| Name: |
| Gary W. Daughdrill |
| Title: |
| Assistant Professor |
| Degree: |
| Ph.D., 1997, University of Oregon |
| Phone: |
| (208) 885-9230 |
| Fax: |
| (208) 885-6518 |
| Email: |
| gdaugh@uidaho.edu |
| Lab/Office Location: |
| Life Science South, Room 155 |
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| Research Interests: |
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I. The relationship between protein flexibility and function
Research in my lab focuses on developing a greater
understanding of how the three dimensional structure of a protein specifies biological function. In particular, I am interested in
the relationship between protein flexibility and biological function. When novel genes are sequenced, their structure and function
can often be reliably predicted based on sequence similarity and evolutionary relationships to proteins with known structures.
Currently, this process assumes that the proteins being compared adopt compact rigid structures and tends to ignore proteins that
have partially collapsed, flexible structures. This is despite the fact that known flexible proteins and protein domains have
important biological functions and that analysis of genome sequence data has revealed proteins with flexible regions longer than
fifty amino acids are common in nature. The lack of information characterizing the partially collapsed flexible structures of
proteins inhibits our ability to predict their existence based on sequence data. It also limits our understanding of how the
sequences of such regions specify function, the presence of residual structure, and the degree of flexibility.
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To begin addressing these questions we are investigating the structure, dynamics, and function of a conserved flexible linker from
the 70 kDa subunit of replication protein A (RPA70). For the handful of sequenced PA70 homologues the similarity of the linker
varies significantly, going from 43% sequence identity between the H. sapien and X. laevis linkers to no significant
similarity between the H. sapien and S. cerevisiae linkers. It is unclear what selective processes have resulted in
the observed sequence variation for the RPA70 linkers. It is also unclear how the observed sequence variation affects the structure
and function of the linkers. If natural selection works to preserve flexible structures then one would expect that the linkers from
different species have acquired the same level of flexibility using different sequences. By testing this hypothesis we will begin
understanding the rules governing the evolution of protein flexibility.
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Protein sequence alignment of the RPA70 flexible linker and a fragment of the first ssDNA binding domain (SSB1) from
H. sapien (hs), X. Laevis (xl), A. thaliana (at), O. Sativa (os), D. melanogaster
(dm), C. elegans (ce), S. pombe (sp), and S cerevisiae (sc). Dark shading indicates identity and
light shading indicates conservative substitutions. The alignment is for residues 111-240 for all eight sequences and
was performed using Clustal 1.8. The alignment shows the stark contrast in sequence identity between SSB1 and the
flexible linker.
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II. The role of replication protein A in eukaryotic DNA repair
To promote the preservation and damage free replication of
genomic DNA a complex system of proteins that can efficiently recognize and repair the most frequently occurring types of DNA damage
has evolved. According to one model, if the repair proteins are not functioning properly then mutations will accumulate in the
genome, disrupting cellular function and eventually leading to cancer. In my lab we use a combination of molecular, biochemical, and
physical methods to investigate the structure, dynamics, and function of the 70 kDa subunit of human replication protein A, hRPA70,
an essential human gene that is a key participant in all human DNA repair mechanisms.
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Our investigations focus on two general areas, the role of flexibility and NTD/protein interactions in regulating the function of
hRPA70 in DNA repair and the structure and dynamics of the macromolecular interactions between hRPA70, p53, and/or constituents of
the excision nuclease. Investigating the role of flexibility in regulating the function of hRPA70 in DNA repair will illuminate a
poorly understood and overlooked structural property for this essential human protein. Investigating the structure and dynamics of
the macromolecular interactions essential for DNA Repair is a necessary step on the way to understanding the mechanisms of DNA
repair and cancer.
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Ribbon diagrams showing the backbone topology for residues 1-105 of the NTD. The backbone positions for NTD residues
involved in binding p53-tad (left) and ssDNA (right) are colored red, showing the binding sites overlap but are not
identical. Side chains and labels are shown for NTD residues that make up the basic cleft. The NTD residues colored red
in the p53-tad diagram are 17, 33, 41, 48, 55-56, 58-59, 61, 68, 87,89,91-93, and 99. The NTD residues colored red in
the ssDNA diagram are 35, 41, 42, 46, 55, 59, 61-62, 86, 88-93, and 98. The ribbon diagram for the NTD was adapted from
the solution structure of hsRPA701-168, PDB accession file 1ewi.
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| Selected Publications: |
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Daughdrill, G.W., Chadsey, M.S., Karlinsey, J.E., Hughes, K.T., and Dahlquist, F. W. (1997) The C-terminal Half of the Anti-Sigma Factor, FlgM, Becomes Structured When Bound to its Target, *28. Nat. Struct. Biol., 4(4) 285-91.
Daughdrill, G.W., Hanely, L.J., and Dahlquist F.W. (1998) The C-terminal Half of the Anti-Sigma Factor FlgM Contains a Dynamic Equilibrium Solution Structure. Biochemistry, 37(4) 1076-83.
Rupert P.B., Daughdrill, G.W., Bowerman, B., and Matthews, B.W. (1998) Structure of the C. Elegans Skn-1 DNA Binding Domain in Complex With DNA. Nat. Struct. Biol., 5(6) 484-91.
Jacobs, D.M., Lipton, A.S., Isern, N.G., Daughdrill, G.W., Gomes, X., Wold, M.S., and Lowry, D.F. (1999) Human Replication Protein A: Global Fold of the N-Terminal RPA-70 Domain Reveals a Basic Cleft and Flexible C-terminal Linker. J. Biomol. NMR, 14 321-331.
Daughdrill, G.W., Ackerman, J., Isern, N.G., Botuyan, M.V., Arrowsmith, C., Wold, M.S. and Lowry D.F. (2001) The Weak Interdomain coupling observed in the 70 kDa Subunit of Human Replication Protein A is unaffected by ssDNA Binding. Nucleic Acids Research, 29(15), 3270-3276.
Daughdrill G.W. (1999) Unfolding and Disassociation of the FlgM/*28 Complex are Thermodynamically Coupled. Prot. & Pep. Let., (6)2 79-86.
Buchko, G.W., Daughdrill, G.W., de Lorimier, R., Rao, S., Isern, N.G., Lingbeck, J.M., Taylor, J.S., Wold, M.S., Gochin, M., Spicer, L.D., Lowry, D.F., and Kennedy, M.A. (1999) Interactions of Human Nucleotide Excisions Repair Protein XPA with DNA and RPA70DC327: Chemical Shift Mapping and 15N NMR Relaxation Studies. Biochemistry, 38(46) 15116-15128.
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