivalis in the inflammatory environment of the periodontal pocket requires an ability to overcome oxidative stress. DNA damage is a major consequence of oxidative stress. Unlike the case for other organisms, our previous report suggests a role for a non-base excision repair mechanism for the removal of 8-oxo-7,8-dihydroguanine (8-oxo-G) in P. gingivalis. Because the uvrB gene is known to be important in nucleotide excision repair, the role of this gene in the repair of oxidative stress-induced DNA damage was investigated. A 3.1-kb fragment containing the uvrB gene was PCR amplified from the chromosomal DNA of P. gingivalis W83. This gene was insertionally inactivated using the ermF-ermAM antibiotic cassette and used to create a uvrB-deficient mutant by allelic exchange. When plated on brucella blood agar, the mutant strain, designated P. gingivalis FLL144, was similar in black pigmentation and beta-hemolysis to the parent strain. In addition, P. gingivalis FLL144 demonstrated no significant difference in growth rate, proteolytic activity, or sensitivity to hydrogen peroxide from that of the parent strain. However, in contrast to the wild type, P. gingivalis FLL144 was significantly sensitive to UV irradiation. The enzymatic removal of 8-oxo-G from duplex DNA was unaffected by the inactivation of the uvrB gene. DNA affinity fractionation identified unique proteins that preferentially bound to the oligonucleotide fragment carrying the 8-oxo-G lesion. Collectively, these results suggest that the repair of oxidative stress-induced DNA damage involving 8-oxo-G may occur by a still undescribed mechanism in P. gingivalis.
Henry
Leroy G
LG
Division of Microbiology and Molecular Genetics, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA. lhenry1@llu.edu
Sandberg
Lawrence
L
Zhang
Kangling
K
Fletcher
Hansel M
HM
D018829:Adhesins, Bacterial / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004249:DNA Damage; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics / Q000528:radiation effects; D005798:Genes, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D016254:Mutagenesis, Insertional; D009154:Mutation; D018384:Oxidative Stress; D016966:Porphyromonas gingivalis / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D012329:RNA, Bacterial / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014466:Ultraviolet Rays
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D012329:RNA, Bacterial; C024829:8-hydroxyguanine; D006147:Guanine; D006861:Hydrogen Peroxide; D003546:Cysteine Endopeptidases; D004265:DNA Helicases