DNA Repair of 8-Oxo-7,8-Dihydroguanine Lesions in Porphyromonas gingivalis
DNA Repair of 8-Oxo-7,8-Dihydroguanine Lesions in Porphyromonas gingivalis
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DOI：
10.1128/jb.00919-08
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发表时间：
2008-12-01
影响因子：
3.2
通讯作者：
Fletcher, Hansel M.
中科院分区：
文献类型：
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作者：
Henry, Leroy G.;Sandberg, Lawrence;Fletcher, Hansel M.
牙龈卟啉单胞菌在牙周袋炎症环境中的持续存在需要克服氧化应激的能力。DNA损伤是氧化应激的主要后果。与其他生物体的情况不同，我们以前的报告表明，牙龈卟啉单胞菌中8-氧代-7，8-二氢鸟嘌呤（8-oxo-G）的去除是一种非碱基切除修复机制。由于已知uvrB基因在核苷酸切除修复中是重要的，因此研究了该基因在氧化应激诱导的DNA损伤修复中的作用。从牙龈卟啉单胞菌W83的染色体DNA中PCR扩增出含有uvrB基因的3.1-kb片段。该基因使用ermF-ermAM抗生素盒插入失活，并用于通过等位基因交换产生uvrB缺陷型突变体。当在布鲁氏菌血琼脂上平板接种时，命名为牙龈卟啉单胞菌FLL 144的突变菌株在黑色色素沉着和β-溶血方面与亲本菌株相似。此外，牙龈卟啉单胞菌FLL 144在生长速率、蛋白水解活性或对过氧化氢的敏感性方面与亲本菌株无显著差异。然而，与野生型相反，牙龈卟啉单胞菌FLL 144对UV照射显著敏感。从双链体DNA中酶促去除8-oxo-G不受uvrB基因失活的影响。DNA亲和分离鉴定了优先与携带8-oxo-G损伤的寡核苷酸片段结合的独特蛋白质。总的来说，这些结果表明，涉及8-oxo-G的氧化应激诱导的DNA损伤的修复可能通过牙龈卟啉单胞菌中尚未描述的机制发生。
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.