Day 1 :
The RNA Institute, SUNY at Albany, USA
Time : 09:15-09:55
Paul F. Agris created and is the founding Director of The RNA Institute, SUNY at Albany. He is known as a Structural Biochemist and innovator in RNA biology, RNA modification science and applications, and nucleic acid design with modified nucleosides. Agris was a Professor and Head of North Carolina State University’s Biochemistry Department, and founded and led the RNA Society of North Carolina for more than a decade. Previously, he was an Assistant, Associate and full Professor in the Division of Biological Sciences and Department of Medicine at the University of Missouri-Columbia. Agris researches into RNA-based therapeutics against infective disease, drug-resistant HIV and MRSA, have been supported continuously by federal agencies since 1974. He is the author of some 170 peer reviewed articles, a number of reviews, chapters and editor of three volumes. Agris founded the RNA-based drug discovery company SIRGA Advanced Biopharma, Inc., Research Triangle Park, NC.
Constantly evolving drug-resistant and multidrug-resistant (MDR) Gram-positive pathogens such as Staphylococcus aureus (methicillin-resistant, MRSA), Streptococcus pneumoniae and Clostridium difficile represent an ever-increasing source of morbidity and mortality in the USA. A new class of topically applied antibiotics against MRSA and other dangerous multi-drug resistant pathogenic infections such as staphylococci and streptococci has been developed. Some bacteria are even developing resistance to vancomycin, an antibiotic drug of last resort. To address these demands, we have selected, screened and characterized a novel putative antibiotic that has little-to-no toxicity, and to which bacteria are slow to develop resistance. This new class of antibiotic targets a ribonucleic acid (RNA) in the cell, rather than a protein. The RNA target is unique to Gram-positive bacteria and is not found in humans, and it is important to basic functions required for the bacteria to live. Our small molecule antibiotic drug turns OFF the RNA function, simultaneously halting expression of as many as 24 genes critical in bacteria, killing the pathogens. Preliminary studies indicate that the new antibiotic is effective against bacteria that had been isolated from humans, is not toxic to human cells in lab cultures at doses effective against bacteria, and is not toxic
in preliminary topical animal studies.
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 : 09:55-10:35
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 : 10:35-11:15
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.
- Workshop on
Zhongwei Li has his expertise in RNA Biology and related biomedical problems. He studies applied biochemical, molecular and genomics approaches to understand how RNA is processed and degraded in various organisms, and to elucidate the role and significance of ribonucleases. He teaches Biomedical Science courses for Under-graduate medical program and Graduate programs. His research primarily focuses on RNA damage control and prevention of human diseases. His goals are to develop RNA damage biomarkers and identifying protective genes to cope with degenerative disorders. In addition, he is studying bacteria aiming to develop treatment of infectious diseases, including identification of bacterial genes involved in infection, different pathways for bacterial RNA metabolism, and methods for quick diagnosis of bacteria in clinical settings. More recently, he has taken the responsibility of faculty affairs administration to help the college grow after obtaining full LCME accreditation.
Statement of the Problem: Oxidation is probably the most common type of damage that occurs in cellular RNA. Oxidized RNA may be dysfunctional and is implicated in the pathogenesis of age-related human diseases. Cellular mechanisms controlling oxidized RNA have begun to be revealed. Currently, many ribonucleases and RNA-binding proteins have been shown to reduce oxidized RNA and to protect cells under oxidative stress. Although information about how these factors work is still very limited, we suggest mechanisms that can be used to minimize oxidized RNA in bacteria and human cells.
Methodology & Theoretical Orientation: RNA oxidation levels are determined by chromatographic separation of nucleosides and detection of 8-oxoG levels. Cell viability was determined by growth rate. Functions of ribonucleases and other proteins were studied by examining mutants lacking genes encoding these proteins, or over-expressing the genes.
Findings: Many ribonucleases and other proteins were found important for maintaining cell viability under oxidative stress. We have also identified proteins that specifically bind oxidized RNA at high affinity. In mutants lacking these activities, oxidized RNA accumulates. Over-expression of these activities reduces oxidized RNA and rescue cells under oxidative stress. Normalized RNA oxidation levels reduce overtime after pulse oxidative challenge.
