Proteolytic disregulation of the S326C mutant OGG1 DNA r

S326C 突变体 OGG1 DNA r 的蛋白水解失调

• 批准号: 7327033
• 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/7327033
• 关键词: Proteolytic; disregulation; S326C; mutant; OGG1

Reactive oxygen species (ROS) are produced as a by-product of cellular metabolism and through exposure to ultraviolet and ionizing radiation and environmental carcinogens. A major base damage produced by ROS is 7,8-dihydro-8-oxoguanine (8-oxoG). Unlike normal guanine, 8-oxoG has the propensity to mispair with adenine during DNA replication, resulting in the fixation of G:C to T:A transversion mutations. Oxidatively modified bases, such as 8-oxoG, are repaired primarily by the base excision repair pathway (BER), the first steps of which are the recognition and excision of the damaged base by a specific DNA glycosylase. The major mammalian enzyme for removing 8-oxoG from DNA is 8-oxoguanine-DNA glycosylase (OGG1). OGG1 is a bifunctional enzyme, having both 8-oxoG excision activity and a weak AP-lyase strand incision activity at abasic sites. Following excision of 8-oxoG by OGG1, the resultant abasic site is further processed in sequential steps by several enzymes to complete repair. Studies of OGG1 knockout mice and immunodepletion experiments suggest that OGG1 is the major mammalian 8-oxoguanine repair activity in non-transcribed DNA. It is widely accepted that accumulation of oxidative DNA damage over time can lead to cancer. A role for OGG1 in tumor suppression is suggested by the frequent loss of the OGG1 chromosomal locus in human lung and renal cancers and by significantly lower OGG1 activity among lung cancer patients compared to controls. Increased late-onset lung tumors in knockout mice deficient in repair of 8-oxoG, elevated 8-oxoG levels in lung tissue of lung cancer patients and decreased repair of 8-oxoG demonstrated in several human cancer cells lines suggest that cancer and 8-oxoG repair capacity may be linked. Changes in the OGG1 coding sequence that result in amino acid substitutions that affect function, abundance, or intracellular location could be anticipated to impact genomic 8-oxoG levels, and thereby influence genomic stability and carcinogenesis. Several OGG1 polymorphisms have been reported and positively correlate with a variety of cancers. A frequently occurring polymorphism results in the substitution of serine for cysteine at position 326 in the C-terminus of OGG1. The allele frequency of S326C OGG1 measured in human populations ranges from 0.13 to as high as 0.62 and varies significantly with ethnicity . Association studies have identified that individuals homozygous for the S326C OGG1 allele have increased incidence of lung, prostate, and orolaryngeal cancers. A previous study found decreased catalytic efficiency (kcat/Km) of purified polymorphic S326C OGG1 , while another study implicated the S326C genotype with decreased 8-oxoguanine repair capacity in vivo. We characterized the glycosylase and AP-lyase activities and DNA damage binding affinity of purified S326C and found novel functional defects in the polymorphic OGG1 and a distinct dimeric DNA binding conformation compared to the wild-type enzyme. Our results confirm that S326C has decreased repair activity towards 8-oxoG paired with C and further show that S326C OGG1 is particularly deficient in 8-oxoguanine excision activity when the lesion is opposite T or G. The stimulation of wild-type OGG1 by APE1 results in increased rates of 8-oxoG excision, and is believed to be an important step in the regulation and coordination of base excision repair in vivo. We show that S326C OGG1 is not significantly stimulated by APE1, unlike the wild-type enzyme, thereby the coordination of BER may be perturbed during repair of 8-oxoguanine by S326C OGG1. We observed decreased repair activity and dimeric conformation of S326C OGG1 expressed in human cells, thus the altered activity and dimeric stoichiometry of the S326C OGG1 variant may be relevant in vivo.

活性氧（ROS）作为细胞代谢的副产品，通过暴露于紫外线和电离辐射和环境致癌物。 ROS产生的主要基本损害为7,8-二氢-8-氧甲烷（8-oxog）。与正常的鸟嘌呤不同，8-oxog在DNA复制过程中倾向于用腺嘌呤混乱，从而导致g：c：c至t：t：a transtression突变。氧化修饰的碱基（例如8-oxog）主要是通过碱基切除修复途径（BER）修复的，其第一步是通过特定的DNA糖基酶对受损碱的识别和切除。从DNA中去除8-氧的主要哺乳动物酶是8-氧气甘氨酸-DNA糖基酶（OGG1）。 OGG1是一种双功能酶，具有8-oxog切除活性，并且在abasic部位具有弱的AP酶链切口活性。通过OGG1切除8-oxog后，通过几种酶进行了顺序处理所得的无asic位点以完成修复。 OGG1基因敲除小鼠和免疫启动实验的研究表明，OGG1是未转录的DNA中主要的哺乳动物8-氧气修复活性。人们普遍认为，随着时间的推移，氧化DNA损伤的积累会导致癌症。与对照组相比，肺癌和肾脏癌患者中OGG1染色体基因座的经常丧失在人类肺和肾脏癌中的频繁丧失，与对照组相比，OGG1染色体基因座的经常丧失明显降低了OGG1染色体位点，与OGG1染色体基因座的频繁丧失相比，与对照组相比，OGG1染色体基因座的频繁丧失明显降低，这表明了OGG1在肿瘤抑制中的作用。在肺癌患者肺组织中肺部修复缺乏8-oxog修复，8-oxog水平升高的敲除小鼠中的晚期肺部肿瘤增加，并且在几个人类癌细胞系列中证明的8-氧气的修复降低表明癌症和8-oxog修复能力可能会联系起来。可以预期会影响功能，丰度或细胞内位置的氨基酸取代的OGG1编码序列的变化会影响基因组8-oxog水平，从而影响基因组稳定性和癌变。 已经报道了几种OGG1多态性，并与各种癌症呈正相关。经常发生的多态性导致丝氨酸在OGG1的C末端326处的半胱氨酸取代。在人类种群中测得的S326C OGG1的等位基因频率范围从0.13到0.62，并且随着种族的差异很大。协会研究已经确定，S326C OGG1等位基因纯合子的个体增加了肺，前列腺和口服癌的发病率。先前的一项研究发现，纯化多态性S326C OGG1的催化效率（KCAT/km），而另一项研究则暗示了S326C基因型，体内8-氧气的修复能力降低。我们表征了纯化的S326C的糖基化酶和AP-溶性活性以及DNA损伤结合亲和力，并在多态性OGG1中发现了新型的功能缺陷，并且与野生型酶相比，具有不同的二聚体DNA结合构象。 Our results confirm that S326C has decreased repair activity towards 8-oxoG paired with C and further show that S326C OGG1 is particularly deficient in 8-oxoguanine excision activity when the lesion is opposite T or G. The stimulation of wild-type OGG1 by APE1 results in increased rates of 8-oxoG excision, and is believed to be an important step in the regulation and coordination of base excision repair in vivo.我们表明，与野生型酶不同，APE1并未显着刺激S326C OGG1，因此在S326C OGG1修复8-氧气的8-氧气时，BER的配位可能会受到干扰。我们观察到在人类细胞中表达的S326C OGG1的修复活性和二聚体构象的降低，因此S326C OGG1变体的活性和二聚体化学计量的变化可能在体内与之相关。