Mechanisms for the repair of oxidative stress-induced DNA damage in Porphyromonas

牙龈卟啉单胞菌作为一种关键病原体，突出了其对严酷炎症的适应能力 牙周袋的状况。因为环境应激反应是决定其 毒力，全面了解其生存策略是我们的长期目标(S)。脱氧核糖核酸 损伤是氧化应激的主要后果。而20多种不同的氧化改变的碱基 可能是由这种压力产生的，8-氧代-7，8-二氢鸟嘌呤(8-oxoG)是 DNA损伤。鸟嘌呤是最容易氧化的碱，由于其低氧化还原作用，主要形成8-oxoG 潜力。在原核细胞中，8-oxoG的存在主要通过碱基切除修复(BER)来修复。一个 对牙龈假单胞菌基因组的调查表明，BER系统的一个重要组成部分缺失。因为 牙龈假单胞菌基因组的平均G+C含量为49%，这是S预防或修复病变的机制 由鸟嘌呤氧化产生的是至关重要的。我们对一种机制的全面认识存在差距(S) 牙龈假单胞菌氧化应激所致DNA损伤的修复我们之前已经在那里证明了 8-oxoG是暴露在过氧化氢诱导的氧化应激下的牙龈假单胞菌染色体上8-oxoG的积累。 在其他菌株中观察到的BER和核苷酸切除修复(NER)似乎都不参与 牙龈假单胞菌8-oxoG损伤的修复DNA亲和层析鉴定保守假说基因PG1037 蛋白质，其中，优先结合到携带8-oxo-G损伤的寡核苷酸片段。 PG1037是牙龈假单胞菌uvrA-pg1037-PcrA操纵子的一部分，已知在H_2O_2作用下上调。 诱导应激。纯化的重组PG1037蛋白可能通过还原功能，具有防止 Fenton化学在体外介导DNA损伤，并在氧化应激条件下减少切割 8-oxoG损伤的大肠杆菌甲酰嘧啶糖基酶(FPG)酶。电子计算机分析中的PG1037 展示了一种含有锌指结构域、两个过氧化物酶同源基序和胞苷酸激酶的蛋白质 域。该提案的目标是检验一种新的牙龈假单胞菌蛋白(PG1037) 携带过氧化物酶基序和锌指结构域参与氧化损伤的修复 DNA我们的目的是确认PG1037在从双链DNA中去除8-oxoG方面的特定作用，并 评估在这一过程中PG1037与其他蛋白质的相互作用。这些数据将提供一个主要的概念性 牙龈假单胞菌DNA氧化应激损伤修复的分子基础研究进展 可能支持一种独特而有效的DNA修复机制，我们建议将其命名为“碱基氧化还原修复”。 它将在未来的RO1应用中为解决关键的结构-功能问题奠定基础 环境暴露中维持厌氧菌基因组稳定性的组分及其协同作用 压力。这些成分可以作为开发新的治疗干预措施的目标。 和预防牙龈假单胞菌相关疾病。

Porphyromonas gingivalis, as a “keystone pathogen”, highlights its ability to adapt to the harsh inflammatory conditions of the periodontal pocket. Because the environmental stress response is a major determinant of its virulence, it is our long-term goal to gain a comprehensive understanding of its survival strategy(s). DNA damage is a major consequence of oxidative stress. While more than 20 different oxidatively altered bases might be generated by this stress, 8-oxo-7,8-dihydroguanine (8-oxoG) is one of the most common product of DNA damage. Guanine is the most susceptible base to oxidation and forms mainly 8-oxoG due to its low redox potential. In prokaryotic cells the presence of 8-oxoG is mainly repaired by base excision repair (BER). A survey of the P. gingivalis genome indicate that an important component of the BER system is missing. Because the average G + C content of the genome of P. gingivalis is 49%, a mechanism(s) to prevent or repair lesions resulting from guanine oxidation is vital. There is a gap in our comprehensive knowledge on a mechanism(s) for the repair of oxidative stress-induced DNA damage in P. gingivalis. We have previously demonstrated that there is an accumulation of 8-oxoG in the chromosome of P. gingivalis exposed to H2O2-induced oxidative stress. Neither BER nor nucleotide excision repair (NER), as observed in other strains, appear to be involved in the repair of the 8-oxoG lesion in P. gingivalis. DNA affinity fractionation identified PG1037, a conserved hypothetical protein, among others, that were preferentially bound to the oligonucleotide fragment carrying the 8-oxo-G lesion. PG1037 is part of the uvrA-pg1037-pcrA operon in P. gingivalis which is known to be upregulated under H2O2- induced stress. The purified recombinant PG1037 protein, likely via a reducing function, has the ability to prevent Fenton chemistry-mediated DNA damage in vitro and, under oxidative stress conditions, reduced the cleavage of the 8-oxoG lesion by the E.coli foramidopyrimidine glycosylase (Fpg) enzyme. In silico analysis of PG1037 shows a protein that contains a zinc finger domain, two peroxidase homologous motifs and a cytidylate kinase domain. The goal of the proposal is to test the hypothesis that a novel P. gingivalis protein (PG1037) carrying peroxidase motifs and a zinc finger domain is involved in the repair of oxidatively damaged DNA. Our aims are to confirm the specific role of PG1037 in the removal of 8-oxoG from duplex DNA and to evaluate any interaction of PG1037 with other proteins in that process. The data will provide a major conceptual advance on the molecular bases for the repair of oxidative stress-induced DNA damage in P. gingivalis and could likely support a unique and effective DNA repair mechanism we propose to designate “base redox repair”. It will set the stage, in a future RO1 application, to address specific structure-function questions on the vital components and their corporation in maintaining genomic stability in anaerobes exposed to environmental stress. These components could be targets for the development of novel therapeutic interventions for the control and prevention of P. gingivalis-associated diseases.