The long-term goals of this proposal are to understand the molecular mechanisms involved in the regulation of the initiation and termination of mammalian DNA replication. Various stress conditions have been found to activate cellular response mechanisms that repress the initiation of DNA replication at various levels. The possibility of damage to the genetic information is thus reduced, and the risk that cells will enter the pathway of carcinogenesis is minimized. We have observed that human nucleolin is involved in at least two pathways that act to inhibit DNA replication, but the mechanisms remain ill-defined. Along with the potential difficulties inherent in stress conditions, cells also have evolved devices (replication fork barriers; RFBs) to reduce the potential for damage caused by the movement of replication forks through critical genomic regions.In this grant, we first propose to characterize the interaction of nucleolin with human replication protein A (hRPA). The nucleolin domains that are critical for physical interaction with hRPA will be identified, and the ability of various stress conditions to induce the hRPA-nucleolin complex will be characterized. We will characterize the effect of nucleolin and hRPA phosphorylation on complex formation in vitro. Second, we characterize the interaction between human p53 and nucleolin. The critical domains on p53 and nucleolin necessary for complex formation in vitro and in vivo will be identified. We will examine the effect of nucleolin on the ability of p53 to transcriptionally transactivate genes involved in cell-cycle control. We will determine the p53 requirements for nucleolin relocalization. Third, we characterize the role of nucleolin phosphorylation in vivo. The critical phosphorylation sites on nucleolin that modulate the interaction with hRPA will be identified. The effects of mutating these sites on various nucleolin activities in vivo will be determined. Lastly, we will characterize the mechanism of replication termination by TTF-l. Using the simian virus 40 (SV4O) DNA replication system, we will analyze the DNA requirements for generation of an RFB in vitro. We will characterize the minimal protein requirements for formation of the RFB, and probe the protein-DNA interactions occurring at a stalled replication fork.
Showing the most recent 10 out of 23 publications
