Research in the Suo laboratory has three major directions:
1. Elucidate kinetic mechanisms of enzymes involved in DNA/RNA replication repair, and lesion bypass
2. Understand Hepatitis C (HCV) replication and regulation of innate immunity.
3. Develop antiviral and anticancer molecules based on rational drug design.
The Suo lab 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.
Pre-Steady State Kinetic Studies of Replicative and Lesion Bypass DNA Polymerase
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).
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 (b) 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.
Design and Synthesis of Novel Nucleoside Analog Inhibitors
Hepatitis C has infected about 2-3% of the 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.
Utility of the Programmable Endonuclease, Cas9
Recently, it was discovered that bacteria have adaptive immunity. Using Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) DNA sequences in conjunction with CRISPR-associated (Cas) genes, bacteria are able to utilize an RNA-guided endonuclease to recognize non-native genetic material and target it for digestion. This system serves to protect bacteria from viruses as well as regulate incorporation of plasmids from other bacteria. The discovery of this bacterial adaptive immune system and the programmable endonucleases involved has revolutionized genomic editing. Cas9, the most popular of programmable endonucleases, has rapidly become one of the most useful tools for modifying cellular genomes. Due to its ease of use and simple programming platform, Cas9 has greatly surpassed other genome editing systems such as Zn-finger endonucleases and TALENs and is constantly being improved. Research in my lab incorporates the utility of Cas9 as a genome editing tool to answer questions regarding genetic mutations, DNA damage, and human diseases.
Collaboration with Biosortia Pharmaceuticals to Screen Microalgal Compounds as Potent, Low toxicity HCV Treatments
Hepatitis C virus (HCV) is a major burden to human health. Chronic HCV infection leads to development of hepatocellular carcinoma, the third leading cause of cancer-related deaths and the sixth most prevalent cancer worldwide. The only current chemotherapy drug proven to increase survival of liver cancer patients is Sorafenib, which only increases the median survival time of patients by three months. Moreover, current drug treatments against HCV are very toxic, expensive, and require long, grueling treatment courses. Notably, several of these anti-HCV drugs, including elbasvir, ledipasvir, daclatasvir, dasabuvir, are natural product-like molecules. We are currently collaborating with Biosortia Pharmaceuticals Inc. to screen their unique library of natural products isolated from aquatic microorganisms. Using cell-based assays, we aim to identify potent inhibitors of HCV replication that have low toxicity. We hope that our work will contribute to development of valuable therapeutics that will reduce the occurrence of HCV-related cancer and improve the survival and quality of life for infected individuals. [Press Release]