Biochemistry of mismatch-repair responses to DNA lesions

描述(由申请人提供)：长期目标是从生物化学的角度解释人类错配修复(MMR)系统如何在体内发挥作用，抑制DNA损伤诱导的突变，同时促进病变触发的细胞周期停滞和凋亡。这里的重点是两类具有代表性的环境诱变和致癌损伤，紫外线光产物和多环芳烃(PAH)加合物。这项探索性工作的具体组成部分的目的是为未来的研究提供一个明确的基础--既包括扩展的生化分析，也包括培养细胞和转基因小鼠的工作。目的1是分析(ATP敏感的)人类MMR识别蛋白hMutS1和hMutS2与含有特定环丁烷-嘧啶-二聚体(CPD)光产物或PAH-嘌呤加合物的DNA的(ATP敏感)结合--相反的“匹配”或“不匹配”核苷酸。这里的MMR靶分别是在不同环境中的T[CPD]T/AG与T[CPD]T/AA，以及B[a]P-N2G：C与B[a]P-N2G：T和B[c]Ph-N6A：T与B[c]Ph-N6A：C。结合两次的靶以及在凝胶迁移率移位阵列中的同源双链DNA将用于进一步的实验。目的2是利用完整的无细胞提取液，确定针对1病变靶点的完整无细胞提取物，该提取物通过必需的MMR蛋白hMutL1激活模型圆形底物(包含非病变链中预先存在的已定义缺口)的切割，并沿着底物缺口到靶点的较短路径同时引发3‘-5’切除。激活MutL1的能力将“I类”MMR目标(碱基错配原型)与“II类”(T[CPD]T/AG原型)目标区分开来。将通过表面等离子体共振技术测试假定的II类目标与(未激活的)hMutL1和hMutS(1/2)形成三元络合物的能力。目的3是测试PAH-嘌呤和原型T[CPD]T/AG靶标的定义II类性质-激活MMR相关的hExoI蛋白，通过纯化蛋白混合物和无细胞提取液5‘-3’切除模型Nick-Circle底物。目的4比较B[a]P和B[C]Ph环氧化物对MMR缺陷细胞和MMR熟练细胞HPRT正向突变的诱导作用。有效的MMR对DNA损伤的反应既可以降低癌症风险，又可以促进某些化疗药物对肿瘤细胞的杀伤。环境诱变剂可能会给携带一种或(特别是)多种多态部分MMR缺陷的个体带来意外的风险。军团除名制度中的其他部分缺陷可能会进一步。未来的生化研究将分析过去模板损伤引起3‘-5’(目标2)或5‘-3’(目标3)切除(无损伤)提取液中的(非损伤)链以及在这些提取液中重新招募(在没有切除的情况下)信号蛋白的效率和准确性。未来对各种转基因小鼠的遗传学研究将检验MMR蛋白“敲除”对B[a]P和B[c]Ph diol epxidos诱导的特定突变途径的影响。公共卫生相关性：这项探索性工作将为以后更广泛的研究提供基础。这些将有助于识别由环境因素引起的癌症风险增加的个人。

DESCRIPTION (provided by applicant): The long-term objectives are to explain biochemically how human mismatch-repair (MMR) systems act in vivo to suppress induction of mutation by DNA lesions, yet promote lesion-triggered cell-cycle arrest and apoptosis. The focus here is on two representative classes of environmentally-induced mutagenic and carcinogenic lesions, UV photoproducts and polycyclicaromatic-hydrocarbon (PAH) adducts. The aim of the specific components of this exploratory work is to provide a well-defined basis for future studies-both expanded biochemical analyses and work with cultured cells and transgenic mice. Aim 1 is to analyze (ATP-sensitive) binding, by the (purified) human MMR-recognition proteins hMutS1 and hMutS2, of DNA containing defined cyclobutane-pyrimidine-dimer (CPD) photoproducts or PAH-purine adducts-opposite "matching" or "mismatching" nucleotides. The MMR targets here are, respectively, T[CPD]T/AG vs. T[CPD]T/AA in various contexts, and B[a]P-N2G:C vs. B[a]P-N2G:T and B[c]Ph-N6A:T vs. B[c]Ph-N6A:C. Targets bound twice as well as homoduplex DNA in electrophoretic-mobility-shift arrays will be used for further experiments. Aim 2 is to determine, using complete cell-free extracts, which Aim 1 lesion targets activate incision, by the essential MMR protein hMutL1, of model circular substrates (containing preexisting defined nicks in non-lesion strands) and concomitantly provoke 3'-5' excision along the shorter paths from substrate nicks to targets. Ability to activate MutL1 distinguishes "Class I" MMR targets (base- mispair prototype) from "Class II" (T[CPD]T/AG prototype) targets. Putative Class II targets will be tested, by surface-plasmon-resonance techniques, for abilities to form ternary complexes with (unactivated) hMutL1 and hMutS(1/2). Aim 3 is to test PAH-purine and the prototypic T[CPD]T/AG targets for the defining Class II property-activation of the MMR-associated hExoI protein for 5'-3' excision of model nicked-circular substrates by purified-protein mixtures and cell-free extracts. Aim 4 is to compare forward Hprt mutation induced in MMR-deficient vs. MMR-proficient cells in culture by B[a]P and B[C]Ph diol epoxides. Efficient MMR responses to DNA lesions can both reduce cancer risk and promote killing of tumor cells by some chemotherapeutic drugs. Environmental mutagens may pose unsuspected risks to individuals harboring one or (especially) multiple polymorphic partial MMR deficiencies. Additional partial deficiencies in legion-removal system may further. Future biochemical studies will analyze the efficiency and accuracy of DNA resynthesis past template lesions that have provoked 3'-5' (Aim 2) or 5'-3' (Aim 3) excision of (non-lesion) strands in extracts and recruitment (in the absence of excision) of signaling proteins in these extracts. Future genetic studies with various transgenic mice will examine effects of MMR-protein "knockouts" on specific mutation pathways induced by B[a]P and B[c]Ph diol epxidos. PUBLIC HEALTH RELEVANCE: This exploratory work will provide the basis for more later more extensive studies. These will help identify individuals at increased risk for cancer induced by environmental agents.