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Room: BO 2022
Mail Stop: 602
Office: WO 4211
Phone: (419) 530-1510
Fax: (419) 530-4033
B.S. Chemistry, Eureka College, 1983;
Ph.D. Chemistry, University of Nebraska-Lincoln, 1989;
Iowa Cardiovascular Center Institutional Research Fellow, University of Iowa, 1992;
NIH Postdoctoral Fellow, University of Iowa, 1993;
NIH IRTA Fellow, NIAMS National Institute of Health, 1993-1998;
NIH Senior Staff Fellow, NIAMS National Institute of Health, 1998-2000.
The structural biology research program of Dr. Timothy C. Mueser, Ph.D. utilizes single crystal X-Ray diffraction methods to determine high-resolution atomic structures of protein and protein-DNA complexes. The major area of research involves the study of proteins associated with viral DNA replication. The study of viral model systems will offer insight into viral invasion and potentially offer new targets for antiviral drug design.
Ofprinciple interest are structural studies of the bacteriophage T4 DNA replication system in collaboration with Dr. Nancy G. Nossal, Chief, Laboratory of Molecular and Cellular Biology, NIDDK National Institutes of Health. The bacteriophage T4, a virus that infects bacteria, is well established as a model system for understanding the complex interactions required for DNA replication, repair, and recombination; fundamental processes that define cellular life cycles. The reconstituted in-vitro T4 DNA replication system requires nine phage-encoded proteins. I have completed the structure determination of two of the nine proteins; the T4 RNase H, a 5' to 3' exonuclease of the RAD2 family of enzymes, and the gene 59 helicase assembly protein, a structure specific DNA binding protein. The two structures provide a framework for the generation of multi-protein and protein-DNA complexes that will ultimately provide a detailed analysis of this intricate system.
T4RNase H is the 5' to 3' RNA/DNA editing exonuclease responsible for processing Okazaki fragments during DNA replication. I have previously reported the structure of native T4 RNase H with fully hydrated magnesium ions bound in the active site (Mueser et al., Cell, 1996, PDB 1TFR). The RAD2 family of DNA repair enzyme contains a clustering of highly conserved acidic residues that interact with the active site metals (Fig.). We have recently produced a metal free crystal form of the native enzyme (T4 RNase H). The metal free crystals can be soaked in different metal solutions, such as manganese and zinc, to determine the binding properties of the catalytic site. In addition, we have a series of site-specific mutations converting individual aspartate residues into asparagines, several of which eliminate catalytic activity but maintain DNA binding properties. We are currently investigating conditions and substrates for RNase H / DNA co-crystallization. In related studies, we are actively pursuing the structure determination of eukaryotic members of the RAD2 family of proteins including mouse, and yeast flap endonuclease1. Most recently, I have established a collaboration with investigators at Third Wave Technologies, Inc. that involves the crystallographic analysis of archaeal flap endonucleases.
The 1.45 Å resolution structure of T4 gene 59 protein has been published ( Mueser, et al. JMB, 2000, PDB 1C1K). The T4 gene 59 helicase assembly protein accelerates the loading of the gene 41 DNA helicase especially in the presence of single stranded DNA binding proteins. We have recently shown that 59 protein binds to fork and flap DNA suggesting that 59 functions to direct the assembly of the helicase specifically at the replication fork. The 59 protein also interacts with the gene 32 single-stranded DNA binding protein suggesting a role in the recruitment of the ssb to the lagging strand of the replication fork. We are currently investigating conditions for co-crystallization of 59 protein with DNA substrates, with gene 41 helicase, and with gene 32 protein.
Ina related study, in collaboration with Dr. Hiroshi Nakai, Georgetown University, we are investigating the structure of priA; an E.coli encoded protein with similar structure specific DNA binding activities to those discovered in the T4 gene 59 protein. In collaboration with Dr. Kevin D. Raney, University of Arkansas for Medical Sciences, we are investigating the structure of the T4 dda helicase, a T4 encoded non-processive DNA helicase involved in origin dependent and recombination dependent DNA synthesis.