SV40 large T antigen (LT) is a viral oncoprotein with diverse biological functions. It is involved in cellular transformation through regulating the activities of tumor suppressors. It also plays an important role in viral DNA replication by assembling around the origin into a double hexamer that function not only as a helicase to open up the origin and unwind the fork DNA, but also as a platform for recruiting the essential cellular replication proteins, such as RPA. Our goals are to understand the structural basis of LT functions in these diverse biological processes by studying LT structures in their various oligomeric and conformational states.
Four specific aims are built upon our recent progress in the biochemistry/protein chemistry of LT and the crystallization of a LT fragment containing the larger C-terminal portion (residues 251-630). (i) Crystal structures of larger LT containing the N-terminal domains will be determined to leaas well as a logical approach with alternatives and potential problems. The reviewers felt that this was a highly improved application that addressed important questions. Success of these aims will not only provide new and exciting information about SV40 T antigen, but also about helicases and DNA replication initiation, in general. Further strengths of this application include the investigator, who is well trained and has been highly productive, the environment, and the excellent collaborators. The only weaknesses in the application include, in some cases, a lack of experimental detail, and a clear description of how the crystallographic data will address mechanism. The reviewers felt that these were minor compared to the strengths of the overall application. In summary, the study section was highly enthusiastic about this application. They felt that based on the preliminary data there was a high likelihood of success, the questions were significant, and the results from this study will have a high impact on both the understanding of SV40 LT and the field of eukaryotic DNA replication. ? ? ?
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