MAINTAINING DNA REPLICATION FORK STABILITY: ROLE OF THE FANCONI ANEMIA PATHWAY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 Fanconi贫血(FA)是一种罕见的疾病，以先天性畸形、骨髓衰竭和癌症易感性为特征。FA细胞对DNA交联剂高度敏感。然而，FA途径的内源性功能仍不清楚。我们最近已经证明，FA通路在细胞对DNA复制中断的反应中是功能所必需的。DNA聚合酶抑制剂APH处理FANCD2蛋白后，FANCD2蛋白通过单一泛素化被强烈激活。此外，在APH处理后，FA途径的取消会导致更多的染色体断裂，包括常见的染色体脆性部位FRA3B和FRA16D的断裂。我们假设FA通路在维持DNA复制分叉稳定性中起着不可或缺的作用。为了深入了解这一功能，本文提出了两个目标。首先，我们将描述FANCD2蛋白与DNA聚合酶处理因子增殖细胞核抗原之间的相互作用。其次，我们将确定APH诱导的FA细胞染色体断裂增加是否导致外源DNA整合增加。最后，我们将通过绘制FA患者肿瘤中人乳头瘤病毒(HPV)整合的基因组位置图，确定FA细胞常见染色体脆性部位断裂增加是否与这些位点上HPV DNA整合频率增加有关。我们建议的研究将导致关于FA通路在体内的生理作用的新发现，并为FA患者增加癌症易感性提供洞察力。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Fanconi anemia (FA) is a rare disease characterized by congenital anomalies, bone marrow failure, and cancer susceptibility. FA cells are hypersensitive to DNA crosslinking agents. The endogenous function of the FA pathway, however, remains unknown. We have recently demonstrated that the FA pathway is functionally required for the cellular response to disruption of DNA replication. The FA pathway is strongly activated via the mono-ubiquitination of the FANCD2 protein following treatment with the DNA polymerase inhibitor aphidicolin (APH). Furthermore, abrogation of the FA pathway leads to increased chromosome breakage, including breakage at the common chromosomal fragile sites FRA3B and FRA16D, following treatment with APH. We hypothesize that the FA pathway plays an integral role in the maintenance of DNA replication fork stability. Two aims are proposed to gain insight into this function. First, we will characterize the interaction between the FANCD2 protein and the DNA polymerase processivity factor PCNA. Second, we will determine if the increased APH-induced chromosome breakage in FA cells leads to increased integration of foreign DNA. Finally, we will determine if the increased common chromosomal fragile site breakage of FA cells is associated with an increased frequency of human papillomavirus (HPV) DNA integration at these loci, by mapping the genomic sites of HPV integration in FA patient tumors. Our proposed studies will lead to new findings regarding the in vivo physiological role of the FA pathway, and provide insight into the increased cancer susceptibility of FA patients.