DNA Repair

项目1将集中在已知参与细胞对紫外线诱导的反应的DMA修复途径 DNA损伤：核苷酸切除修复(NER)、复制后修复(PRR)和DNA双链 中断(DSB)修复特定目标1将研究从黑素细胞中获得的功能性NER能力 来自健康的高加索新生儿，在黑色素瘤细胞系中，在具有基因改变的黑素细胞中 常见于黑色素瘤。主要目标是确定NER容量是否衰减或丢失 在黑色素瘤的发展阶段。此外，AIM 1将包括纯化的体外检测 对NER的动力学校对模型进行了蛋白质力学检验。具体目标2将决定 黑素细胞和黑色素瘤细胞系在复制过程中对未修复的DNA复制产物的耐受能力。它 将检验一项创新的新假说，即紫外线诱导的DNA损伤触发一条信号通路， 导致活性复制子中DNA链延长的反式抑制。这是由积极的 在直接接触模板之前DNA复制分叉的进展速度的降低 损伤。DNA纤维梳理和免疫染色分析将使复制动态可视化成为可能 单独的复制体。在其他感兴趣的蛋白质中，Timless和Tipin的敲除将决定 复制叉保护复合体是否调节DNA复制叉在 紫外线损伤的细胞。特殊目标3将检查染色体异常和等位基因的诱导。 紫外线处理细胞中的缺失。染色体异常被认为与DNADSB有关 在折叠的复制叉处产生，以及在复制过程中形成的其他单链DNA区域 紫外线损伤的DNA。磷酸-组蛋白H_2AX/磷酸-ATM/Mre11阳性核灶将被量化为 监测DNA DSB的形成和修复。研究将确定是否会导致 重建的人类皮肤中的黑色素瘤增强了培养的黑素细胞中紫外线断裂的产生。该项目将 确定DNA修复缺陷是否会在皮肤中产生紫外线-染色体-突变表型 黑素细胞。

Project 1 will focus on the pathways of DMA repair that are known to participate in cellular responses to UVinduced DMA damage: nucleotide excision repair (NER), post-replication repair (PRR) and DMA doublestrand break (dsb) repair. Specific aim 1 will investigate functional NER capacity in melandcytes derived from healthy Caucasian newborns, in melanoma cell lines, and in melanocytes with genetic alterations commonly found in melanoma. The main goal is to determine whether NER capacity is attenuated or lost during stages of development of melanoma. In addition, aim 1 will include in vitro assays with purified proteins for a mechanistic test of the kinetic proofreading model of NER. Specific Aim 2 will determine the capacity of melanocytes and melanoma lines to tolerate unrepaired DNA phptoproducts during replication. It will examine an innovative new hypothesis that UV-induced DNA damage triggers a signaling pathway that results in trans-inhibition of DNA chain elongation in active replicons. This is achieved by the active reduction in the rate of progression of DNA replication forks before the direct encounter with a template lesion. A DNA fiber-combing and immuno-staining assay will enable visualization of replication dynamics in individual replicons. Knockdown of Timeless and Tipin, among other proteins of interest, will determine whether the replication fork protection complex regulates the rate of displacement of DNA replication forks in UV-damaged cells. Specific aim 3 will examine the induction of chromosomal aberrations and allelic deletions in UV-treated cells. Chromosomal aberrations are thought to be associated with DNA dsb generated at collapsed replication forks and other single-strand DNA regions formed during replication of the UV-damaged DNA. Phospho-histone H2AX/phosphd-ATM/MRE11 -positive nuclear foci will be quantified to monitor the formation and repair of DNA dsb. Studies will determine whether genetic alterations that induce melanoma in reconstructed human skin enhance UV-clastogenesis in cultured melanocytes. This project wil determine whether defects in DNA repair produce a UV-chromosomal-mutator phenotype in skin melanocytes.