DNA Repair

项目1将集中于已知参与紫外线诱导的细胞反应的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 断裂（DSB）修复。具体目标1将研究黑色素细胞衍生的功能性NER能力。 在黑色素瘤细胞系和遗传改变的黑色素细胞中， 常见于黑色素瘤主要目标是确定净入学率能力是否减弱或丧失 在黑色素瘤的发展阶段。此外，目标1将包括体外试验， 蛋白质的NER的动力学校对模型的机制测试。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耐受未修复的DNA phptoproducts的能力。它 将研究一个创新的新假设，即紫外线诱导的DNA损伤触发了一个信号通路， 导致活性复制子中DNA链延长的反式抑制。这是由积极的 在直接遇到模板之前，DNA复制叉的进展速度降低 损伤。DNA纤维梳理和免疫染色测定将使复制动力学可视化， 单个复制子。在其他感兴趣的蛋白质中，Timeless和Tipin的敲除将决定 复制叉保护复合物是否调节DNA复制叉的置换速率， 紫外线损伤的细胞具体目标3将检查染色体畸变和等位基因的诱导 UV处理的细胞中的缺失。染色体畸变被认为与DNA双链断裂有关 在折叠的复制叉和其他单链DNA区域产生的复制过程中形成的 紫外线损伤的DNA将定量磷酸化组蛋白H2 AX/磷酸化ATM/MRE 11阳性核灶， 监测DNA双链断裂的形成和修复。研究将确定基因改变是否会导致 重建的人皮肤中的黑色素瘤增强培养的黑色素细胞中的UV-染色体断裂。该项目将 确定DNA修复缺陷是否在皮肤中产生UV染色体突变子表型 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.