Proteolytic disregulation of the S326C mutant OGG1 DNA repair enzyme

活性氧(ROS)是细胞代谢的副产品，暴露在紫外线、电离辐射和环境致癌物中。ROS产生的一个主要碱基损伤是7，8-二氢-8-氧鸟嘌呤(8-oxoG)。与正常的鸟嘌呤不同，8-oxoG在DNA复制过程中容易与腺嘌呤错误配对，导致G：C到T：A颠倒突变的固定。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，第一步是通过特定的DNA糖基酶识别和切除受损的碱基。哺乳动物中去除DNA中8-oxoG的主要酶是8-oxoguanine-DNA糖基酶(OGG1)。OGG1是一种双功能酶，在碱性部位既有8-oxoG切割活性，又有微弱的解链切割活性。在8-oxoG被OGG1切除后，所产生的基本位点被几种酶按顺序进一步处理以完成修复。 对OGG1基因敲除小鼠的研究和免疫耗竭实验表明，OGG1是哺乳动物非转录DNA中主要的8-氧鸟嘌呤修复活性。人们普遍认为，随着时间的推移，DNA氧化损伤的积累会导致癌症。在人类肺癌和肾癌中，OGG1染色体位点的频繁丢失，以及肺癌患者中OGG1活性显著低于对照组，表明OGG1在肿瘤抑制中发挥了作用。OGG1编码序列的改变导致氨基酸替换，从而影响功能、丰度或细胞内位置，预计会影响基因组8-oxoG水平，从而影响基因组的稳定性和致癌作用。 已有多个OGG1基因多态被报道，并与多种癌症呈正相关。OGG1 C末端326位丝氨酸取代半胱氨酸是一种常见的多态现象。我们对纯化的S326C的糖基酶和解酶活性以及DNA损伤结合亲和力进行了表征，发现与野生型酶相比，OGG1的多态存在新的功能缺陷和明显的二聚体DNA结合构象。我们的结果证实了S326C对与C配对的8-oxoG的修复活性降低，进一步表明当病变与T或G相反时，S326C OGG1尤其缺乏8-oxoguanine切除活性。我们鉴定了R229Q多态的酶活性，并确定了R229Q表达对暴露于DNA损伤剂后KG-1存活的影响。我们的结果表明，R229Q OGG1在体外和体内都是高度不热的，在生理温度下都能迅速失活。在甲苯二酮和8-oxodG作用下，R229Q OGG1的核和线粒体表达均可使KG-1细胞发生凋亡，因此R229Q可促进ROS和氧化核苷暴露后的细胞凋亡。我们还发现人的8-氧鸟嘌呤-DNA糖基酶1(OGG1)是钙依赖的蛋白水解酶Calain I的特异性靶标。在遭受慢性氧化应激、缺血/再灌注和其他细胞应激的组织中，Calain对OGG1的降解可能导致8-氧鸟嘌呤修复活性降低和8-氧鸟嘌呤水平升高，这些应激因素可引起细胞内钙稳态的破坏，从而激活Calain。 今年，我们已经开始解决这样一个问题，即可能对识别和处理氧化诱导的DNA损伤至关重要的其他蛋白质是否与OGG1的多态形式相互作用不同。我们已经在基线上检测了野生型OGG1与DNA损伤传感蛋白的结合。这使我们能够更直接地了解OGG1及其多态变体在细胞氧化DNA损伤的处理和修复中的可能作用，这些细胞可能更容易受到氧化应激的影响。在BER途径中发生了多个蛋白质-蛋白质相互作用，以协调该途径的高度复杂的过程。 我们正在使用一种无偏见的生化方法来确定OGG1的功能结合伙伴。利用这一方法，我们初步确定了PARP-1与OGG1特异性相互作用。PARP-1是DNA断裂的分子感受器，它通过与多种核蛋白、组蛋白、单链断裂修复蛋白(SSBR)、BER蛋白和PARP-1本身的结合或聚合(ADP-核糖基)在DNA断裂修复中发挥关键作用。此外，PARP-1在DNA损伤时被激活，使用敲除细胞和PARP-1抑制剂的研究表明，PARP-1对于维持基因组的完整性是重要的。我们正在寻求实验的重点是结合的类型和这种结合到OGG1的功能结果。

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. 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. 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. We characterized the enzymatic activity of the R229Q polymorphism and determined the effect of R229Q expression on KG-1 survival following exposure to DNA damaging agents. Our results showed that R229Q OGG1 is highly thermolabile and rapidly inactivated at physiological temperatures both in vitro and in vivo. Expression of both nuclear and mitochondrial R229Q OGG1 sensitized KG-1 cells to killing via an apoptotic pathway following exposure to menadione and 8-oxodG, thus R229Q promotes apoptosis following ROS and oxidized nucleoside exposure. We have also identified human 8-oxoguanine-DNA glycosylase 1 (OGG1) as a specific target of the Ca2+-dependent protease Calpain I. The degradation of OGG1 by calpain may contribute to decreased 8-oxoguanine repair activity and elevated levels of 8-oxoguanine reported in tissues undergoing chronic oxidative stress, ischemia/reperfusion and other cellular stressors known to produce perturbations of intracellular calcium homeostasis which activate calpain. This year we have begun to address the question of whether other proteins that may be vital to recognition and processing of oxidatively induced DNA damage interact differently with polymorphic forms of OGG1. We have proceeded to examine at baseline the binding of wild type OGG1 to DNA damage sensing proteins. This has enabled us to understand more directly the possible role of OGG1 and its polymorphic variants in the processing and repair of oxidative DNA damage in cells from individuals who may be more vulnerable to the effects of oxidative stress. Multiple protein-protein interactions occur during the BER pathway in order to coordinate the highly intricate process of this pathway. We are using an unbiased biochemical approach in order to determine functional binding partners for OGG1. Using this approach, we preliminarily have determined that PARP-1 specifically interacts with OGG1. PARP-1 is a molecular sensor of DNA breaks and it plays a key role in repair of these breaks by either physically associating with or also by poly(ADP-ribosyl)ation of partner proteins including various nuclear proteins, histones, single-strand break repair proteins (SSBR), BER proteins and on PARP-1 itself. Furthermore, PARP-1 is activated in response to DNA damage and studies using knockout cells and PARP-1 inhibitors show that PARP-1 is important for maintaining genomic integrity. We are pursing experiments focused on the type of binding and the functional outcomes of this binding to OGG1.