Interaction of 8-oxoguanine-DNA glycosylase with PCNA

8-氧代鸟嘌呤-DNA 糖基化酶与 PCNA 的相互作用

• 批准号: 6815199
• 负责人: michele k evans
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6815199
• 关键词: DNA repair; DNA replication; N glycosidase; enzyme activity; guanine; human tissue; immunofluorescence technique; immunoprecipitation; oxidative stress; phosphoester ligase; proliferating cell nuclear antigen; protein protein interaction

Human 8-oxoguanine-DNA glycosylase (OGG1) is the major enzyme for repairing 7-8, dihydro-8-oxoguanine (8-oxoG), a pre-mutagenic guanine base lesion produced by reactive oxygen species (ROS). The mutagenicity of 8-oxoguanine lies in its propensity to mispair with adenine during DNA replication. The importance of 8-oxoguanine and its repair by OGG1 are underscored by the frequent absence of the OGG1 allele in human lung tumors and the increased incidence of lung tumors in mice lacking a functional OGG1. 8-oxoguanine can occur in DNA by the oxidation of guanine in a G:C pair and by the incorporation of 8-oxoG into the newly synthesized nascent strand opposite cytosine or adenine during DNA replication or repair synthesis. Mispairings of 8-oxoG, when repaired by OGG1, could fix mutations if 8-oxoG in the parental strand is removed from a mispair with adenine. Accordingly, OGG1 should act only to remove 8-oxoG formed in DNA in situ and newly incorporated 8-oxoG in the nascent strand. If 8-oxoG in the parental strand becomes mispaired during DNA replication and is subsequently removed by OGG1, a G to T transversion mutation could result. Using co-immunoprecipitation, we identified an interaction between OGG1 and proliferating cell nuclear antigen (PCNA). PCNA is a multi-functional protein involved in DNA replication, repair synthesis and cell cycle regulation. The interaction of OGG1 with PCNA is of particular interest because known PCNA-binding proteins, such as DNA polymerases and components of the mismatch repair system, perform their functions on newly synthesized DNA are directed to the nascent strand via a directional interaction with PCNA. Using an in vitro binding assay and mutant OGG1 proteins, we have identified a functional consensus PCNA binding motif in the C-terminus of OGG1. Additionally, using immunofluorescence, we have shown that OGG1 and PCNA co-localize at sites of DNA synthesis in vivo. The association of OGG1 and PCNA suggests a bimodal mechanism of OGG1-mediated repair of 8-oxoguanine. In non-dividing cells, OGG1 and perhaps other DNA repair proteins may indiscriminately remove 8-oxoguanine as it occurs in DNA. During replication however, the OGG1-PCNA interaction may serve to direct OGG1 to the nascent strand in order to prevent fixation of mutations in the parental strand. The functional consequences of the interaction of OGG1 with PCNA, which are likely to be highly significant in vivo, are currently being investigated.

人8-氧代鸟嘌呤-DNA糖基化酶（OGG 1）是修复7-8，二氢-8-氧代鸟嘌呤（8-oxoG）的主要酶，8-oxoG是由活性氧（ROS）产生的致突变前鸟嘌呤碱基损伤。8-氧代鸟嘌呤的致突变性在于其在DNA复制过程中与腺嘌呤错配的倾向。8-氧代鸟嘌呤的重要性及其修复OGG 1强调了OGG 1等位基因在人类肺肿瘤中的频繁缺失和缺乏功能性OGG 1的小鼠肺肿瘤发病率的增加。8-氧代鸟嘌呤可以通过G：C对中鸟嘌呤的氧化和通过在DNA复制或修复合成期间8-氧代G掺入到与胞嘧啶或腺嘌呤相对的新合成的新生链中而在DNA中出现。当被OGG 1修复时，如果亲本链中的8-oxoG从与腺嘌呤的错配中去除，8-oxoG的错配可以修复突变。因此，OGG 1应该仅用于去除原位DNA中形成的8-oxoG和新生链中新掺入的8-oxoG。如果亲本链中的8-oxoG在DNA复制过程中错配，随后被OGG 1去除，则可能导致G至T颠换突变。使用免疫共沉淀，我们确定了OGG 1和增殖细胞核抗原（PCNA）之间的相互作用。PCNA是一种参与DNA复制、修复合成和细胞周期调控的多功能蛋白。OGG 1与PCNA的相互作用特别令人感兴趣，因为已知的PCNA结合蛋白，如DNA聚合酶和错配修复系统的组分，在新合成的DNA上执行其功能，通过与PCNA的定向相互作用被定向到新生链。使用体外结合试验和突变OGG 1蛋白，我们已经确定了一个功能性的共识PCNA结合基序的C-末端的OGG 1。此外，使用免疫荧光，我们已经表明，OGG 1和PCNA共定位在体内DNA合成的网站。OGG 1和PCNA的相关性表明OGG 1介导的8-氧代鸟嘌呤修复的双峰机制。在非分裂细胞中，OGG 1和其他DNA修复蛋白可能会不加选择地去除DNA中的8-氧代鸟嘌呤。然而，在复制过程中，OGG 1-PCNA的相互作用可能会直接OGG 1的新生链，以防止固定突变的亲本链。目前正在研究OGG 1与PCNA相互作用的功能后果，这可能在体内非常重要。