Malignant transformation of human cells involves a multistep process of progressive genetic alterations. While research progress has been outstanding in defining alterations in the structure and expression of specific genes, our knowledge on changes occuring at the level of DNA replication is still extremely poor. The goal of this research proposal is to evaluate the unique details of cancer-related replication misfunctions in the human beings. Cancer cells possess uncontrolled and indefinite replication capacity, indicating that replication in human tumor cells differ in significant ways from replication in normal cells. Replication of the human genome involves a highly organized process of activation of individual replicons, which can be somatically an genetically disrupted during carcinogenesis. The studies proposed here will help us test certain critical aspects of this hypothesis, using as model replicon the human ribosomal RNA (rRNA) locus. The power and originality of this proposal derives from the modification of the human genome via a photocatalyzed DNA crosslinking reaction with psoralen. First, because the process may depend on the sensitivity of individual replicons to chemical carcinogenic damage, it is proposed to investigate the effect of DNA crosslinking on the replication of the rRNA replicon. Specifically, we will characterize the initiation and termination sites of the rRNA replicon by analyzing the transient inhibition in DNA replication and by identifying the replication structures blocked by PUVA. We will complement this topological mapping by studying the episomal replication of plasmid DNA carrying the initiation and termination regions of the rRNA locus in untreated and PUVA-treated cells. Second, because stable, somatically acquired alterations in the activation of specific replicons is involved in malignant transformation of human cells, it is proposed to identify alterations in the replication timing of the rRNA replicons in human tumor cells. Specifically, we will analyze initiation and elongation of the rRNA replicon throughout the S phase of untreated and PUVA-treated cells. We will compare the temporal pattern of replication of the rRNA replicon in the S phase of normal and tumor human cells with various degree of chromosomal ploidy. Third, since genetically acquired alteration of DNA replication are associated with cancer-proneness, it is proposed to analyze transient disruptions of the rRNA replicon in cells from the cancer-prone human syndrome xeroderma pigmentosum variant. Specifically, we will determine if DNA replication in XPV is uniquely sensitive the PUVA-induced intrastrand monoadducts. We will study the replication defect of XPV toward PUVA damage in distinct functional regions of the rRNA replicon, including the initiation and termination sites. The long-term aim of the proposed study is that the unraveling of replication misfunctions in the ribosomal RNA locus may be seen as a paradigm for understanding alteration in other critical human replicons, and hopefully may provide new therapies for cancer.
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