研究领域

Biochemistry/Organic/Physical/Chemical Physics

The research in the Suo laboratory has three major directions: to elucidate kinetic mechanisms of enzymes involved in DNA/RNA replication, repair, and lesion bypass; to understand Hepatitis C (HCV) replication and regulation of innate immunity; to develop antiviral and anti-cancer molecules based on rational drug design. The Suo laboratory utilizes a variety of multi-disciplinary techniques to investigate intriguing questions in enzymology and to pursue rational drug design. Pre-steady state kinetic methods are employed using rapid chemical quench-flow and stopped-flow. These methods allow us to quench reactions on the millisecond time scale and to extract more kinetic information than the traditional steady-state kinetic methods. We also use protein engineering methods including site-directed mutagenesis and domain-swapping to study structure-function relationships of DNA polymerases. Single molecule FRET spectroscopy is used to investigate the kinetics and dynamics of individual protein molecules to reveal molecular details undetectable in bulk ensemble experiments. X-ray crystallography is being used to examine interesting enzyme-substrate complexes. These multi-disciplinary approaches will allow us to develop new methods and to advance enzymology into unprecedented territory. Our goals are to understand the elementary steps of conformational changes and chemical reactions occurring at the active site of enzymes. Our understanding of the kinetics, structure and dynamics of these enzymes is used for rational drug design. The designed enzyme inhibitors are being synthesized and tested in vitro and in vivo. Currently, we are investigating several systems described below. (1) Pre-Steady State Kinetic Studies of Replicative and Lesion Bypass DNA Polymerases DNA lesions often block DNA replication, so cells possess specific, often error-prone, DNA polymerases to bypass such lesions and to promote replication of damaged DNA. More than 220 DNA lesion bypass polymerases have been discovered. Most of these polymerases, which share sequence similarity and catalyze DNA polymerization with low fidelity and poor processivity, are classified into a new family: the Y-family. Human polymerases eta (η), iota (ι), kappa (κ) and Rev1 are examples of DNA lesion bypass Y-family enzymes. Pol η, encoded by hRAD30A, bypasses cis-syn pyrimidine- pyrimidine dimers efficiently and accurately. Mutations in hRAD30A inactivate Pol η and lead to UV-induced mutagenesis and skin cancer. The Suo laboratory is using pre-steady state kinetic methods to decipher the detailed mechanisms of correct and incorrect nucleotide incorporations opposite undamaged or damaged DNA templates by Dpo4, a thermostable polymerase from Sulfolobus solfataricus, Pol η, Pol ι, Pol κ, and Rev1. We have developed a novel assay, short oligonucleotide sequencing assay (SOSA), to determine the DNA sequence of lesion bypass products synthesized by Y-family enzymes. We are employing single molecule and stopped-flow FRET spectroscopy to probe the DNA binding properties and conformational dynamics of individual DNA polymerase molecules with undamaged and damaged DNA substrates. Furthermore we are working to crystallize binary and ternary complexes of Dpo4 bound to various types of damaged DNA and nucleoside analogs. Our studies will establish a general kinetic, thermodynamic, and structural mechanism for DNA translesion synthesis. More importantly, a better knowledge of the Y-family polymerases based on our results will facilitate the understanding of cancer formation and the development of anticancer drugs. Additionally, we are undertaking similar studies to investigate the kinetic mechanisms of replicative polymerases including Sulfolobus solfataricus polymerase B1 and Human DNA polymerase epsilon (ε). (2) Kinetic and Protein-Protein Interaction Studies of Human DNA Repair and Antibody Generation enzymes. Genomic DNA in every cell of the human body is spontaneously damaged