HOT SPOTS AND GENOMIC INSTABILITY

描述：这是一个修改后的应用程序，用于研究人类基因突变。 可能由脱氧核苷酸前体[dNTP]库失衡引起的细胞 在人类细胞的DNA复制过程中，并找出它们的机制。 （最近的证据表明，增变基因表型发挥着重要的作用， 在结肠癌和其他癌症的发展中起作用。）最近的证据 表明由dNTP池不平衡引起的致突变性损伤， 改变核苷酸代谢的环境因子可能具有 在癌症发展中的重要作用。 （核苷酸库失衡） 在人体细胞中可以由营养缺乏引起， 用于癌症化疗的药物和暴露于各种 致癌的环境遗传毒素）。本提案的目的是 剖析可能导致脱氧核苷酸前体[dNTP]池的机制 人类细胞中DNA复制过程中的不平衡引起的错误。 这些 研究将使用携带lacI基因的可转座EBV载体， 确定抗叶酸诱导突变的突变特异性， 正常和修复缺陷的人结肠癌细胞系。 的机制 导致这些热点的形成，然后将通过使用 其中围绕突变热点的DNA序列已被 通过位点特异性方法改变DNA序列背景， 突变位点 这些研究的基本原理是， 长期以来，突变体以异常高的频率被恢复， 最显著的特征是由广泛的 各种破坏DNA的物质。 这些网站有可能成为 最容易识别的诱变剂指纹 尽管我们知道 然而，DNA序列背景作用的直接证据 突变的原因主要是从体外研究中推断出来的 在活细胞和有机体中， 调查事务所 在这一建议中，机制导致形成一个热 将探索由dNTP池不平衡引起的斑点突变。 这些 调查将检查易感DNA序列的反应， 具有正常和有缺陷的失配修复的细胞 复制后修复能力的改变对诱导的 需要多种表达机制相互作用的热点。 了解DNA序列如何导致dNTP池不平衡诱导 热点将有助于评估环境中的癌症风险 暴露于有毒物质，并有助于合理选择治疗， 在保证过程中存在遗传或后天缺陷的个人 忠实地复制自己的DNA

DESCRIPTION: This is a revised application to study mutations in human cells which may result from deoxynucleotide precursor [dNTP] pool imbalance during DNA replication in human cells, and to ferret out their mechanisms. (Recent evidence has demonstrated that mutator phenotypes play an important roll in the development of colon and other cancers.) Recent evidence suggests that mutagenic damage from dNTP pool imbalances caused by environmental agents which alter metabolism of nucleotides may have important roles in the development of cancers. (Nucleoide pool imbalances can be caused in human cells by nutritional deprivation, by a number of agents used in cancer chemotherapy and by exposure to a variety of carcinogenic environmental genotoxins.) The goal of this proposal is to dissect mechanisms which may result in deoxynucleotide precursor [dNTP] pool imbalance-induced errors during DNA replication in human cells. These studies will employ transposable EBV vectors carrying the lacI gene to determine the mutational specificity of anti-folate induced mutations in normal and repair deficient human colon cancer lines . The mechanisms leading to formation of these hot spots will then be dissected by employing vectors in which DNA sequence surrounding hot spots for mutation have been altered by site specific approaches to change the DNA sequence context of the mutation site. The rationale for these studies is that sites where mutants are recovered at exceptionally high frequency have long been the most distinctive feature of the spectra of mutations caused by a wide variety of agents which damage DNA. Such sites have the potential to be the most accessible fingerprint of mutagens. In spite of our knowledge of this relationship, however, direct evidence for the role of DNA sequence context in causation of mutations has largely been inferred from studies in vitro and supported in living cells and organisms by relatively few investigations. In this proposal, mechanisms leading to formation of a hot spot mutation caused by dNTP pool imbalances will be explored. These investigations will examine the response of the susceptible DNA sequence in cells with both normal and defective mismatch repair in order to determine the impact of alterations in post-replicative repair capacity on inducible hot spots that require an interplay of multiple mechanisms for expression. Understanding how DNA sequence contributes to dNTP pool imbalance-induced hot spots will facilitate assessment of cancer risk from environmental exposure to toxic agents and aid in a rational choice of therapy for individuals with inherited or acquired defects in processes assuring faithful duplication of their DNA.