Conclusion & Significance: The results demonstrate mechanisms for selective removal of oxidized RNA in bacteria and cultured human cells. Selective reduction of RNA oxidation depends on the activities of ribonucleases, suggesting that RNA degradation plays a major role in this process. The finding of specific proteins with high affinity to oxidized RNA implies that oxidized RNA is recognized by these proteins, targeting the RNA to effective degradation or repair. The findings demonstrate an important mechanism for maintaining RNA quality for normal function of the cell, and for prevention of related human diseases.
- 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
Time : 12:20-12:45
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.
University of Texas Medical Branch, USA
Time : 12:45-13:10
Junji Iwahara’s current research focuses on the dynamic processes whereby transcription factors scan DNA and recognize their target sites. His group developed some novel methods for investigating the dynamics and kinetics of the protein-DNA interactions at atomic and molecular levels. Using the biophysical and biochemical approaches, Prof. Iwahara’s group is trying to better understand how proteins scan and recognize DNA to regulate genes.
In eukaryotic genomes, there are numerous nonfunctional high-affinity sequences for transcription factors. These sequences potentially serve as natural decoys that sequester transcription factors. We have previously shown that the presence of sequences is like the target sequence could substantially impede association of the transcription factor Egr-1 with its targets. More recently, using a stopped-flow fluorescence method, we examined the kinetic impact of DNA methylation of decoys on the search process of the Egr-1 zinc-finger protein. We analyzed its association with an unmethylated target site on fluorescencelabeled DNA in the presence of competitor DNA duplexes, including Egr-1 decoys. DNA methylation of decoys alone did not affect target search kinetics. In the presence of the MeCP2 methyl-CpG-binding domain (MBD), however, DNA methylation of decoys substantially (~10-20-fold) accelerated the target search process of the Egr-1 zinc-finger protein. This acceleration did not occur when the target was also methylated. These results suggest that when decoys are methylated, MBD proteins can block them and thereby allow Egr-1 to avoid sequestration in nonfunctional locations. This effect may occur in vivo for DNA methylation outside CpG islands and could facilitate localization of some transcriptional activators within regulatory CpG islands, where DNA methylation is rare. Our recent studies to examine this model will be presented.
University of Texas at Austin, USA
Title: Stereochemical Course of Site-specific DNA Recombination Revealed by Methylphosphonate-substituted DNA substrates and active site variants of Flp and Cre Recombinases
Time : 14:00-14:25
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.
National Institute on Aging, NIH, USA
Title: Analyses of patient-derived missense mutations in Fanconi anemia group J (FANCJ) DNA helicase
Time : 14:25-14:50
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.
University of Saskatchewan, Canada
Time : 14:50-15:15
Yuliang Wu obtained his BSc and MSc from Zhejiang University, China in 1995 and 1998 respectively, and Ph.D. from International Centre of Genetic Engineering and Biotechnology (ICGEB), Delhi, India in 2002. In the following eight years, Dr. Wu did his postdoc training at the University of Alberta, Canada and the National Institute on Aging-NIH, where he studied the molecular and cellular basis of human genetic diseases characterized by genomic instability. Dr. Wu joined the Department of Biochemistry at the University of Saskatchewan, Canada in May 2011. Dr. Wu’s lab focuses on DNA repair proteins, including helicase, single strand DNA binding protein, and recombinase. Through structural and functional studies of these DNA repair proteins, we try to understand the molecular mechanisms underlying genomic instability. Ultimately the molecular information derived from these projects may be exploited to advance diagnosis, prognosis, and treatment of human diseases and cancers.