more than 500,000 times every day. DNA repair plays a major role in maintaining the integrity of genomic DNA in cells. Human DNA polymerase lambda (λ) shares sequence similarity with the well-known DNA repair polymerase beta (β) and is thereby believed to catalyze base excision repair (BER). Human DNA polymerase mu (μ) shares sequence and functions similar with the well-known human deoxynucleotidyl transferase (TdT) which participates in antibody generation. My group has purified three X-family polymerases and is employing pre-steady state kinetic methods to characterize the kinetic mechanisms of these polymerases as well as those for other human enzymes involved in BER including an 8-oxoG Glycosylase (OGG1), AP endoculease (APE1) and DNA ligase 1. In addition, Pol λ and Pol μ have an N-terminal BRCT domain which interacts with cell-cycle checking proteins, such as the tumor suppressor p53. We are trying to identify these interacting proteins by employing immuno-precipitation assay and mass spectroscopy analysis. Moreover, we are trying to crystallize both Pol λ and Pol μ in the presence of DNA and dNTP substrates. (3) Design and Synthesis of Novel Nucleoside Analog Inhibitors Hepatitis C has infected about 2-3% of human population. Viral genome replication is crucial for viral life cycles and has been studied intensively. NS5B, the RNA-dependent RNA polymerase, which is at the center of viral replication, is one of major antiviral drug targets. Although there are extensive biochemical and steady-state kinetic studies on this polymerase, the elementary steps of nucleotide incorporation catalyzed by NS5B are still undefined. Using pre-steady state kinetic methods, we are studying the kinetic mechanism, processivity, fidelity, drug susceptibility, and drug resistance. The knowledge gained from these studies has severed as the basis for our rational design of nucleoside inhibitors. Currently, we are testing more than 140 nucleoside analogs which we have synthesized or obtained through collaboration in our cell-based assays. (4) Developing Anti-HCV Peptide-Based Inhibitors The non-structural proteins NS3, NS4A, NS4B, NS5A, and NS5B of HCV are processed from viral polyprotein precursor C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B by viral protease complex NS3/NS4A. NS3 has an N-terminal protease domain and a C-terminal helicase domain. The crystal structure of the NS3 protease domain shows that the N-terminus 28 residues are unfolded. In the complex with NS4A, the NS3 N-terminus folds into a beta sheet and an alpha helix, and the active site residues are slightly rearranged to form a catalytically favorable conformation. The NS3 protease is 995-fold more active in the presence than in the absence of NS4A. We are using the Stopped-Flow technology to study these conformational changes in NS3 after NS4A binding. We are also searching for tighter binding peptides to inhibit NS4A binding to NS3. The peptide inhibitors are then tested in the liver cell-line Huh 7-based HCV replicon assay. The inhibitory mechanism of the best peptide inhibitors will be studied further using confocal microscopy and multiphoton imaging. (5) Effects of HCV Protease NS3/4A on Human Kinases Involved in Immune Response Virus infections signal antiviral response through transcription factors, nuclear factor kB (NFkB) and interferon regulatory factors (IRFs). Current treatment includes interferon-a (IFN-a) based therapy that amplifies host antiviral response. In contrast, HCV has evolved unknown mechanisms to disrupt the host response to IFN-a. To examine the effect of HCV protease NS3/4A on these pathways, we are collaborating with Dr. Hao Wu at Cornell University Medical School to elucidate these novel pathways.

