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)是一种罕见的隐性遗传性疾病，以骨髓衰竭和明显的癌症易感性为特征。FA通路是细胞对DNA交联剂如丝裂霉素C作出反应所必需的。然而，FA通路在体内的生理功能尚不清楚。我们最近发现，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。在APH治疗后，FA途径的取消会导致染色体断裂的增加，包括染色体脆性部位FRA3B和FRA16D的断裂。我们假设FA通路在维持DNA复制分叉稳定性方面发挥着不可或缺的作用，而DNA复制分叉稳定性是预防癌症的关键功能。为了进一步深入了解这一角色，本文提出了两个目标。首先，我们将描述FANCD2和DNA聚合酶处理因子增殖细胞核抗原之间的相互作用。利用定点突变技术，我们将突变最近发现的两个FANCD2-增殖细胞核抗原相互作用基序的关键氨基酸残基，并确定FANCD2-增殖细胞核抗原相互作用的功能意义。其次，利用整合报告质粒，我们将确定DNA复制增加应激诱导的FA细胞染色体断裂是否导致外源DNA整合增加。最后，我们将尝试建立这种表型的临床推论：我们将通过绘制FA头颈部癌中HPV整合的基因组位置，来确定FA细胞染色体脆性部位断裂增加是否与这些位点上HPV DNA整合的频率增加有关。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 recessive disorder characterized by bone marrow failure and pronounced cancer susceptibility. The FA pathway is required for the cellular response to DNA cross-linking agents, e.g. mitomycin C. However, the in vivo physiological function of the FA pathway remains unknown. We have recently discovered 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). Abrogation of the FA pathway leads to increased chromosome breakage, including breakage at the 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 cancer. Two aims are proposed to gain further insight into this role. First, we will characterize the interaction between FANCD2 and the DNA polymerase processivity factor PCNA. Using site-directed mutagenesis we will mutate the critical amino acid residues of two recently identified FANCD2 PCNA interaction motifs and determine the functional significance of the FANCD2-PCNA interaction. Second, using integration reporter plasmids, we will determine if the increased DNA replication stress-induced chromosome breakage of FA cells leads to increased integration of foreign DNA. Finally, we will attempt to establish a clinical corollary for this phenotype: We will determine if the increased 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 head and neck cancers. 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.