Dr. R. Stephen Lloyd received his BS in Biology from Florida State University in 1975, majoring in marine pollution biology. His research interests turned to cancer chemotherapy and in 1979, he earned his Ph.D. in Molecular Biology from the University of Texas Graduate School of Biomedical Sciences in Houston, TX. After learning about mechanisms by which DNA can be damaged, he began his career in DNA repair as a postdoctoral fellow at Stanford University in the laboratory of Dr. Philip Hanawalt. Following two years at Stanford, he worked for two more years for a genetic engineering company, before joining the Biochemistry Department at Vanderbilt University in 1983. In his ten-year stay at Vanderbilt, Dr. Lloyd rose through the ranks to Full Professor, and his research focused on both DNA repair and molecular mutagenesis. He was then recruited to the Center for Molecular Science at the University of Texas Medical Branch in which the faculty exclusively specialized in DNA repair mechanisms. During his twelve years at UTMB, he also became the Director of two Centers in Environmental Toxicology. In August 2003, he, along with his wife, Dr. Amanda K. McCullough was recruited to join the CROET faculty at OHSU. Together they have both separate and joint research projects in the research areas described below.

DNA repair processes and high fidelity DNA replication represent the major mechanisms to maintain genomic stability. Our laboratory uses multi-disciplinary approaches to focus on:

1. strategies to prevent sunlight-induced skin cancer via the topical introduction of repair enzymes into human cells;
2. the mechanisms by which the loss of a DNA repair enzyme can lead to the clinical manifestations of Metabolic Syndrome - a constellation of diseases (obesity, fatty liver disease, insulin resistance and hypertension) that affect >45 million Americans;
3. the molecular mechanisms by which environmental toxicants create mutations in DNA; and
4. the cellular response mechanisms for the repair of DNA-protein crosslinks.

These are discussed in more detail below.

Structure-Function Analyses of Pyrimidine Dimer Glycosylases: Investigations Toward Preventing Skin Cancer

Figure 1. Co-crystal structure of the covalent linkage of T4-pyrimidine dimer DNA glycosylase with abasic DNA

Exposure to short wave ultraviolet light has been demonstrated to be the causal factor in nonmelanoma skin cancers and a strong risk factor in melanomas. While human cells only use nucleotide excision repair to repair the UV-induced dipyrimidine DNA photoproducts, other organisms initiate the base excision repair pathway by DNA glycosylases that catalyze incision at the 5' base of pyrimidine dimers. Developing an understanding of the function of enzymes is critical, since T4 pyrimidine dimer glycosylase (T4-Pdg) is being used in human clinical trials. Although topical delivery of wild-type T4-Pdg on xeroderma pigmentosum patients has demonstrated efficacy in cancer reduction, all investigations to date using wild-type mammalian cells reveal that T4-Pdg results in decreased, rather than increased survival after UV. It is hypothesized that the ability of T4-Pdg to incise all dimer sites within DNA domains leads to cytotoxic double-strand breaks in which dimers are in close proximity in complementary strands. Thus, we have hypothesized that forms of T4-Pdg that have lost the ability to incise dimers that are formed in clusters will enhance repair and decrease mutagenesis without creating cytotoxic double-strand breaks. In order to rationally engineer T4-Pdg to achieve this objective, we have recently solved the co-crystal structure of T4-Pdg covalently complexed with DNA (see Figure 1 & Golan et al, 2006). Based on these data and analysis of nucleotide flipping and enzyme catalysis current investigations are as follows: 1) engineer T4-Pdg to be less efficient in the precatalytic steps of DNA bending and nucleotide flipping, with the net result being a decrease in the ability of these altered enzymes to form a Michaelis complex and incise dimers in clusters; 2) express control and mutant T4-Pdgs in keratinocytes to determine effects on double-strand break formation, survival, and mutagenesis; and 3) activate base excision repair of UV-photoproducts in mitochondria and determine the role of dimers in cytotoxicity.

