Day 1 :
Johns Hopkins University, USA
Keynote: What 40-plus years of study have taught us about the DNA-looping protein AraC and its regulation of the L-arabinose operon in Escherichia coli.
Time : 10:00 :11:00
Robert Schleif, Professor of Biology and Biophysics, Johns Hopkins University, received graduate training in Physics and Molecular Biology at the University of California, Berkeley, and post doctoral training at Harvard University with Drs. Gilbert and Watson. After 18 years in the Biochemistry Department at Brandeis University, he moved to Johns Hopkins. Current interests are directed towards obtaining a sufficiently deep and detailed understanding of the principles by which AraC functions, that new regulatory proteins utilizing the same principles could be designed and built.
The AraC protein both positively and negatively regulates expression of the L-arabinose operon in Escherichia coli. More than three hundred person-years of research spread over more than four decades has revealed much about gene regulation and transcription factors. This work included the discovery of the phenomenon of DNA looping in gene regulation, and has stimulated development of a number of techniques used in molecular biology including DNA gel retardation assays and missing contact footprinting. The talk will summarize current understanding of the mechanism by which the binding of arabinose to AraC shifts the protein from preferring to loop DNA and repressing the pBAD promoter by binding to two DNA sites separated by 210 base pairs to preferring to bind to two adjacent DNA sites and activating the promoter. Several recent experiments will be described including elucidation of the role of the N-terminal arm of the protein in controlling the protein’s DNA binding properties and experiments demonstrating that arabinose binding to one subunit affects the N-terminal arm of only the opposite subunit
University of Nebraska Medical Center, USA
Time : 11:00-12:00
Tahir Tahirov has received his Master of Science degree in metallophysics from Kiev Polytechnic Institute (Ukraine). During doctoral training in crystallochemistry in the Chernogolovka branch of the Semenov Institute of Chemical Physics (Russia) he discovered a new class of organic superconductors with two incommensurate crystal lattices. His career in structural biology has started during postdoctoral training in Tsing Hua University (Taiwan) where he solved the first crystal structure of protein. He continued his studies as a researcher at the Himeji Institute of Technology, Osaka University and the Yokohama City University School of Medicine, and then as a team leader at the RIKEN Harima Institute. At the age of 43 Dr. Tahirov became a Full Professor at the Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center. He has published over 120 research articles (50 in physics and chemistry and the rest in structural biology). He is one of the leading experts in structural studies of human DNA replication machinery.
All eukaryotes possess four paralogous B family DNA polymerases: Pola, Pold, Pole and Polz. Pola functions in initiation and early elongation steps of replication. Pola is tightly associated with a primase and hence is the only DNA polymerase that can initiate the synthesis of DNA by extending RNA primers laid by primase. Pold plays a central role in DNA replication and DNA repair in eukaryotic cells. Pole is involved in the initiation of replication at origins and in leading-strand synthesis in the vicinity of the origins, whereas Polz is involved in translesion DNA synthesis. When Pold encounters replication-blocking lesions, it switches from replication to translesion synthesis by recruiting damage bypass polymerases, including Polz. Polz is responsible for nearly all mutations induced by DNA damaging agents in human cells and model organisms. Accumulation of mutations in cellular genetic material causes various diseases, including cancer.In spite of key role of B family DNA polymerases in replication and genome maintenance, only limited data are available regarding mechanisms of their functions. Our aim is to explore the structural features beyond the polymerase catalytic core and reveal how the intersubunit interactions and conformational changes regulate the function of these polymerases. We started with crystal structure-based characterization of the role of the second B-subunits in human B-family DNA polymerases. In particular, we determined the crystal structures of B-subunit complexes for all four DNA polymerases and discovered that Pold and Polz are sharing the same second and third subunits. We will briefly review our recent achievements and focus our presentation on novel crystal structure of entire human primase-Pola complex. The structure reveals how the primase and Pola are acting in a highly coordinated fashion during the initiation of RNA primer synthesis, extension and counting by primase and transfer of primer-template duplex for further extension by Pola.
- Track 1: Molecular Medicine
Track 2: DNA Replication & Recombination
Track 3: Recombinant DNA Technology
Track 4: RNA Editing and Interference
Track 5: Epigenetics
Track 6: RNA Processing and Protein Synthesis
Liberty University, USA
McClintock is a Professor and Director of Forensic Sciences in the Department of Biology and Chemistry at Liberty University in Lynchburg, VA where he teaches undergraduate and graduate courses in forensic sciences (forensic DNA analysis, trace evidence) and microbiology. The forensic DNA course focuses on current laboratory methods and applications in forensic DNA profiling and effective presentation of DNA evidence at trial. His latest book, entitled "Forensic Analysis of Biological Evidence: A Laboratory Guide for Serological and DNA Typing" focuses on the newest techniques available for the analysis of biological material. Dr. McClintock is also the founder of DNA Diagnostics, Inc., a forensic service company that provides DNA testing and the scientific review and analysis of DNA test results performed in forensic casework. In 2013, Dr. McClintock, was named among the top 15 DNA analysts in the country by ForensicsColleges.com, a leading website on forensics programs across the nation.
