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上解释人类错配修复（MMR）系统如何在体内发挥作用，以抑制DNA损伤诱导突变，但促进损伤触发的细胞周期停滞和凋亡。这里的重点是两个有代表性的类环境诱导的诱变和致癌病变，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，包括扩大的生化分析和培养细胞和转基因小鼠的研究。目的1是分析（ATP敏感的）结合，由（纯化的）人MMR识别蛋白hMutS 1和hMutS 2，DNA含有定义的环丁烷嘧啶二聚体（CPD）的光产物或PAH嘌呤加合物相反的“匹配”或“错配”的核苷酸。这里的MMR靶点分别是各种情况下的T[CPD]T/AG与T[CPD]T/AA，以及B[a] P-N2 G：C与B[a] P-N2 G：T和B[c] Ph-N6 A：T与B [c] Ph-N6 A：C。结合两倍的靶标以及在双折射迁移率移动阵列中的同源双链DNA将用于进一步的实验。目的2是使用完全无细胞提取物，确定Aim 1损伤靶点通过必需的MMR蛋白hMutL 1激活模型圆形基质（在非损伤链中含有预先存在的限定切口）的切口，并伴随引起沿着从基质切口到靶点的较短路径的3 '-5'切除沿着。激活MutL 1的能力区分“I类”MMR靶标（碱基错配原型）与“II类”（T[CPD]T/AG原型）靶标。将通过表面等离子体共振技术检测推定的II类靶标与（未活化的）hMutL 1和hMutS（1/2）形成三元复合物的能力。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-通过纯化的蛋白质混合物和无细胞提取物活化MMR相关的hExoI蛋白以5 '-3'切除模型缺口环状底物。目的4是比较由B[a]P和B[C]Ph二醇环氧化物在培养物中的MMR缺陷细胞与MMR熟练细胞中诱导的正向Hprt突变。对DNA损伤的有效MMR反应可以降低癌症风险并促进某些化疗药物对肿瘤细胞的杀伤。环境诱变剂可能对一种或（特别是）多种多态性部分MMR缺陷的个体造成意外风险。军团清除系统的其他部分缺陷可能会进一步。未来的生物化学研究将分析DNA再合成的效率和准确性，这些模板损伤已经引起提取物中（非损伤）链的3 '-5'（Aim 2）或5 '-3'（Aim 3）切除以及这些提取物中信号蛋白的募集（在没有切除的情况下）。未来对各种转基因小鼠的遗传学研究将检查MMR蛋白“敲除”对B[a]P和B[c]Ph二醇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。这些将有助于识别因环境因素诱发的癌症风险增加的个体。

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.