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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 范可尼贫血（FA）是一种罕见的隐性疾病，其特征是先天性异常，骨髓衰竭和明显的癌症易感性。FA患者细胞对DNA交联剂如丝裂霉素C和顺铂过敏。然而，FA途径的内源性功能仍然未知。我们最近已经证明，FA途径是功能上所需的细胞响应DNA复制中断。FA途径通过用DNA聚合酶抑制剂阿非迪霉素（APH）处理后FANCD 2蛋白的单泛素化而被强烈激活。此外，废除FA途径导致增加的染色体断裂，包括在常见的染色体脆性位点FRA 3B和FRA 16 D断裂，用APH处理后。我们假设FA通路在维持DNA复制叉稳定性中起着不可或缺的作用，这是预防肿瘤转化的关键功能。提出了两个目标，以进一步了解这一职能。首先，我们将描述FANCD 2蛋白和主要的DNA聚合酶合成因子PCNA之间的相互作用。具体来说，我们将研究最近发现的FANCD 2 PCNA相互作用基序突变的功能后果。其次，我们将确定是否增加的APH诱导的FA细胞中的染色体断裂导致外源DNA的整合增加。最后，我们将试图建立一个临床推论FA途径的作用，在维持DNA复制叉稳定性：我们将确定是否增加常见的染色体脆性位点断裂FA细胞与增加频率的人乳头瘤病毒（HPV）DNA整合在这些位点，通过映射的基因组位点的HPV整合在FA患者肿瘤。我们提出的研究将导致新的发现，在体内的生理作用的FA途径，并提供洞察FA患者的癌症易感性增加。

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 recessive disorder characterized by congenital anomalies, bone marrow failure, and pronounced cancer susceptibility. FA patient cells are hypersensitive to DNA crosslinking agents such as mitomycin C and cisplatin. 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, a function critical for the prevention of neoplastic transformation. Two aims are proposed to gain further insight into this function. First, we will characterize the interaction between the FANCD2 protein and the major DNA polymerase processivity factor PCNA. Specifically, we will examine the functional consequences of mutation of a recently identified FANCD2 PCNA-interaction motif. Second, we will determine if the increased APH-induced chromosome breakage in FA cells leads to increased integration of foreign DNA. Finally, we will attempt to establish a clinical corollary for a role of the FA pathway in the in the maintenance of DNA replication fork stability: 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.