Bloodstain pattern analysis has been an integral part of criminal investigations for over a century. The use of chemiluminescent reagents such as luminol or Bluestar® to visualize presumed bloodstains in criminal investigations has provided useful investigative information. Newer technologies and recent advances in forensic DNA analysis have gained much notoriety over the past two decades as a tool in human identification and parentage verification. More recently, bloodstain analysis, coupled with methods to generate DNA profiles, have been applied to investigate samples of historical significance. This study investigated samples collected from America’s past conflicts to determine the biological origin and nature of the samples/stains using classic and various state-of-the-art approaches as well as isolate the genetic material for forensic DNA analysis. Specifically, samples were collected from the Hillsman House in Rice, VA that served as a Union field hospital during the last battle of the Civil War. Approximately 358 Union and 161 Confederate soldiers were treated over a twenty-four hour period during the battle at Sailor’s Creek. The prominent “bloodstains” on the floorboards under the single surgical table and two post-surgical beds provides evidence of the vast number of soldiers treated. These presumed bloodstains also found their way through the cracks in the wood floors onto the supporting floor joists. The presumed bloodstains were subjected to various presumptive blood tests (e.g., luminol and Bluestar®, leucomalachite green, phenolphthalein, and RSID™ Blood Competitive Analysis Kit), the DNA isolated, quantitated, and subjected to genetic analysis using capillary electrophoresis. The generation of partial or complete DNA profiles will confirm the presence of human DNA, as well as demonstrate the ability of DNA profiling to reveal a part of history from a battle fought over 150 years ago. Other presumed bloodstain samples from the Korean War era and tissue and hair samples collected from burial sites from a civilization long extinct have been analyzed in an attempt to generate DNA profiles and to corroborate historical documentations of accounts that occurred many decades ago.
Makkuni Jayaram is a Professor of Molecular Biosciences at the University of Texas at Austin. His primary research interest is in the biochemical mechanisms of site-specific DNA recombination. Over the past three decades, his research group has used the Flp site-specific recombinase as a template for understanding the chemistry, conformational dynamics and topological features of strand breakage/exchange reactions in nucleic acids. A second interest of the Jayaram laboratory concerns strategies devised by selfish DNA elements for moderating their selfishness so as to establish long-term peaceful coexistence with their host genomes.
Phosphoryl transfer reactions in RNA and DNA abound in living cells, and are central to biological information processing. A common feature of self-catalyzed or protein-catalyzed phosphoryl transfer in nucleic acids is the role of divalent metal ions in stabilizing the penta-coordinate phosphate transition state. Most systems appear to follow the classical ‘two-metal ion’ paradigm or its variations, while recent evidence suggests the potential involvement of a third metal ion, at least in some systems. By contrast, members of the serine- and tyrosine-family site-specific recombinases exemplify metal-free mechanisms for mediating phosphoryl transfer associated with the DNA strand cleavage and strand joining steps that they perform. In the tyrosine family, the positively charged side-chains of two highly conserved arginine residues appear to functionally bypass metal ion requirement. By using Flp and Cre recombinases as representatives of the tyrosine family, we probed the individual roles of this arginine duo (Arg-I and Arg-II) in transition state stabilization. We find that Flp or Cre variants lacking either Arg-I or Arg-II can be rescued by replacing the scissile phosphate with methylphosphonate, thereby eliminating the negative charge on one of the non-bridging oxygen atoms in the transition state. Stereochemically pure RP and SP forms of the methylphosphonate substrates in conjunction with recombinase variants lacking either Arg-I or Arg-II have enabled us to dissect the stereochemical contributions of the individual arginines to the recombination reaction. The general strategies employed by us are of broad utility in the analyses of other recombination systems.
Beijing Institute of Technology, China
Feng Qu received her PhD degree of Analytical Chemistry from Chemistry and Molecular Engineering College, Peking University in 1997. She has expertise in bioanalysis based on capillary electrophoresis. Her research focus on: capillary electrophoresis in biological and biomedical application; protein and nucleic acid interaction analysis; aptamers selection strategy and methodology for protein, cell, bacteria, small molecule targets .
Nucleic acid aptamers are short, single-stranded DNA (ssDNA) or RNA molecules that are selected for binding to a specific target. Aptamers can be used as recognition probes in biomedical, food and environment analysis. Moreover, they have great potential in disease diagnosis and treatment, drug discovery, medicine research, as well as bioimaging, which are expected to bring huge economic benefits. However, current aptamers application are far from satisfactory, and have not yet been fully developed. The complicated selection process with high cost and low efficiency is one of the bottlenecks of their application. There is still not universally accepted standard selection methods are accepted.
