Department of Biochemistry
Iowa City, IA 52242-1109 USA
fax: (319) 335-9570
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Marc Wold, PhD
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.
Figure Legend: Model of a Eukaryotic replication fork. Protein interactions important for coordination of leading and lagging strand synthesis are shown. RPA interactions with the DNA template, DNA polymerase alpha/primase and cellular helicases are all thought to be important for efficient DNA synthesis.
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.
Sparks, J.L., Kumar, R., Singh, M., Wold M.S., Pandita, T.K., Burgers, P.M. (2012) “Human Exonuclease 5 is a Novel Sliding Exonuclease Required for Genome Stability J. Biol. Chem. 287, Epub Oct 24. PMID: 23095756.
Lindsey-Boltz, L.A., Reardon, J.T., Wold, M.S., and Sancar, A. (2012) “In Vitro Analysis of the Role of RPA and RPA Phosphorylation in ATR-Mediated Checkpoint Signaling” J. Biol. Chem. 287, 36123-36131. PMID: 22948311.
Hass, C.S., Chen, R. and Wold, M.S. Detection of Post-translational Modifications of Replication Protein, in Methods in Molecular Biology (James L Keck, Ed.) Humana Press, 2012, Vol 922, 193-204. PMID: 22976188.
Hass, C.S., Lam, K., and Wold, M.S. (2012) “Repair-specific functions of Replication Protein A”. J Biol Chem. 287, 3908–3918. PMID: 22179778.
Neal, J.A., Dang, V., Douglas, P., Wold, M.S., Lees-Miller, S.P., and Meek, K. (2011) “Inhibition of HR by DNA-PK requires kinase activity, is titratable, and is modulated by autophosphorylation.” Mol. Cell Biol. 31, 1719-1733. PMID: 21300785.
Machwe, A., Enerlyn Lozada, E., Wold, M.S., Guo-Min Li, G-M., and David K. Orren (2011) “Molecular cooperation between the Werner syndrome protein (WRN) and replication protein A (RPA) in relation to replication fork blockage” J. Biol. Chem. 286, 3497-3508. PMID: 21107010.
Choi, J.H., Lindsey-Boltz, L.A., Kemp, M., Mason, A.C., Wold, M.S., and Sancar, A. (2010) “Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling” Proc Natl Acad Sci U S A. 107, 13660-5. PubMed PMID: 20616048.
Mason, A.C., Roy, R., Simmons, D.T., and Wold, M.S. (2010) “Functions of alternative Replication Protein A (aRPA) in initiation and elongation” Biochemistry 49,5919-28. PMID: 20545304.
Hass, C.S., Gakhar, L. and Wold, M.S. (2010) “Functional Characterization of a cancer causing mutation in human Replication Protein A” Mol Cancer Res, 8, 1017-26. PMID: 20587534.
Kemp, M.G., Mason, A.C., Carreira, A., Reardon, J.T., Haring, S.J., Borgstahl, G.E.O., Stephen C. Kowalczykowski,, S. C., Sancar, A., and Wold, M.S. (2010) “An alternative form of RPA (aRPA) expressed in normal human tissues supports DNA repair.” J. Biol. Chem. 285, 4788-4797. PMID: 19996105.
Haring, S.J., Humphreys, T.D. and Wold, M.S. (2010) “A naturally-occurring human RPA subunit homolog does not support DNA replication or cell cycle progression” Nucl. Acids. Res. 37 846-858. PMID: 19942684.
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