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Molecular mechanisms of DNA replication
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abstract
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ABSTRACT DNA replication is a fundamental process for all organisms to precisely duplicate genetic material prior to cell division. Central to the process is a helicase enzyme that uses ATP-hydrolysis to separate base-paired DNA to allow polymerases to gain access to synthesize complementary strands and also to drive the replication machinery along the DNA. In human and other eukaryotic cells, the helicase engine is the 6- protein MCM complex. The mechanism for MCM-mediated manipulation of DNA is poorly understood at the molecular level because of a long-standing lack of structural information for several discrete functional states. The proposed research will fill a knowledge gap by providing detailed pictures of MCM proteins interacting with ATP compounds and with different forms of DNA. These will be studied at the molecular level by a coordinated approach involving structural studies by X-ray crystallography and in vitro methods to study their functions and interactions. A large body of preliminary data has been obtained for this project, including MCM:ATP co-crystal structures, important preliminary diffracting crystals of human MCMs with single-stranded DNA, and several purified proteins with confirmed biochemical activity.
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