The K-homology (KH) domain is a nucleic acid–binding domain present in many proteins, but has not been reported in helicases. DDX43, also known as HAGE (helicase antigen gene), is a member of the DEAD-box protein family. It contains a helicase core domain in its C-terminus and a potential KH domain in its N-terminus. DDX43 is highly expressed in many tumors, and is therefore considered a potential target for immunotherapy. Despite its potential as a therapeutic target, little is known about its activities. Here, we purified recombinant DDX43 protein to near homogeneity and found that it exists as a monomer in solution. Biochemical assays demonstrated that it is an ATP-dependent RNA and DNA helicase. Although DDX43 was active on duplex RNA regardless of the orientation of the single-stranded RNA tail, it preferred a 5' to 3' polarity on RNA and a 3' to 5' direction on DNA. Truncation mutations and site-directed mutagenesis confirmed that the KH domain in DDX43 is responsible for nucleic acid binding. Compared with the activity of the full-length protein, the C-terminal helicase domain had no unwinding activity on RNA substrates and had significantly reduced unwinding activity on DNA. Moreover, the full length DDX43 protein, with single amino acid change in the KH domain, had reduced unwinding and binding activates on RNA and DNA substrates. Our results demonstrate that DDX43 is a dual helicase and the KH domain is required for its full unwinding activity.
University of the Witwatersrand, South Africa
Patrick Arbuthnot is currently a Personal Professor and Director of the Wits/SAMRC Antiviral Gene Therapy Research Unit at the University of the Witwatersrand in South Africa. After graduating with a Medical degree, he has completed his PhD in 1992 then carried out his Post-Doctoral work at Necker Hospital in Paris, France. On returning to South Africa, he established the Antiviral Gene Therapy Research Unit, which has now published widely on HBV infection, liver cancer, HIV-1 infection and developing new methods of treating these diseases. His main research interest is in advancing use of biological and synthetic nanoparticles to carry potentially therapeutic nucleic acids (DNA or RNA) that are capable of permanently disabling HBV.
Chronic infection with hepatitis B virus (HBV) remains and is an important global health problem. Carriers of the virus are at high risk for cirrhosis and liver cancer. Available treatment only has modest curative efficacy and improved therapy is a priority to prevent the life-threatening complications that accompany the infection. The viral replication intermediate comprising covalently closed circular DNA (cccDNA) exists as a stable mini-chromosome in infected hepatocytes. Licensed treatment has no effect on cccDNA and devising methods based on gene therapy to disable this replication intermediate has considerable potential. Previous work from our laboratory demonstrated effective inhibition of HBV replication and targeted disruption of cccDNA by Transcription Activator-Like Effector Nucleases (TALENs). Although this approach is promising, unintended mutagenesis may occur in chronic carriers because of TALEN activity at HBV sequences that are integrated into the host genome. To circumvent this problem, we have produced repressor TALEs (rTALEs) that were designed to induce transcriptional repression at essential HBV transcriptional regulatory elements: the basic core promoter/enhancer II and preS2 promoter sequences. KRAB-encoding sequences were fused to the N-terminal regions of TALEs contain sequence-specific DNA binding domains derived from the AvrBs4 N1 Xanthomas TALE. Each rTALE was expressed from the CMV promoter and engineered to interact with an HBV-specific 18bp target. The repressors were incorporated into recombinant adenoassociated viral vectors which were used to deliver the antiviral elements. Inhibition of HBV replication was observed in cell culture models of HBV replication and in vivo. No evidence of toxicity was detected and inhibitory effects were sustained over a period of at least 2 months. Collectively these data indicate that rTALEs are effective against HBV and provide an efficient means of disabling HBV cccDNA without causing mutations that result from target DNA cleavage.
Saint Petersburg State University, Russia
Time : 15:40-16:05
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: Phenomenology; the source of variation and the underlying mechanism. For instance, any mutation is considered as an alteration which is stable and hereditable, stochastically arising, and 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.
The Hong Kong University of Science and Technology, Hong Kong
Time : 16:25-16:50
Guang Zhu is a Professor of Division of Life Science in Hong Kong University of Science and Technology. He has obtained his BSc and MSc in Physics. He has completed his PhD degree from University of Maryland and National Institutes of Health, USA, specialized in Biomolecular NMR Spectroscopy. Currently, his research focuses on structure-functional study of human and viral proteins in DNA replication initiation. He has published more than 79 peer-reviewed reports. He has served on the editorial boards of International Journal of Spectroscopy, Chinese Journal of Magnetic Resonance and Scientific Report.
