Molecular genetics; Protein Function and Structure; DNA Replication, Recombination and Repair; Bacterial Pathogenesis
My associates and I use a combination of biochemical and biophysical approaches to study the molecular basis of stalled DNA replication fork rescue. Our model organism is the well-characterized bacterium Escherichia coli (E. coli), since the majority of the proteins thought to be involved in fork rescue are known. Most of our experimental work is concerned with the function and regulation of the complexes that control fork rescue, with studies focused primarily on the role of the single-strand DNA binding protein (SSB) and several recombination DNA helicases. Comparative studies are also underway using selected components of some medically relevant bacterial organisms. We collaborate with scientists from the National Institutes of Health (NIH) and other research institutions. The team working in my lab consists of undergraduate and graduate students, postdoctoral fellows and a technician. We seek to understand fork rescue utilizing both bulk-phase and single molecule techniques. Typically, studies focus initially on purification and characterization of the various proteins (there are now more than 10 being studied). We study DNA binding, unwinding and the hydrolysis of adenosine triphosphate (ATP) using a combination of modern spectroscopic (both ultraviolet–visible and fluorescence) and equilibrium binding methods. The goal of these initial studies is to understand the range of DNA substrates on which an enzyme can act, as a means to understanding its role in vivo. This is followed by careful single molecule studies using a technique I pioneered that combines optical tweezers, microfluidics and high-resolution fluorescence microscopy. My research team is also pursuing a new area of research targeted at developing small molecule inhibitors. These are aimed at disrupting binding between SSB and the 12-14 proteins comprising the SSB-interactome. As SSB is an essential protein and its binding to interactome partners is required for viability, the goal of these studies is to identify inhibitors that will be further developed into novel antibiotics.
Allergy and Immunology; Medical Microbiology; Infectious Disease; Microbiology; Genomics and proteomics; Immunology; Microbial Pathogenesis; Molecular and Cellular Biology; Molecular Basis of Disease; Molecular genetics; Gene Expression; Signal Transduction; Protein Function and Structure; Bacterial Pathogenesis
Research efforts in my laboratory are focused in the fields of immunology and bacterial pathogenesis, two diverse fields of biomedical research for which I have two separate research groups. Projects in both fields are performed by undergraduates, doctoral and master’s degree students, postdoctoral fellows and senior research associates. One major focus of my laboratory is studying the regulation of mucosal immune responses. We investigate the cellular and molecular events by which Type II heat-labile enterotoxins (HLTs), produced by certain strains of Escherichia coli, modulate immune responses. We have demonstrated that LT-Ilia, LT-IIb and LT-IIc, when co-administered with an antigen, have the capacity to enhance antibody and cellular immune responses to that antigen. Using a variety of immunological and cellular technologies, including flow cytometry, fluorescence resonance energy transfer (FRET) detection, cytokine multiplex analysis, mutagenesis, quantitative Reverse Transcription PCR (qRT-PCR), RNA-sequencing (RNA-Seq) and a variety of transgenic mice, we are investigating the mechanisms by which these immunomodulators productively interact with various immunocompetent cells (T cells, B cells, dendritic cells, macrophages) to induce or suppress cytokine production, costimulatory ligand expression and cellular proliferation. A practical outgrowth of these experiments is the potential to engineer novel recombinant vaccines by genetically fusing antigens from different pathogens to the enterotoxins. Recent experiments have shown that these HLT are lethal for triple-negative breast cancer cells, which has opened a new area of oncological research for the lab. A second focus of my laboratory is to investigate the molecular mechanisms by which adherent-invasive Escherichia coli (AIEC) induce, exacerbate or prolong the symptoms of inflammatory bowel disease (IBD) and Crohn’s disease, two acute and chronic inflammatory diseases of the human gut. In vitro, AIEC strains invade into the cytoplasm of several epithelial cell lines. Using recombinant screening methods and RNA-Seq technologies, we are identifying the genes of AIEC that are required to attach and to invade gut cells.
