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）是细胞代谢的副产物，通过暴露于紫外线和电离辐射以及环境致癌物质而产生。由ROS产生的主要碱基损伤是7，8-二氢-8-氧代鸟嘌呤（8-oxoG）。与正常鸟嘌呤不同，8-oxoG在DNA复制过程中倾向于与腺嘌呤错配，导致G：C到T：A颠换突变的固定。氧化修饰的碱基，如8-oxoG，主要通过碱基切除修复途径（BER）修复，其第一步是通过特异性DNA糖基化酶识别和切除受损碱基。从DNA中去除8-oxoG的主要哺乳动物酶是8-oxoguanine-DNA糖基化酶（OGG 1）。OGG 1是一种双功能酶，在脱碱基位点具有8-oxoG切除活性和弱AP-裂解酶链切割活性。在OGG 1切除8-oxoG后，所得脱碱基位点通过几种酶在连续步骤中进一步加工以完成修复。 OGG 1基因敲除小鼠的研究和免疫耗竭实验表明，OGG 1是哺乳动物非转录DNA中主要的8-氧代鸟嘌呤修复活性。人们普遍认为，随着时间的推移，氧化DNA损伤的积累可能导致癌症。OGG 1在肿瘤抑制中的作用是由人肺癌和肾癌中OGG 1染色体位点的频繁丢失以及肺癌患者中与对照组相比显著较低的OGG 1活性所暗示的。在8-oxoG修复缺陷的敲除小鼠中迟发性肺肿瘤的增加、肺癌患者肺组织中8-oxoG水平的升高以及在几种人类癌细胞系中证明的8-oxoG修复的降低表明癌症和8-oxoG修复能力可能是相关的。OGG 1编码序列的变化导致影响功能、丰度或细胞内位置的氨基酸取代，预计会影响基因组8-oxoG水平，从而影响基因组稳定性和致癌作用。 已经报道了几种OGG 1多态性，并与多种癌症呈正相关。一种常见的多态性导致OGG 1 C-末端326位丝氨酸取代半胱氨酸。在人群中测得的S326 C OGG 1等位基因频率范围为0.13至高达0.62，并随种族而显著变化。相关研究已经确定，S326 C OGG 1等位基因纯合子个体肺癌、前列腺癌和口咽癌的发病率增加。先前的一项研究发现纯化的多态性S326 C OGG 1的催化效率（kcat/Km）降低，而另一项研究表明S326 C基因型体内8-氧代鸟嘌呤修复能力降低。我们的特点是糖基化酶和AP-裂解酶的活性和DNA损伤的结合亲和力纯化S326 C和发现新的功能缺陷的多态性OGG 1和一个独特的二聚体DNA结合构象相比，野生型酶。我们的结果证实了S326 C对与C配对的8-oxoG具有降低的修复活性，并且进一步表明当病变与T或G相对时，S326 C OGG 1特别缺乏8-oxoguanine切除活性。APE 1对野生型OGG 1的刺激导致8-oxoG切除率增加，并且被认为是体内碱基切除修复的调节和协调中的重要步骤。我们发现，S326 C OGG 1是不显着刺激APE 1，不像野生型酶，从而BER的协调可能会受到干扰，在修复8-oxoguanine由S326 C OGG 1。我们观察到人细胞中表达的S326 C OGG 1的修复活性和二聚体构象降低，因此S326 C OGG 1变体的活性和二聚体化学计量的改变可能与体内相关。