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Sherrer, S.M., Taggart, D.J., Pack, L.R., Malik, C.K., Basu, A.K, and Suo, Z.* (2012) Quantitative Analysis of the Mutagenic Potential of 1-Aminopyrene-DNA Adduct Bypass Catalyzed by Y-Family DNA Polymerases, Mutat. Res. In press. Wang, Y., Chu, X., Suo, Z., Wang, E., Wang, J.* (2012) Multi-domain Protein Solves the Folding Problem by Multi-funnel Combined Landscape: Theoretical Investigation of a Y-Family DNA Polymerase, JACS. 134 (33), 13755-13764. Sherrer, S.M., Maxwell, B.A., Pack, L.R., Fiala, K.A., Fowler J.D., Zhang, J. and Suo, Z.* (2012) Identification of an Unfolding Intermediate for a DNA Lesion Bypass Polymerase, Chem. Res. Toxicol. 25 (7), 1531-1540. Gowda, A.P., Krishnegowda, G., Suo, Z., Amin, S. and Spratt, T.E.* (2012) Low Fidelity Bypass of O2-(3-Pyridyl)-4-oxobutylthymine, the Most Persistent Bulky Adduct Produced by the Tobacco Specific Nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone by Model DNA Polymerases, Chem. Res. Toxicol. 25 (6), 1195-1202. Maxwell, B.A., and Suo, Z.* (2012) Kinetic Basis for Differing Response to an Oxidative Lesion by a Replicative and a Lesion Bypass DNA Polymerase from Sulfolobus Solfataricus, Biochemistry. 51, 3485−3496. Maxwell, B.A., Xu, C., and Suo, Z.* (2012) A DNA Lesion Alters the Global Conformational Dynamics of a Y-Family DNA Polymerase during Catalysis, J. Biol. Chem. 287 (16), 13040–13047. Sherrer, S.M., Sanman, L.E., Xia, C.X., Bolin, E.R., Malik, C.K., Efthimiopoulos, G., Basu, A.K., and Suo, Z.* (2012) Kinetic Analysis of the Bypass of a Bulky DNA Lesion Catalyzed by Human Y-family DNA Polymerases, Chem. Res. Toxicol. 25, 730-740. Song, Q., Sherrer, S.M., Suo, Z., and Taylor, J.-S. (2012) Preparation of a site-specific T=mCG cis-syn cyclobutane dimer-containing template and its error-free bypass by yeast and human polymerase eta, J. Biol. Chem. 287(11), 8021-8028. Brown, J.A., Pack, L.R., Fowler, J.D., and Suo, Z.* (2012) Pre-Steady State Kinetic Investigation of the Incorporation of Anti-Hepatitis B Nucleotide Analogs Catalyzed by Non-Canonical Human DNA Polymerases, Chem. Res. Toxicol. 25, 225-233. Ma, D., Fowler, J.D., and Suo Z.*(2011) Backbone Assignment of the Little Finger Domain of a Y-Family DNA Polymerase, Biomolecular NMR Assignments. 5(2), 195-198. Kirouac, K.N., Suo, Z., and H. Ling* (2011) Structural mechanism of ribonucleotide discrimination by a Y family DNA polymerase, J. Mol. Biol. 407(3), 382-390. Brown, J.A. and Suo, Z.* (2011) Unlocking the Sugar 'Steric Gate' of DNA Polymerases, Biochemistry, invited review 50, 1135-1142. Brown, J.A., Pack, L.R., Fowler, J.D. and Suo Z.* (2011) Pre-Steady State Kinetic Analysis of the Incorporation of Anti-HIV Nucleotide Analogs Catalyzed by Human X- and Y-family DNA Polymerases, Antimicrob. Agents Chemother. 55, 276-283. Brown, J.A., Pack, L.R., Sanman, L.E. and Suo Z.* (2011) Efficiency and Fidelity of Human DNA Polymerases Lambda and Beta during Gap-Filling DNA Synthesis, DNA Repair 10, 24-33. Sherrer S.M., Fiala, K.A., Fowler J.D., Newmister, S.A., Pryor, J.M. and Suo Z.* (2011) Quantitative Analysis of the Efficiency and Mutagenic Spectra of Abasic Lesion Bypass Catalyzed by Human Y-family DNA Polymerases, Nucleic Acids Research 39, 609–622. Sherrer S.M., Beyer, D.C., Xia, C.X., Fowler J.D., and Suo Z.* (2010) Kinetic Basis of Sugar Selection by a Y-family DNA Polymerase from Sulfolobus solfataricus P2, Biochemistry 49, 10179-10186. Brown, J.A., Pack, L.R., Sherrer, S.M., Kshetry, A.K., Newmister, S.A., Fowler, J.D., Taylor, J.-S. and Suo Z.* (2010) Identification of Critical Residues for the Tight Binding of Both Correct and Incorrect Nucleotides to Human DNA Polymerase Lambda, J. Mol. Biol. 403, 505-515. Ma, D., Fowler, J.D., Yuan, C. and Suo Z.* (2010) Backbone Assignment of the Catalytic Core of a Y-family DNA Polymerase, Biomolecular NMR Assignments 4, 207-209 DOI: 10.1007/s12104-010-9244-7. Brown, J.A., Fowler, J.D. and Suo Z.* (2010) Kinetic Basis of Nucleotide Selection Employed by a Protein Template-Dependent DNA Polymerase, Biochemistry 49, 5504-5510. Wong, J.H., Brown, J.A., Suo Z., Blum, P., Nohmi, T. and Ling, H.* (2010) Structural Insight into Dynamic Bypass of the Major Cisplatin-DNA Adduct by Y-family Polymerase Dpo4, EMBO J. 29, 2059-2069.