Protein Function and Structure; Proteins and metalloenzymes; Vitamins and Trace Nutrient
Cytochrome P450 enzymes are powerful catalysts that play integral roles in biochemical pathways throughout nature. In mammals, members of this class of enzyme serve a variety of functions that include drug metabolism, steroid biosynthesis and the activation and deactivation of vitamin D, to name a few. Cytochrome P450 enzymes are also heavily involved in bacterial and plant biochemistry. The overall goal of our research is to use a combination of biochemical and biophysical tools to investigate structure and function in class I cytochrome P450 enzymes, thereby contributing toward an understanding of how this important class of enzymes work as well as informing the design of therapeutics. This goal is divided between two efforts. First, we are interested in characterizing the substrate and redox partner interactions of the enzyme CYP24A1, the P450 responsible for deactivating vitamin D. Describing the interaction between CYP24A1 and vitamin D has the potential to illuminate how the vitamin D structure becomes modified at a particular site. This insight could impact the design of vitamin D analogs with benefits for an array of human health conditions, including bone density disorders, diabetes and chronic kidney disease (CKD). A parallel effort in our group is a structural study of the enzyme CYP121 of Mycobacterium tuberculosis, the disease-causing pathogen in tuberculosis (TB). The resurgence of standard TB and the rise of drug-resistant forms of TB are quickly becoming a global pandemic, with TB claiming more lives worldwide in 2014 than HIV. CYP121 is essential for survival of the bacterium and thus has emerged as one of the more promising antitubercular drug targets. Students and postdocs joining my lab will be exposed to a multidisciplinary set of research tools, including expression and purification of recombinant membrane protein, nuclear magnetic resonance, protein X-ray crystallography and P450 ligand binding assays.
Protein Function and Structure
My research focus is the development of new methods of analysis in structural biology. My lab uses tools such as X-ray crystallography and small angle scattering to probe the structure and dynamics of biological macromolecules such as proteins and nucleic acids. Our approaches include developing new computational algorithms to analyze data and using cutting edge technologies such as X-ray free electron lasers (XFELs) to collect data on ultrafast time-scales. By developing these tools, we can build pictures and movies of biological molecules to help understand how they perform their functions in the cell. These insights not only help drive advancements in basic science but also help in the design of new drugs for treating diseases.
Structural Biology; X-ray Crystallography; Microbial Pathogenesis; Microbiology; Protein Function and Structure; Proteins and metalloenzymes
My research program aims to understand how bacteria produce natural products, small molecules that are secreted from the cell to adapt to diverse environments. These molecules allow the bacteria to compete with other microbes or, in the host-pathogen setting, to establish or exacerbate an infection. Natural product biosynthesis may therefore serve as a target for antimicrobial development. My lab uses a variety of techniques to examine these pathways. A core approach is to use X-ray crystallography to determine the molecular structure of proteins that catalyze important steps in natural product biosynthesis. Structural observations are tested and validated using biochemical techniques to examine the catalytic reactions. Finally, molecular and cellular techniques are used to examine biosynthetic gene cluster activity in the cell. These studies will inform efforts to engineer enzymes to produce novel natural product and identify new products of previously uncharacterized pathways. I have a long-standing interest in the Nonribosomal Peptide Synthetases (NRPSs), a family of large, multidomain enzymes that produce important peptide natural products like the antibiotic vancomycin or the anticancer agent bleomycin. NRPSs operate like an assembly line in which the nascent peptide is attached to a carrier domain that shuttles the synthetic intermediates to neighboring catalytic domains. The carrier and catalytic domains are often joined in a single polypeptide that is thousands of residues in length. By examine the crystal structures of large NRPS proteins, we have determined some of the features that enable this fascinating biosynthetic mechanism. Many NRPS products are siderophores, small molecules that bind iron and are required for growth in the pathogenic environment. My lab also studies aerobactin, an NRPS-independent siderophore pathway that is a virulence factor for hypervirulent Klebsiella pneumoniae. We have biochemically and structurally characterized the aerobactin biosynthetic pathway and have developed an approach to find inhibitors of aerobactin biosynthesis that may be tools to probe the pathway chemically to inhibit growth of this human pathogen.