Capillary electrophoresis (CE) is one of the most powerful methods for aptamers sieving (known as CE-SELEX), which has the advantages of fast, high resolution, low sample consumption and smart separation modes of capillary zone electrophoresis (CZE), affinity capillary electrophoresis (ACE) and capillary isoelectric focus (CIEF). Moreover, the binding of target and synthetic single stranded DNA (ss-DNA) occurs in free solution, which eliminates the biases caused by stationary support and linker. Some important protein aptamers have been successfully obtained based on CE, which greatly improves the selection efficiency.
Saint Petersburg State University, Russia
Oleg Tikhodeyev is the author of the original approach for resolving multiple ambiguities and contradictions in current genetic concepts. He has shown that the key source of such ambiguities and contradictions is the erroneous belief that the same genetic term (for example, mutation) is able to comprise both specific phenomenology and the underlying mechanisms (Tikhodeyev, 2015). This belief became widely accepted after 1952, when the hereditary role of DNA had been demonstrated. In modern genetic concepts, the terms describing molecular mechanisms should be clearly distinguished from those describing phenomenology because there is no strict correlation between phenomenology and molecular mechanisms
Statement of the Problem: During last 30 years, the majority of basic genetic terms (mutation, recombination, genotype, gene, allele, etc.) became fuzzy due to discovery of multiple “non-canonical” phenomena like inheritance of acquired traits, protein inheritance, paramutations, and genotrophy. As a result, there is a significant gap between factual material and current genetic concepts, thus reflecting the need for a paradigm shift. Modern genetic concepts are required, which will be equally valid for all known canonical and “non-canonical” genetic phenomena. Methodology & Theoretical Orientation: We accomplished a critical analysis of current genetic concepts (gene theory, mutation theory, the chromosome theory of inheritance, the DNA theory of inheritance) to find the key origins of the terminological fuzziness. Findings: The current concepts stand on the idea that any genetic term simultaneously describes three following aspects: (i) phenomenology, (ii) the source of variation, and (iii) the underlying mechanism. For instance, any mutation is considered as an alteration which is (i) stable and hereditable, (ii) stochastically arising, and (iii) affecting DNA sequences. We name this idea “the integral concept of variability”. Meanwhile, the available factual material clearly demonstrates that the abovementioned aspects are autonomous from each other and thus cannot be covered by the same term. In particular, some hereditable alterations gradually decline, some are clearly predictable under certain environmental influences, some do not affect DNA sequences, and some alterations of DNA sequences are not heritable. We propose that each genetic term should describe only one aspect of variability. This idea (we name it “the differential concept of variability”) was already shown to be successful for a lot of genetic terms. Conclusion & Significance: The way to resolve the fuzziness of genetic terminology and the crisis of current genetic concepts is a paradigm shift based on the differential concept of variability.
National Institute on Aging, NIH, USA
Robert Brosh has his expertise in DNA repair and genome stability maintenance. He leads a research group at the National Institute on Aging, NIH that is focused on characterizing the roles of clinically relevant human DNA helicases in cellular nucleic acid metabolism. This work has yielded insights into how DNA repair helicases promote phenotypes consistent with healthy aging and cancer resistance.
Statement of the Problem: Fanconi Anemia (FA) is a rare genetic DNA repair disorder characterized by progressive bone marrow failure, congenital abnormalities, and cancer. Of the 21 genes linked to FA, the FA Group J (FANCJ) gene is unique that it encodes an ATP-dependent DNA helicase. Mutations in FANCJ are not only genetically linked to FA, but also associated with breast and ovarian cancer. Consistent with its known role in homologous recombination (HR) repair, FANCJ-/- cells are sensitive to DNA interstrand cross-linking (ICL) agents and are also hypersensitive to agents that induce replication stress. Methodology & Theoretical Orientation: We characterized two FA patient-derived FANCJ mutations, R707C and H396D, which reside in the conserved helicase core domain. Genetic and biochemical analyses were performed to delineate the molecular defects underlying the genetic disease. Findings: FANCJ-R707C retained partial (~30%) helicase activity, whereas FANCJ-H396D was nearly completely inactive. Single-turnover kinetic assays, ATPase measurements, and DNA binding determinations confirmed the differential effects of FANCJ missense mutations on helicase activity. Expression of either FANCJ-R707C or FANCJ-H396D in fancj-/- cells completely failed to rescue cisplatin sensitivity. In striking contrast, expression of FANCJ-R707C in fancj-/- cells restored resistance to the DNA polymerase inhibitor aphidicolin, whereas FANCJ-H396D completely failed. Single-molecule replication tract analysis confirmed that FANCJ-R707C, but not FANCJ-H396D, restored fork rates after cellular exposure to aphidicolin. Thus, a quantitatively lower threshold of FANCJ catalytic activity is required for the aphidicolin-induced replication stress response compared to cisplatin-induced damage. Conclusion & Significance: The catalytic requirement of FANCJ to reconstruct broken replication forks after ICL-induced damage is distinct from that required to remodel stalled replication forks. These findings provide new insight to FANCJ’s role in DNA repair and molecular phenotypes of clinically relevant FANCJ missense mutations that are relevant to human disease and cancer.