Disease Consequences Resulting from the Knockout of the DNA Glycosylase, NEIL 1

Figure 2. neil1 knockout mouse on left, compared with wild type control on right

The growing epidemic of human obesity is currently estimated to affect over 65 million adult Americans, with secondary consequences including but not limited to, decreased life span, non-alcohol-induced fatty liver disease, increased cardiovascular disease, increased incidence of stroke and type 2 diabetes. The majority of the total obese population (> 45 million Americans) has a combination of at least four of these disorders (obesity, insulin resistance, dyslipidemia, and hypertension), collectively known as the Metabolic Syndrome. The underlying causes of these diseases are not well established but have been investigated using genetic models and/or exposure to conditions of exogenous stress. Although wild-type cells maintain overall energy homeostasis by minimizing the cellular damage from reactive oxygen species (ROS), a variety of conditions that result in high levels of ROS produce these diseases. DNA is one of the major targets of ROS-induced damage, and the possible interrelationship between defective DNA repair and Metabolic Syndrome has not been explored in depth. However, we recently demonstrated that mice carrying a deletion of the DNA glycosylase NEIL1, develop symptoms consistent with Metabolic Syndrome: severe obesity, fatty liver, dyslipidemia, and insulin resistance (See Figure 2 & Vartanian et al, 2006). Disease is manifested primarily in male knockout mice and is observed in mice extensively backcrossed to C57BL/6 and heterozygotes. To understand the relationship between the loss of an enzyme that repairs oxidative-stress-induced DNA damage and the development of the Metabolic Syndrome, our central hypothesis is that the threshold of DNA damage required to initiate events leading to Metabolic Syndrome is significantly reduced in NEIL1-deficient organisms. Evidence supporting this model is that mitochondrial DNA contains significantly elevated levels of unrepaired damage and deletions. Goals of future investigations include the following: To discern the role that NEIL1 plays in cells, modulation of survival, mutagenesis and mitochondrial function will be evaluated. Additionally, due to its central role in maintaining mtDNA integrity, aims are designed to determine the identity and role of the mitochondrial- versus the nuclear-targeted forms of the enzyme. Since preliminary data show that some human polymorphic variants of NEIL1 are catalytically inactive, these variants will be characterized for their ability to initiate base excision repair and the ability to reverse the phenotype of the neil1-deficient mice.

Repair, Replication Bypass, and Mutagenesis of DNAs Containing Interstrand and Intrastrand Crosslinks

The stability of an organism's genome is governed by complex mechanisms that involve 1) the repair of DNA damage, 2) the regulation of progression through and transition between phases of the cell cycle and 3) the ability to reorganize chromatin structure. Each of these mechanisms is affected by the nature of the DNA damage and the signal transduction stimuli that alter transcriptional and post-translational activities. Defects in key components within these pathways can manifest in a variety of human disease, such as xeroderma pigmentosum with defects in NER repair and lesion bypass, ataxia telangiectasia, with defects in cell cycle control and intracellular signaling, Fanconi anemia with defective chromatin remodeling and replication restart and colon cancer with defects in mismatch or base excision repair. Thus, integrated responses to maintain of genomic stability are critical for long-term survival and organismal fitness. A major focus of our laboratory centers on investigations of bis-electrophiles, a class of environmental and endogenous chemicals that upon exposure to DNA, form a complex mixture of lesions that can contribute to the initiation of cancer and premature aging. Our current studies test a series of hypotheses that will accomplish the following: 1) determine the mutagenic and cytotoxic consequences of replication of DNAs containing site-specific base lesions that spontaneously form interstrand and intrastrand DNA crosslinks and the role that DNA repair plays in the modulation of the mutagenic potential of these lesions; 2) ascertain key genetic components of and the biochemical basis for a pathway to repair of interstrand crosslinks that occurs with high fidelity in the absence of homologous recombination; and 3) ascertain the extent to which the structure of specific DNA lesions that exist in a dynamic equilibrium between various species (monofunctional adduct, intra- and interstrand DNA crosslinks and DNA-protein crosslinks) affect nucleic acid transactions such as repair and replication.

These investigations have direct application to human health since exposure to these compounds, whether from sources produced internally or as an environmental pollutant, contribute to cancer and premature aging. Investigations on the identity of the mutagenic and cytotoxic DNA lesions responsible for these diseases and their mechanisms of repair can ultimately lead to improved rational therapeutic designs.

Formation and Repair of Protein-DNA Crosslinks

The majority of the DNA repair and mutagenesis literature focuses on biochemical and cellular responses to relatively simple modifications to DNA bases and the sugar-phosphate backbone. Base modifications that include deamination, saturation of double bonds, oxidation, alkylation and ring fragmentation are generally restored via base excision repair (BER). Bulky chemical additions such as polycyclic aromatic hydrocarbons and covalent linkage of adjacent bases, such as ultraviolet light (UV)-induced pyrimidine dimers and 6-4 photoproducts, and interstrand crosslinks are corrected by nucleotide excision repair (NER). However, there exists another abundant class of DNA lesions that has received relatively little experimental investigation, the DNA-protein crosslink (DPC). DPCs are formed in DNA from exposure to various chemical agents such as formaldehyde, transplatin, and bifunctional electrophiles, as well as from exposure to ionizing radiation (IR) and UV. A potential reason for a paucity of data on how cells process these lesions and their role in mutagenicity and carcinogenesis is that up until very recently, it has not been possible to create site-specific and protein-specific DPCs. Recent advances in our laboratory and those of our colleagues now permit the construction of defined DNAs that contain model DPCs in which the site of attachment to the DNA can be either through the major or minor groove or along the sugar-phosphate backbone. The types of proteins that can be crosslinked through these sites can range in size from a few amino acids (MW~600 Da) to large enzymes, such as topoisomerase II (monomer MW~120,000 Da). With the availability of a wide spectrum of adduct sizes and sites of attachments, the opportunity now presents itself to test hypotheses concerning the replication, repair and mutagenesis of DNAs containing DPCs. To address these goals, our laboratories are adopting the following strategies.