Pathophysiology; Cytoskeleton and cell motility; Endocrinology; Eukaryotic Pathogenesis; Gene Expression; Molecular Basis of Disease; Molecular and Cellular Biology; Molecular genetics; Protein Function and Structure; Regulation of metabolism; Transcription and Translation
In my laboratory, we are interested in structural components of the cell, their role in establishing and regulating cellular functions, and how this regulation translates into physiological consequences in health and disease. We have two major focus areas: 1) The role of cytoskeletal elements in prostate cancer development and progression and 2) The role of nucleoskeletal elements in establishing and maintaining nuclear structure and function. 1) The majority of death from cancer is caused by metastasis, the spreading of cancer cells from the site of a primary tumor to other body parts. We use a combination of biochemical, cell biological, physiological, and translational approaches to elucidate the mechanisms that are involved in the acquisition of metastatic phenotypes. Specifically, we focus on the role that myosins play in this process. We are also interested in how dietary fats can contribute to the development of metastatic phenotypes in prostate cancer cells. 2) Aberrations in nuclear structure and dynamics are the underlying cause of diseases ranging from cancer to premature aging. We are interested in the role of nuclear actin and myosins in regulating dynamic nuclear processes such as nucleolar assembly and functions in health and disease.
Neuroimmunology; Behavioral pharmacology; Gene therapy; Immunology; Molecular and Cellular Biology; Molecular Basis of Disease; Neurobiology; Gene Expression; Signal Transduction; Protein Function and Structure; Neuropharmacology
My research spans three interrelated fields: chronic pain, depression and inflammation. Experiments in my laboratory focus on how brain-derived pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF), function as modulators of brain-body interactions during neuropathic pain and how brain-TNF is involved in the mechanism of action of antidepressant drugs. My overall goal is to advance knowledge of, and therapeutic efficacy for pain, depression, neuro-inflammation and drug addiction. This research is based on my earlier work showing that neurons produce the pro-inflammatory cytokine TNF and that the production of TNF by macrophages is regulated by neurotransmitters. Cytokines and neurotransmitters are principal signaling molecules that mediate bidirectional communication between the nervous and immune systems--the crosstalk important in maintaining homeostasis. Consequently, aberrant production of either of these two classes of mediators could profoundly affect signaling by the other, thereby impacting health. A shift in balanced cytokine-neuron interactions that regulate neurotransmitter release in the central nervous system (CNS), and that have potential behavioral consequences, manifest themselves as states of depression and chronic pain. My research uses both cell systems and animal models to test these hypotheses. Colleagues and I use a combination of imaging techniques to localize cytokine production, bioassays and ELISA (enzyme-linked immunosorbent assays) for pharmacological and functional analyses, electrophysiological (brain slice stimulation) and molecular methods for our studies. In addition to investigating neuron functioning in the brain, trainees in my laboratory also study the peripheral macrophage, a major source of TNF during inflammation. Specifically studying neurotransmitter regulation of TNF production in the periphery is enhancing our knowledge of how the brain controls a peripheral inflammatory lesion. Our studies are designed to investigate the mechanisms of centrally mediated pain as associated with immune dysfunction and to elucidate mechanisms of drugs used to treat such pain states. My projects are evolving to investigate the mechanisms and neural pathways involved in TNF neuromodulator functions during chronic pain (due to peripheral nerve injury and diabetes) and stress-induced depressive behavior. We also study mechanisms contributing to the comorbidity of chronic pain and depression. I collaborate with researchers in several UB departments and at other institutions. Our projects include using noninvasive methods for delivery of anti-TNF therapeutics for chronic pain, elucidating the neural-immune mechanisms involved in the rapid recovery afforded by centrally administered anti-TNF therapy and using nanotechnology-mediated, targeted gene silencing within the CNS. I am invested in helping my undergraduate and graduate students, medical residents and postdoctoral fellows realize their potential and achieve their goals. Previous students have advanced professionally and hold clinical, academic and industrial positions.
