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Professional Background:
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.
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Research Synopsis:
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.
Of principle 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.
T4 RNase 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.
In a 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.
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