An initial focus is on the formation and biophysical characterization of DPCs in which the linkage between the protein and the DNA is a reduced Schiff base. The site of attachment to the DNA is through exocyclic amino groups that in normal B-form DNA are located in either the major groove (N⁶-adenine) or the minor groove (N²-guanine). In both scenarios, the chemical linkage between the DNA and the protein/peptide is made using an acrolein-derived γ-hydroxy propanodeoxyguanine (γ-HO PdG) or an acrolein-derived γ-hydroxy propanodeoxyadenine (γ-HO PdA). Additionally, DPCs are created along the sugar phosphate backbone using an abasic (AP) site. A variety of peptides and proteins, ranging from ~600 to 120,000 Da have been attached through these sites in DNA and the resulting duplex DNAs characterized for DNA-protein induced bending, DNA footprinting and modulation of the thermal stability of the adducted DNAs. These data will be correlated with repair, replication and mutagenesis studies in the following studies.

We will test the hypothesis that both the size and site of linkage of the DPC will determine the ability of 1) in vitro DNA repair systems to excise DPCs, 2) DNA polymerases to bypass DPCs and 3) DNA helicases to unwind DNAs associated with DPCs. Additionally, we will test hypotheses concerning the in vivo processes that modulate mutagenic processing of the DPC lesions. Both single- and double-stranded (ss and ds, respectively) DNA mammalian shuttle vectors will be created with a single site-specific DPC. These will be replicated in mammalian cells that vary in repair competency and mutagenesis will be assessed. Together, these aims will test our model hypothesis that a subset of DPCs are directly repairable by NER, while others must undergo proteolytic degradation prior to repair or replication bypass. These investigations will constitute the first comprehensive evaluation of cellular processing of DPCs.

Fernandes PH, Kanuri M, Nechev LV, Harris TM, Lloyd RS. Related Articles, Links Mammalian cell mutagenesis of the DNA adducts of vinyl chloride and crotonaldehyde. Environ Mol Mutagen. 45: 455-9, 2005.   Abstract

Minko IG, Kurtz AJ, Croteau DL, Van Houten B, Harris TM, Lloyd RS. Initiation of repair of DNA- polypeptide cross-links by the UvrABC nuclease. Biochemistry. 44: 3000-3009, 2005.   Abstract

Lloyd RS. Investigations of pyrimidine dimer glycosylases--a paradigm for DNA base excision repair enzymology. Mutat Res. 577: 77-91, 2005.   Abstract

Kanuri M, Nechev LV, Kiehna SE, Tamura PJ, Harris CM, Harris TM, Lloyd RS. Evidence for Escherichia coli polymerase II mutagenic bypass of intrastrand DNA crosslinks. DNA Repair (Amst). 4(12):1374-80, 2005.   Abstract

Sanchez AM, Kozekov ID, Harris TM, Lloyd RS. Formation of inter- and intrastrand imine type DNA-DNA cross-links through secondary reactions of aldehydic adducts. Chem Res Toxicol. 18(11):1683-90, 2005.   Abstract

Vartanian V, Lowell B, Minko IG, Wood TG, Ceci JD, George S, Ballinger SW, Corless CL, McCullough AK, Lloyd RS. The metabolic syndrome resulting from a knockout of the NEIL1 DNA glycosylase. Proc Natl Acad Sci U S A. 103(6): 1864-9, 2006.   Abstract

Harbut, MB, Meador, M, Dodson, ML and Lloyd, RS. Modulation of the turnover of formamidopyrimidine DNA glycosylase. Biochem. 45(23): 7341-6, 2006.   Abstract

Fernandes, PH, Hackfield, LC, Kozekov, ID, Hodge, RP, Lloyd, RS. Synthesis and mutagenesis of the butadiene-derived N3 2'-deoxyuridine adducts. Chem Res Toxicol. 19(7): 968-76, 2006.   Abstract

Golan, G, Zharkov, DO, Grollman, AP, Dodson, ML, McCullough, AK, Lloyd, RS, Shoham, G. Structure of T4 pyrimidine dimer glycosylase in a reduced imine covalent complex with abasic site-containing DNA. J Molec Biol 362(2): 241-58, 2006.   Abstract

Dr. Lloyd on PubMed