Membrane Transport (Ion Transport); Neurobiology; Protein Function and Structure; Proteins and metalloenzymes; Vitamins and Trace Nutrient
The long term goal of the research conducted in my lab is to learn about the general principles that organisms use to acquire and metabolize the essential nutrients iron, manganese and copper. Since in eukaryotes, iron metabolism, for example, depends on the activity of copper-containing enzymes called ferroxidases, we examine the trafficking copper in cells as well. In addition, as divalent metal ions, manganese and ferrous iron share many of the same trafficking pathways. The first challenge for a cell is to scavenge these metals from the environment. This is true for a yeast cell in culture, for an epithelial cell in your intestine, an endothelial cell in the capillaries in the brain, or a neuron. The second challenge is to efficiently and correctly partition these metals in the cell for subsequent utilization and storage. Ultimately the cell or organism will have to regulate the accumulation of these metals and to ensure that they are not allowed to roam "free" since all three are toxic. Yet all are essential micronutrients, as well. They are required in fundamental cellular processes such as cellular respiration in all organisms, and for vital physiologic functions such as oxygen transport in blood and muscle. The brain has a strong requirement for iron and copper to support the elevated energy metabolism needed to support neuronal function; manganese is essential to neurotransmitter synthesis. This essentiality is contrasted by cytotoxicity that results from their strong tendency to generate oxygen radicals which in turn destroy key cellular components. For example, iron uptake into the brain must be tightly regulated, a process we focus in our research. Failure of this regulation can result in a variety of brain pathologies particularly those that result in degeneration of neuronal function. We study in detail the role of the amyloid precursor protein and alpha-synuclein in iron and manganese trafficking and how these functions are related these proteins‘ roles in neurodegenerative disease.
Genomics and proteomics; Protein Function and Structure; Proteins and metalloenzymes
The Malkowski Laboratory is focused on understanding the structure and function of integral membrane enzymes involved in the conversion of lipid precursors into potent bioactive signaling molecules. We utilize a myriad of methods and techniques to characterize these enzymes, including X-ray crystallography, electron spin resonance spectroscopy, protein chemistry, biochemistry, molecular biology, cell biology, and kinetics.
Pulmonary & Critical Care Medicine; Immunology; Protein Function and Structure; Proteins and metalloenzymes
I am engaged in clinical, teaching and research responsibilities related to the evaluation and treatment of patients with pulmonary disease or patients who are critically ill. My inpatient practice situated at the medical intensive care unit (MICU) at the Buffalo General Medical Center (BGMC) positions me to provide ongoing medical care to patients who are critically ill and require significant life support therapies to sustain life or vital bodily functioning. I am specifically interested in asthma, COPD, interstitial lung disease, pleural disease, pulmonary hypertension and lung cancer, but deal with a variety of lung disease. I evaluate patients with pulmonary disorders including shortness of breath, lung masses, abnormal chest imaging, abnormal pulmonary function tests, chronic obstructive pulmonary disease (COPD), asthma, pleural disease, interstitial lung disease, pulmonary hypertension, and lung cancer, at the UBMD practice location at Conventus. As a member of the UBMD pulmonary division, I provide inpatient pulmonary consultation at both BGMC and Roswell Park Cancer Institute. Currently, I am focusing on the analysis of Big Data in the medical/healthcare fields. I am mainly focused on the application of drug repurposing in translation and clinic research. Additionally, I am engaged in the study of the human airways microbiome and metagenome. The human microbiome is the collection of all the microbial organisms in a human body, and the corresponding metagenome is the collection of the genes, and gene products of the microbes. Due to the potential impact of the microbiome on human health and disease, I am interested in studying the putative effects the interaction with human hosts, specifically innate immunity interaction with the metagenome in lung disease. My collaborators include the Division of Allergy and Immunology. We endeavor to elucidate immune cell function in airway diseases such as asthma and COPD. Our research focuses on the development of therapeutics aimed at novel targets identified as necessary in the molecular basis of pulmonary disease; efficacious laboratory results will generate more effective treatment plans for patients. I am actively involved in teaching medical students, residents, and fellows about the appropriate care of the patient with either pulmonary disease and critical illness.
