Cross-links At Abasic Sites in Duplex DNA

描述(由申请人提供)：来自双链DNA基本站点的交叉链接。在癌症研究和环境毒理学方面正在进行的一项重大努力涉及识别重要的dna损伤损伤并表征其诱导细胞的能力。 死亡或致癌突变。这项拟议的工作将描述一类新的DNA损伤，这些损伤源于双链DNA中的无嘌呤/碱性(AP)位点。AP位点是由多种过程产生的，可能是细胞DNA遭受的最常见类型的损伤。这一建议建立在我们最近观察到的AP位点可以通过与双螺旋相反链上的核苷酸反应产生链间DNA-DNA交联链的基础上。令人惊讶的是，AP位点可以产生链间交叉链接，这通常被认为是所有DNA损伤中最有害的。交联链对人类细胞中的DNA修复机制提出了特殊的挑战。交联链的修复可能容易出错，由此产生的遗传密码突变可能会导致自发性癌症和化学诱导癌症的病因。如果不加以修复，交叉链接可能会阻止DNA转录和复制。细胞对这些交叉连接的反应(S)的确切性质可能会受到个人基因构成的影响，特别是关于他们的DNA损伤反应和修复机制中的缺陷或多态。这项拟议的工作意义重大，因为它将表征交叉链接如何有助于内源性或化学诱导的基本位点在人类中导致突变、癌症、细胞功能障碍、衰老和衰老的倾向。我们的研究是描述这些最近发现的DNA损伤的形成和生物学后果的第一次努力。拟议的工作将：(目标1和2)表征双链DNA中两种不同AP来源的交联链的形成和化学结构，(目标3)使用质谱学方法定量测量在暴露于诱导AP位点的药物(包括辐射和临床使用的烷化剂)的人类细胞中AP来源的交联链的形成和修复，以及(目标4)使用穿梭载体方法评估人类细胞中DG-AP交联链的转录旁路、复制和修复。 与公共卫生相关：-来自双链DNA中碱性位点的交叉链接碱性位点是由细胞DNA中遗传密码的核苷酸碱基“字母”丢失而产生的。DNA碱性位点在细胞中自发存在，也可以被各种天然毒素、工业化学品、杀虫剂和抗癌药物诱导。这项拟议的工作将研究从双链DNA中的基本位点衍生的一种新的链间DNA交联链的发生和修复。交联链对细胞来说是一个特殊的挑战，因为它们阻止了双螺旋的两条链的分离。基础位点衍生的交叉链接的修复可能容易出错，由此产生的遗传密码突变可能导致自发性癌症和化学诱导性癌症的病因。如果不加以修复，这些交叉链接可能会阻止DNA转录和复制，导致细胞功能障碍、细胞死亡、衰老和衰老。细胞对这些交叉连接的反应(S)的确切性质可能会受到个人基因构成的影响，特别是关于他们的DNA损伤反应和修复机制中的缺陷或多态。这项工作具有重要意义，因为它将表征AP衍生的交联链如何有助于非常常见的DNA损伤(基本部位)在人类中导致突变、癌症、细胞功能障碍、衰老和衰老的倾向。

DESCRIPTION (provided by applicant): Cross-links From Abasic Sites in Duplex DNA. A significant ongoing endeavor in cancer research and environmental toxicology involves the identification of important DNA-damage lesions and characterization of their ability to induce cell death or cancer-causing mutations. The proposed work will characterize a novel family of DNA lesions that are derived from apurinic/abasic (AP) sites in duplex DNA. AP sites are generated by a wide variety of processes and may be the most common type of damage sustained by cellular DNA. This proposal builds upon our recent observations that AP sites can generate interstrand DNA-DNA cross-links via reactions with nucleobases on the opposing strand of the double helix. It is striking that AP sites can generate interstrand cross-links, which generally ar thought to be the most deleterious of all DNA lesions. Cross-links present an exceptional challenge to the DNA-repair machinery in human cells. The repair of cross-links may be error- prone and the resulting mutations in the genetic code could contribute to the etiology of both spontaneous and chemical-induced cancers. Left unrepaired, cross-links may block DNA transcription and replication. The exact nature of the cellular response(s) to these cross-links may be influenced by an individual's genetic makeup, especially with regard to defects or polymorphisms in their DNA damage response and repair machinery. The proposed work is significant because it will characterize how cross-links contribute to the propensity for endogenous or chemically-induced abasic sites to cause mutagenesis, cancer, cell dysfunction, senescence, and aging in humans. Our studies represent the first efforts to characterize the formation and biological consequences of these recently discovered DNA lesions. The proposed work will: (Aims 1 and 2) characterize the formation and chemical structures of two different AP-derived cross-links in duplex DNA, (Aim 3) use mass spectrometric methods to quantitatively measure the formation and repair of AP-derived cross-links in human cells exposed to agents that induce AP sites, including radiolysis and clinically-used alkylating agents and, (Aim 4) assess transcriptional bypass, replication, and repair of the dG-AP cross- link in human cells using a shuttle-vector methodology. PUBLIC HEALTH RELEVANCE: - Cross-links From Abasic Sites in Duplex DNA Abasic sites are generated by the loss of a nucleobase "letter" from the genetic code in cellular DNA. DNA abasic sites occur spontaneously in cells and also can be induced by a variety of natural toxins, industrial chemicals, pesticides, and anticancer drugs. The proposed work will examine the occurrence and repair of a novel family of interstrand DNA cross-links derived from abasic sites in duplex DNA. Cross-links present an exceptional challenge to cells because they prevent separation of the two strands of the double helix. The repair of the abasic-site-derived cross-links may be error prone and the resulting mutations in the genetic code could contribute to the etiology of both spontaneous and chemical-induced cancers. Left unrepaired, these cross-links may block DNA transcription and replication, leading to cell dysfunction, cell death, senescence, and aging. The exact nature of the cellular response(s) to these cross-links may be influenced by an individual's genetic makeup, especially with regard to defects or polymorphisms in their DNA damage response and repair machinery. This work is significant because it will characterize how AP-derived cross-links contribute to the propensity for a very common DNA lesion (the abasic site) to cause mutagenesis, cancer, cell dysfunction, senescence, and aging in humans.