HOT SPOTS AND GENOMIC INSTABILITY

描述：这是一个用于研究人类突变的修订应用程序 可能由脱氧核苷酸前体[dNTP]池失衡引起的细胞 在人类细胞中复制DNA的过程中，并找出它们的机制。 (最近的证据表明，突变子的表型在 在结肠癌和其他癌症的发展过程中发挥作用。)最近的证据 表明dNTP池失衡引起的突变损害是由 改变核苷酸新陈代谢的环境物质可能有 在癌症的发展中扮演着重要的角色。(核化物池不平衡 可由营养缺乏在人体细胞中引起，由许多 在癌症化疗中使用的药物以及暴露于各种 致癌环境基因毒素。)这项提议的目标是 剖析可能导致脱氧核苷酸前体[dNTP]池的机制 人类细胞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基因的转座EBV载体来 确定抗叶酸诱导的小鼠肝细胞突变的特异性 正常和修复缺陷的人结肠癌细胞系。其作用机制 导致这些热点形成的原因将通过使用 突变热点周围的DNA序列已被 由特定部位的方法改变，以改变DNA序列上下文 突变部位。这些研究的基本原理是， 突变体以极高的频率恢复长期以来一直是 广谱基因突变的最显著特征 破坏DNA的各种药剂。这样的网站有可能成为 最容易找到的诱变剂指纹。尽管我们知道这一点 然而，DNA序列上下文的作用的直接证据 突变的原因很大程度上是从体外研究中推断出来的 并在活细胞和生物体中由相对较少的 调查。在这一建议中，导致热形成的机制 将探索由dNTP池不平衡引起的斑点突变。这些 调查将检查敏感的DNA序列在 同时具有正常和缺陷错配修复的细胞，以便确定 复制后修复能力的改变对诱导性的影响 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池失衡 热点地区将有助于评估环境中的癌症风险 暴露于有毒物质并帮助合理选择治疗方法 在过程中有遗传或后天缺陷的个人 忠实地复制他们的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.