Inherited Metabolic Disorders; Membrane Transport (Ion Transport); Molecular Basis of Disease; Molecular and Cellular Biology; Molecular genetics; Protein Function and Structure; Transgenic organisms; Vision science
Most physiological processes and numerous disease states influence or are influenced by pH. Even relatively small deviations in whole body pH can have devastating consequences for our health. Our bodies are subject to a constant challenge from dietary and metabolic acids, thus it is critical for the body to have mechanisms that tightly regulate pH. Blood plasma pH is maintained at a value close to 7.4, predominantly thanks to the buffering action of 24 mM bicarbonate (HCO3-). HCO3- neutralizes acid, generating carbon dioxide and water (HCO3- + H+ to CO2 + H2O), preventing lethal acidosis. I study the SLC4 family of membrane proteins that move acid/base equivalents across cell membranes. Notable members include  the Na/2HCO3 cotransporter NBCe1-A that reclaims HCO3- from filtered blood plasma in kidney tubules (preventing loss of vital plasma HCO3- to the urine),  NBCe1-B that promotes fluid removal from the corneal stroma (preventing corneal edema and vision loss),  the Cl-HCO3 exchanger AE1 that promotes O2-CO2 exchange in red blood cells, and  SLC4A11 that conducts H+ and promotes corneal clarity. Dysfunction of SLC4 family members is associated with renal tubular acidosis, blindness, cancer, deafness, epilepsy, and hypertension. Course Director for PGY405/505 (Cellular and Molecular Physiology) Course Co-Director for IMC512 (Renal Module)
Endocrinology; Molecular Basis of Disease; Neurobiology; Regulation of metabolism; Inherited Metabolic Disorders; Protein Function and Structure; Transgenic organisms
One research goal is to investigate the structure-function relationships and regulation of the human pyruvate dehydrogenase complex (PDC). We investigate the catalytic mechanism of the pyruvate dehydrogenase (PDH) component and its interactions with the dihydrolipoamide acetyltransferase (E2) component of PDC. We also determine the loci of interactions between PDH kinases (four PDK isoenzymes) and the lipoyl domains of E2. Using a PDC-knockout mouse line we investigate the importance of glucose metabolism as a source of energy for fetal development as well as the role of PDC in glucose-stimulated insulin secretion by pancreatic beta cells. Another research goal is to investigate diet-induced metabolic programming during early life. We investigate (i) the effects of an altered nutrition during the immediate postnatal life on development of adult-onset obesity and (ii) the effects of maternal obesity on fetal programming. Current research focuses on the role of the hypothalamic signaling pathways in rodents with diet-induced obesity and also in the progeny of obese mothers.
Gene Expression; Molecular Basis of Disease; Molecular and Cellular Biology; Molecular genetics; Protein Function and Structure; Transcription and Translation
Our laboratory uses genetic, biochemical and molecular biological approaches to study the molecular mechanisms of eukaryotic transcription initiation and regulation. Studies in our laboratory utilizing both the budding yeast Saccharomyces cerevisiae and human cells have resulted in the identification and biochemical characterization of mutants of RNA polymerase II (RNAPII) and the general transcription factors TFIIB and TFIIF that coordinately affect transcription start site utilization and transcript elongation. These studies support a model where yeast and human TFIIF induce conformational changes in RNAPII that result in structural and functional changes in the polymerase active center.
