Research Interests 

Defining the kinetic mechanism of an enzyme means discovering the sequence of molecular events that occur during a single turnover from substrate binding through conformational changes, catalysis and finally product release. We use presteady-state techniques like stopped-flow and rapid chemical quenched flow to capture the reaction in the millisecond time scale in order to study these molecular events within the first turnover of a reaction. Currently, we are focusing on two DNA processing enzymes:
1) a DNA repair enzyme, MutY, which catalyzes the repair of a mismatch that forms with an oxidatively damaged guanine,
8-oxo-guanine, and 2) retroviral DNA integrase, an essential enzyme in the replication of retroviruses such as HIV. The goal of any presteady-state analysis is to define the reaction mechanism in order to correlate structural information, e.g. from X-ray crystallography or NMR, with actual molecular events in the time domain. Identification of key reaction intermediates and conformational states in turn will lead to greater understanding of pathological mutations and/or identification of structure-function-based inhibitors. We also use atomic force microscopy (AFM) to acquire detailed topological images of individual protein-DNA complexes. We are experimenting with following structural changes during the reaction by this technique and correlating the these structural changes directly with more traditional kinetic time courses.
 

Publications  

Patel, S.S., Wong, I., and Johnson, K.A. (1990) Presteady-State kinetic Analysis of Processive DNA Replication Including Complete Characterization of an Exonuclease-Deficient Mutant.  Biochemistry, 30, 511-525.

Wong, I., Patel, S.S., and Johnson, K.A. (1990) An Induced-Fit Kinetic Mechanism of DNA Replication Fidelity: Direct Measurement of Single-Turnover Kinetics.  Biochemistry, 30, 529-537.

Wong, I., Chao, K., Bujalowski, W., and Lohman, T.M. (1992) DNA-Induced Dimerization of the Escherichia coli Rep Helicase.  The Journal of Biological Chemistry, 267, 7596-7610.

Wong, I., and Lohman, T.M., (1992) Allosteric Effects of Nucleotide Cofactors on Escherichia coli Rep Helicase-DNA Binding.  Science, 256, 350-355.

Runyon, T.R., Wong, I., and Lohman, T.M. (1993) Overexpression, Purification, DNA Binding, and Dimerization of the Escherichia coli UvrD Gene Product (Helicase II).  Biochemistry, 32, 602-612.

Wong, I., and Lohman, T.M. (1993) A Double-Filter Method for Nitrocellulose-Filter Binding: Application to Protein-            Nucleic Acid Interactions.  Proc. Natl. Acad. Sci., U.S.A., 90, 5428-5432.

Wong, I., Amaratunga, M., and Lohman, T.M. (1993) Heterodimer Formation between Escherichia coli Rep and UvrD Proteins.  The Journal of Biological Chemistry, 268, 20386-20391.

Wong, I., and Lohman, T.M. (1995) Effect of Protein Assembly on Protein-DNA Binding.  Methods in Enzymology, G.K. Ackers and M. Johnson, eds., 259, 95-127.

Wong, I., Moore, K.J.M., Hsieh, J., and Lohman, T.M. (1996) ATPase Activity of Escherichia coli Rep Helicase is Dramatically Dependent on DNA Libation and Protein Oligomeric States.  Biochemistry, 35, 5726-5731.

Wong, I., Bjornson, K.J., and Lohman, T.M. (1996) ATPase Activity of Escherichia coli Rep Helicase Crosslinked to Single-Stranded-DNA: Implications for ATP Driven Helicase Translocation.  Proc. Natl. Acad. Sci. U.S.A., 93, 10051-10056.

Bjornson, K.J., Wong, I., and Lohman, T.M. (1996) ATP Hydrolysis Stimulates Binding and Release of ss-DNA from Alternating Subunits of the Dimeric Rep Helicase.  Implications for ATP-Driven Helicase Translocation.  Journal of Molecular Biology, 263, 411-422.

Wong, I., and Lohman, T.M. (1997) A Two-Site Mechanism for ATP Hydrolysis by the Asymmetric Rep Dimer P₂S as Revealed by Site-Specific Inhibition with ADP-AlF₄.  Biochemistry, 36, 3115-3125.

Bao, K.K., Skalka, A. M. & Wong, I. (2002) Presteady-State Analysis of ASV Integrase: I. Splicing Activity and Structure-Function Implications for Cognate Site Recognition. The Journal of Biological Chemistry, 277(14), 12089-12098.

Bao, K.K., Skalka, A. M. & Wong, I. (2002) Presteady-State Analysis of ASV integrase: II. Reverse-Polarity Substrates Identify Preferential Processing of the U3-U5 Pair.  The Journal of Biological Chemistry, 277(14), 12099-12108.

Wong, I., Lundquist, A.J., Bernards, A.S., and Mosbaugh, D.W. (2002) Presteady-State Analysis of a Single Catalytic Turnover by Escherichia coli Uracil-DNA Glycosylase Reveals a "Pinch-Pull-Push" Mechanism. The Journal of Biological Chemistry, 277(22), 19424-19432.

Bernards, A.S., Miller, J.K., Bao, K.K., and Wong, I. (2002) Flipping Duplex DNA Inside-Out: A Double Base-Flipping Reaction Mechanism by Escherichia coli MutY Adenine Glycosylase. The Journal of Biological Chemistry, 277(23), 20960-20964.

Bao, K.K., Wang, H., Erie, D.A., Skalka, A.M., and Wong, I. (2003) Functional Oligomeric State of Avian Sarcoma Virus Integrase.  The Journal of Biological Chemistry, 278(2), 1323-1327.

Bernards, A.S., Miller, J.K., Wirz, J.A., and Wong, I. (2003) A Dimeric Model for Contextual Target Recognition by MutY Glycosylase.  The Journal of Biological Chemistry, 278(4), 2411-2418.

Bao, K.K., Miller, J.K., Skalka, A.M., and Wong, I.  The C-terminal Domain of ASV-Integrase is Required for Disintegration Activity.  Manuscript in preparation.