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Department of Biochemistry

Research Interests

The human genome is composed of ~4x10⁹ base pairs of DNA. In order to grow, a human cell must precisely duplicate this entire genome each cell cycle and continuously protect it from damage or modification. Defects in DNA replication or repair result in higher rates of mutation, chromosome instability and have a direct role in a number of human diseases. Impaired responses to DNA damage and defects in DNA replication directly contribute to the development of cancer and are responsible for genetic diseases such as Fragile X and Xeroderma Pigmentosum.

Replication protein A (RPA) is a multi-functional, single-stranded DNA-binding protein composed of subunits of 70-, 32- and 14-kDa. RPA is essential for DNA replication, DNA repair, recombination and coordination of the cellular response to DNA damage. RPA is composed of six structurally conserved DNA binding domains that are all essential for life (see figure). The functions of these domains are poorly understood. RPA is phosphorylated during S-phase and in response to DNA damage. Recent studies have shown that RPA hyper-phosphorylation modulates RPA activity and plays a role in the cellular response to DNA damage; however, the mechanism of this regulation is also poorly understood.

Our current research focuses on utilizing a combination of in vitro and in vivo approaches to define functions of the domains of RPA and to elucidate the role of RPA in regulating cellular DNA metabolism. We are also studying the mechanism by which phosphorylation of RPA modulates the cellular response to DNA damage. We use variety of experimental approaches in our research. We use molecular and genetic approaches to generate specific mutant forms of RPA. Biochemical and structural analyses are used to define RPA interactions with DNA substrates and protein complexes found in cellular DNA metabolism. We have also have recently established a system that uses RNAi and plasmid directed expression of exogenous genes to examine the activity of wild-type and mutant RPA genes in human cells. These studies will lead to a better understanding of the molecular mechanisms of cellular DNA metabolism and will contribute to developing new treatments for diseases associated with defects in DNA repair or replication.

Recent Publications

Mason, A.C., Haring, S.J., Pryor, J.M., Staloch, C.A., Gan, T.F., and Wold, M.S. (2009) An alternative form of replication protein A prevents viral replication in vitro. J. Biol. Chem. 284:5324-5331. PMID: 19116208.

Dickson, A.M., Krasikova, Y., Pestryakov, P., Lavrik, O., and Wold, M.S. (2009) Essential functions of the 32-kDa subunit of yeast Replication Protein A. Nucl. Acids. Res. 37:2313-2326. http://dx.doi.org/10.1093/nar/gkp090.

Bartos, J.D., Willmott, L.J., Binz, S.K., Wold, M.S., and Bambara, R.A. (2008) Catalysis of Strand Annealing by Replication Protein A Derives from Its Strand Melting Properties. J. Biol. Chem. 283:21758-21768.

Binz, S.K. and Wold, M.S. (2008) Regulatory Functions of the N-terminal Domain of the 70-kDa Subunit of Replication Protein A (RPA). J. Biol. Chem. 283:21559-21570.

Kuhn-Parnell, E.J., Helou, C., Marion, D.J., Gilmore, B.L., Parnell, T.J., Wold, M.S., and Geyer, P.K. (2008) Investigation of the Properties of Non-gypsy Suppressor of Hairy-wing Binding Sites. Genetics 179: 1263-1273.

Haring, S.J., Mason, A.C., Binz, S.K., and Wold, M.S. (2008) Cellular functions of human RPA1: Multiple roles of domains in replication, repair, and checkpoints. J. Biol. Chem. 283:19095-19111.

Haring, S.J., and Wold, M.S. (2007) A common means to an end. Nature Struct. & Molec. Biol. 14:176-177.

Majka, J., Binz, S.K., Wold, M.S., and Burgers, P.M. (2006) RPA directs loading of the DNA damage checkpoint clamp to 5'-DNA junctions. J. Biol. Chem. 28:27885-27862.

Parnell, T.J., Kuhn, E.J., Gilmore, B.L., Helou, C., Wold, M.S. and Geyer, P.K. (2006) Identification of genomic sites that bind the Drosophila Suppressor of Hairy-wing protein. Mol. Cell. Biol. 26:5983-5993.

Binz, S.K., Dickson, A.M., Haring, S.J., and Wold, M.S. (2006) Functional assays for replication protein A (RPA). Meth. of Enzymol. Vol. 409, DNA Repair, Part B, J.L. Campbell & P. Modrich, Ed., pp. 11-38.

Olson, K.E., Narayanaswami, P., Vise, P.D., Lowry, D.F., Wold, M.S., Daughdrill, G.W. (2005) Secondary structure and dynamics of an intrinsically unstructured linker domain. J. Biomolecular Structure & Dynamics 23:113-124.

Binz, S.K., Sheehan, A.M., and Wold, M.S. (2004) Replication protein A phosphorylation and the cellular response to DNA damage. DNA Repair 3: 1015-1024.

Vassin, V.M., Wold, M.S., and Borowiec, J.A., (2004) Replication protein A (RPA) phosphorylatio prevents RPA association with replication centers. Mol. Cell. Biol. 24: 1930-1943.

Wyka, I.M., Dhar, K., Binz, S.K., and Wold, M.S. (2003) Replication protein A interactions with DNA: Differential binding of the core domains and analysis of the DNA interaction surface. Biochemistry 42: 12909-12918.

Daughdrill, G.W., Buchko, G.W., Botuyan, M.V., Arrowsmith, C., Wold, M.S., Kennedy, M.A., and Lowry, D.F. (2003) Chemical shift changes provide evidence for overlapping single-stranded DNA- and XPA-binding sites on the 70 kDa subunit of human replication protein A. Nuc. Acids. Res. 31: 4176-4183.

Binz, S.K., Lao, Y., Lowry, D.F., and Wold, M.S. (2003) The phosphorylation domain of the 32-kDa subunit of replication protein A (RPA) modulates RPA-DNA interactions. J. Biol. Chem. 278: 35584-35591.

Shen, J.-C., Lao, Y., Kamath-Loeb, A., Wold, M.S., and Loeb, L.A. (2003) The N-terminal domain of the large subunit of human replication Protein A binds to Werner Syndrome protein and stimulates helicase activity. Mechanism of Aging & Devel. 124: 921-930.

Jackson, D., Dhar, K., Wahl, J.K., Wold, M.S., and Borgstahl, G.E. (2002) Analysis of the human replication protein A:Rad52 complex: Evidence for crosstalk between RPA32, RPA70, Rad52 and DNA. J. Mol. Biol. 321(1): 133-148.

Mass, G. Nethanel, T., Lavrik, O.I., Wold, M.S., and Kaufmann, G. (2001) Replication protein A modulates its interface with primed DNA template during RNA-DNA primer elongation in replicating SV40 chromosomes. Nuc. Acids Res. 29: 3892-3899.

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. Nuc. Acid Res. 29: 3270-3276.

Kolpashchikov, D.M., Khodyreva, S.N., Khlimankov, Wold, M.S., Favre, A. and Lavrik, O.I. (2001) Polarity of human replication protein A binding to DNA. Nuc. Acids. Res. 29: 373-379.

Affiliations

Biochemistry Department
Molecular and Cellular Biology Program

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