Neurodegenerative disorders; Ion channel kinetics and structure; Membrane Transport (Ion Transport); Molecular Basis of Disease; Neurobiology; Neuropharmacology; Protein Function and Structure; Signal Transduction
We investigate the activation mechanisms of fast neurotransmitter receptors. We seek to define the activation pathway, modulatory mechanisms and structure-function relationships of the N-methyl-D-aspartate (NMDA) receptor to better understand the roles played by this protein in the brain. NMDA receptors are the most abundant glutamate-stimulated, Ca2+-conducting ion channels in brain and spinal cord. They are the predominant molecular devices for controlling synaptic development and plasticity and govern memory and learning processes. Understanding the mechanisms that control their activity may lead to more effective strategies to treat neuropathies including stroke, neurodegenerative conditions, chronic pain and addiction as well as mental disorders such as schizophrenia and epilepsy.
Ion channel kinetics and structure; Membrane Transport (Ion Transport); Molecular Basis of Disease; Molecular and Cellular Biology; Protein Folding; Protein Function and Structure; Signal Transduction
Work in my lab seeks to elucidate the transduction mechanisms of ion channels involved in thermal sensation and pain, such as the heat-activated vanilloid receptors (TRPV1-4) and the cold-activated TRPM8 – the so-called thermal TRP channels. Expressed in peripheral afferent nerve endings, these channels function as an array of thermometers for sensing ambient temperature from noxious cold to noxious hot. While all proteins are thermally sensitive, thermal TRP channels are gated by temperature and possess unprecedentedly high temperature dependence. But the mechanisms of their temperature gating has remained mysterious, in contrast to our abundant knowledge on other types of ion channel gating (e.g. voltage or ligand-driven). Thermal TRP channels are also distinct for their polymodal responsiveness. TRPV1, for example, is responsive to heat, voltage, pH, capsaicin (i.e. the hot ingredient of chili peppers) among many other irritant compounds. The channels are thus informative for deciphering how biological proteins achieve multitasking. Thermal TRP channels also have receptor-like roles in mediating intracellular signaling. The calcium influx through the channels has potentially a broad spectrum of functional consequences, one of which is the desensitization of the channels themselves, a phenomenon that is believed to underlie peripheral analgesics. Our research is centered on problems like these, and we approach them by a combination of techniques such as recombinant mutagenesis, patch-clamp recording, fluorescence measurements, quantitative modeling, etc, which together allow us to draw insights into functions of the channels at mechanistic levels. Complementing our experimental studies, we are also interested in development of methodology to ever extend experimental resolutions. For example, to time-resolve temperature-dependent activation of thermal TRP channels, we have developed a laser diode-based temperature clamp apparatus, which achieves for the first time a submillisecond resolution (>105 oC/s) while capable of clamping temperature constant. For the past decade we have also been developing sophisticated algorithms for statistical analysis of single-molecule measurements such as single-channel patch-clamp recordings, which can help unravel the richness of data pertaining to molecular mechanisms at high resolutions. Together, these approaches provide us with unique abilities for in-depth studies of structure-mechanisms of ion channels.
Eukaryotic Pathogenesis; Gene Expression; Genomics and proteomics; Infectious Disease; Microbial Pathogenesis; Microbiology; Molecular and Cellular Biology; Molecular genetics; Protein Function and Structure; RNA
Trypanosoma brucei is a eukaryotic pathogen that causes human African trypanosomiasis, a disease that is invariably fatal if not treated. Essential and novel processes in this parasite may serve as starting platforms for new chemotherapeutics, which are urgently needed. Our laboratory combines biochemical, genetic, genomic and proteomic approaches toward understanding gene regulation and protein modification in this pathogenic eukaryote. One focus in my laboratory is RNA editing, a novel mechanism for regulating mitochondrial gene expression in which sequence information is added to mRNAs after transcription by specific insertion and deletion of uridine residues. RNA editing is essential for creating translatable open reading frames (ORFs). We are performing functional and biochemical characterization of the large, dynamic RNA-protein complex termed MRB1, which coordinates multiple aspects of the RNA editing process. A second focus is on regulating RNA stability and translational control in T. brucei, which constitute the major methods of gene regulation in this organism. We identified an RNA binding protein, DRBD18, that impacts the stabilities of hundreds of mRNAs. Our data support a model in which posttranslational modification of DRBD18 by arginine methylation acts as a switch to change DRBD18 from an mRNA destabilizer to an mRNA stabilizer by regulating specific protein-protein and protein-RNA interactions. We are testing this model in vitro and in vivo using reporter assays, in vivo protein-RNA cross-linking and protein-protein interaction assays. A third focus is on understanding the mechanisms by which protein arginine methylation modulates trypanosome biology. We performed a global proteomic analysis of the arginine methylome of T. brucei, identifying >1100 methylproteins spanning most cellular compartments and a wide array of functional classes. We are now analyzing novel mechanisms of protein arginine methyltransferase regulation and defining the physiological and molecular functions of arginine methylmarks on selected proteins. I foster a collaborative and flexible laboratory environment, and I encourage my students to explore the research topics that interest them.
