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.
STATEMENT This project focuses on DNA replication, a fundamental event that is required in all life forms, and its disruption can lead to several forms of disease, including cancer. The central engine of the replication machinery is the hexameric MCM complex that unwinds DNA at the replication fork once the tightly regulated replication process begins. MCMs and the kinases that activate them are logical, promising targets for anti-cancer therapies, and inhibitors of the Cdc7 kinase that phosphorylates MCMs to initiate its DNA unwinding function are currently in clinical trials.
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|Miller, Justin M; Enemark, Eric J (2016) Fundamental Characteristics of AAA+ Protein Family Structure and Function. Archaea 2016:9294307|
|Miller, Justin M; Enemark, Eric J (2015) Archaeal MCM Proteins as an Analog for the Eukaryotic Mcm2-7 Helicase to Reveal Essential Features of Structure and Function. Archaea 2015:305497|
|Froelich, Clifford A; Nourse, Amanda; Enemark, Eric J (2015) MCM ring hexamerization is a prerequisite for DNA-binding. Nucleic Acids Res 43:9553-63|
|Miller, Justin M; Arachea, Buenafe T; Epling, Leslie B et al. (2014) Analysis of the crystal structure of an active MCM hexamer. Elife 3:e03433|
|Froelich, Clifford A; Kang, Sukhyun; Epling, Leslie B et al. (2014) A conserved MCM single-stranded DNA binding element is essential for replication initiation. Elife 3:e01993|