Mechanisms of Replication-Dependent Microsatellite Instability in Human Disease

染色体断裂是最危险的DNA损伤形式，因为它们会导致多种类型的 突变和染色体重排。DNA在复制过程中对断裂最敏感， 难以复制的非规范DNA结构导致复制叉停滞。非规范DNA结构 强烈暗示为染色体断裂和易位的内源性来源，导致 发育缺陷和癌症，然而，复制叉停滞导致DNA 双链断裂（DSB）是未知的。 尽管在整体或单分子研究中对DNA损伤反应蛋白进行了大量分析， 损伤的位点未被识别，复制依赖的DNA链的分子机制 人类细胞中特定位点的断裂和修复尚不完全清楚。为了解决这个问题， 缺口，我们将研究两种类型的自然复制障碍（CTG/CAG三核苷酸重复和不对称 嘌呤-嘧啶（Pu/Py）镜像重复序列）整合在人类基因组的异位位点， 结构和对复制的影响可以被操纵。我们还研究了几个内源性复制叉 在DNA复制过程中诱导DSB的屏障。我们将使用PCR，DNA测序，染色质 免疫沉淀，质谱和流式细胞术显示（1）聚合酶如何在 非规范DNA结构导致DSB，（2）DNA修复蛋白如何重塑停滞的复制 叉重新启动合成，和（3）DSB重组的机制和基因组后果， 结构诱导的分叉屏障。 我们将检验非规范DNA结构通过阻断DNA的进展而诱导DSB的假设 聚合酶，促进核酸酶敏感性叉回归，并抑制DNA末端加工所需的 重组从这项工作的概念进展将包括确定的分子机制， DSB在特定停滞叉附近的形成，复制叉逆转的生物化学分析， 结构诱导的DSB的加工如何不同于核酸酶诱导的“干净”DSB。我们的长期目标 是为了确定DNA结构诱导的基因不稳定性在人类疾病中的作用。 目的1将揭示叉停滞和损伤信号之间的关系，叉的生物化学 逆转，结构特异性核酸内切酶在停滞叉的功能，以及DNA二级结构的影响。 分叉切除和修复结构。目的2将建立在我们的证明，范可尼贫血型J 蛋白（FANCJ）是维持整个基因组的非典型DNA结构所必需的， 复制应激，以确定FANCJ依赖性微卫星稳定化的机制。在目标3中， 将描述FANCJ缺陷的基因组后果。我们的实验会证明 重复的DNA序列会导致染色体断裂和突变，从而导致遗传疾病。

Chromosome breaks are the most dangerous form of DNA damage because they result in multiple types of mutations and gross chromosome rearrangements. DNA is most sensitive to breakage during replication, when hard-to-replicate noncanonical DNA structures cause replication fork stalling. Noncanonical DNA structures are strongly implicated as endogenous sources of chromosome breaks and translocations leading to developmental defects and cancers, however, the mechanisms by which replication fork stalling causes DNA double strand breaks (DSBs) are not known. Despite significant analyses of DNA damage response proteins in global or single molecule studies where the sites of damage are not identified, the molecular mechanisms of replication-dependent DNA strand breakage and repair at specific sites in human cells are incompletely understood. To address this knowledge gap, we will study two types of natural replication barriers (CTG/CAG trinucleotide repeats and asymmetric purine-pyrimidine (Pu/Py) mirror repeats) integrated at an ectopic site in the human genome where their structure and effect on replication can be manipulated. We also examine several endogenous replication fork barriers that induce DSBs during DNA replication. We will use PCR, DNA sequencing, chromatin immunoprecipitation, mass spectrometry and flow cytometry to show (1) how polymerase stalling at noncanonical DNA structures causes DSBs, (2) how DNA repair proteins act to remodel stalled replication forks to restart synthesis, and (3) the mechanisms and genomic consequences of DSB recombination at structure-induced fork barriers. We will test the hypothesis that noncanonical DNA structures induce DSB by blocking the progress of DNA polymerases, promoting nuclease-sensitive fork regression, and inhibiting DNA end processing required for recombination. Conceptual advances from this work will include determination of the molecular mechanisms of DSB formation near specific stalled forks, biochemical analysis of replication fork reversal, and identification of how the processing of structure-induced DSB differs that of nuclease-induced `clean' DSB. Our long-term goal is to define the role of DNA structure-induced g e n o m e instability in human disease. Aim 1 will disclose the relationship between fork stalling and damage signaling, the biochemistry of fork reversal, the function of structure-specific endonucleases at stalled forks, and the impact of DNA secondary structure on fork resection and repair. Aim 2 will build on our demonstration that the Fanconi anemia type J protein (FANCJ) is essential for the maintenance of noncanonical DNA structures across the genome during replication stress, to determine the mechanisms of FANCJ dependent microsatellite stabilization. In Aim 3 we will characterize the genomic consequences of FANCJ deficiency. Our experiments will show how hard-to- replicate DNA sequences cause chromosome breaks and mutations that lead to genetic disease.