Geriatric Medicine; Nutrition; Bioinformatics; Gene Expression; Molecular and Cellular Biology; Molecular genetics; Protein Folding; Protein Function and Structure; RNA; Regulation of metabolism; Stem Cells; Vitamins and Trace Nutrient
DNA Replication, Recombination and Repair; Genome Integrity; Molecular and Cellular Biology; Molecular genetics; Protein Function and Structure
In my laboratory, we are interested in the general problem of maintaining genome stability. To this end, we focus on two distinct aspects of genome stability: 1) the roles of mismatch (MMR) proteins in multiple pathways for DNA repair and 2) the manner in which regulation of dNTP pools, through the regulation of ribonucleotide reductase (RNR) activity, impacts genome integrity. 1) MMR proteins recognize many different types of DNA lesions and then target the lesion for the appropriate repair pathway. We are interested in the mechanism(s) by which recognition of a lesion is translated into the appropriate DNA repair pathway, using the yeast Saccharomyces cerevisiae as a model system. Is it through differential protein-nucleic acid or protein-protein interactions? To address these questions as well as the regulation of DNA repair pathway selection, we use a combination of genetic, biochemical and biophysical approaches. 2) RNR activity modulates the level of dNTPs that are available in a cell at a given time. Higher levels of dNTPs lead to higher mutation rates. We are interested in the various ways in which misregulated dNTP pools might affect cellular metabolism and affect the stability of the genome.
DNA Replication, Recombination and Repair; Gene Expression; Genome Integrity; Microbiology; Molecular and Cellular Biology; Protein Function and Structure; Signal Transduction
We are interested in developing an integrated mechanistic view of how organisms coordinate the actions of their DNA replication machinery with those of other cellular factors involved in DNA repair and damage tolerance. Failure to properly coordinate these functions leads to mutations, genome instability, and in extreme cases, cell death. We utilize a combination of biochemical, biophysical, and genetic approaches to investigate the molecular mechanisms of DNA replication, DNA repair, and error-prone DNA damage tolerance functions in Escherichia coli. The primary mechanism for damage tolerance involves direct bypass of damaged bases in the DNA. This process is inherently error-prone, and is the basis for most mutations. Current efforts are focused on understanding the mechanisms by which the actions of high fidelity and error-prone lesion bypass DNA polymerases are coordinated with each other, as well as other proteins involved in DNA metabolism. Our goal in this work is to develop methods that enable us to control the fidelity of DNA repair for therapeutic gain. We are also interested in understanding the mechanisms that contribute to DNA mutagenesis in the opportunistic human pathogen, P. aeruginosa. P. aeruginosa is a particular problem for individuals afflicted with cystic fibrosis. Persistent colonization of cystic fibrosis airways with P. aeruginosa serves as a major source of morbidity and mortality for these patients. The ability of P. aeruginosa to persist in the airways relies in part on its ability to adapt to the continuously changing environment within the diseased airways. We are particularly interested in determining the contribution of mutagenesis and DNA repair to adaptive mutations that contribute to clonal expansion and pathoadaptation of P. aeruginosa during colonization of cystic fibrosis airways.