Oligonucleotides play many functional roles in cells. G-rich DNA and RNA sequences can form stable four-stranded structures termed G-quadruplexes, in which four guanine bases associate through Hoogsteen hydrogen bonding to form a square planar structure or guanine tetrad (G4). G4 motifs are evolutionarily conserved in certain regions and associated with a specific subset of the genome. Two or more guanine tetrads stack to form a G-quadruplex, which may differ in how the DNA strand(s) are folded. G4 DNA are found in telomeric sequences such as d(GGTTAG)n, and in the promoter regions of many other genes. Genome-wide search identify 370,000 potential quadruplex sequences in the human genome. It was suggested that G-quadruplex formation in a promoter may block transcription of the gene. It has also shown that RNAs also form G-quadruplex and play an important role in transcription and translation processes. We are interested in structure-functional study of G-quadruplex of DNA and RNA in human DNA replication initiation and related diseases. Our biochemical and structural study showed that human Cdc6 binds G4 DNA directly supporting a role for G4 DNA in the recruitment of Pre-RC to replication origins. In analyzing the structure of G4 DNA that Cdc6 binds, we revealed a novel structural fold of G-quadruplex of human telomeric DNA. We also investigated the role of G-rich RNA in latent DNA replication of Epstein-Barr virus. These mechanistic studies will provide insight on the molecular mechanism for origin selection in human and human viruses.
Jining Medical University, Jining, China, University of Illinois at Chicago, USA
Wancai Yang is the Dean of the Institute of Precision Medicine and School of Basic Medical Sciences, Jining Medical University, China, and a Professor of Pathology at University of Illinois at Chicago, USA. He is also an Adjunct Professor of Biological Sciences at University of Texas, El Paso, USA. He obtained his MD degree and was trained a Pathologist, and received Post-doctoral training from Rockefeller University and Albert Einstein Cancer Center. In 2006, he moved to the Department of Pathology, University of Illinois at Chicago. He is serving as Grant Reviewer for the National Institutes of Health (USA) and the National Nature Science Foundation of China. His research focuses on: (1) mechanisms of gastrointestinal carcinogenesis, (2) identification of biomarkers for cancer detection and patient selection for chemotherapy, (3) implication of precision medicine in cancers. He has published about 90 articles and has brought important impact in clinical significance.
Chronic colitis malignant transformation is one of major causes to colorectal cancer, but the mechanisms of colitis development and malignant transformation is largely unknown. Using a unique mouse model, we have demonstrated that the mice with targeted disruption of the intestinal mucin gene Muc2 spontaneously developed chronic inflammation at colon and rectum at early age, whose histopathology was similar to ulcerative colitis in human. In the aged mice, Muc2-/- mice developed colonic and rectal adenocarcinoma accompanying severe inflammation. To determine the mechanisms of the malignant transformation, we conducted miRNA array on the colonic epithelial cells from Muc2-/- and +/+ mice. MicroRNA profiling showed differential expression of miRNAs (i.e. lower or higher expression enrichments) in Muc2-/- mice. Based on relevance to cytokines and cancer, the miRNAs were validate and were found significantly downregulated or upregulated in human colitis and colorectal cancer tissues, respectively. The targets of the miRNAs were further characterized and their functions were investigated. More studies from the Muc2-/- mice showed disorder of gut microbiota. Moreover, a novel tumor suppressor PRSS8 also plays a critical role in colorectal carcinogenesis and progression, for instance, tissue-specific deletion of the PRSS8 gene resulted in intestinal inflammation and tumor formation in mice. Gene set enrichment analysis showed that the colitis and tumorigenesis were linked to the activation Wnt/beta-catenin, PI3K/AKT and EMT (epithelial-mesenchymal transition) signaling pathways. Taken above, the disorder of gut microbiota could result in genetic mutations, epigenetic alterations, leading to the activation of oncogenic signaling, in colorectal epithelial cells, and finally, to colitis development, promoting malignant transformation and mediating colorectal cancer metastasis.