Genome Integrity Discussion Group April 2014
Monday, April 7, 2014
The New York Academy of Sciences
Presented by the Genome Integrity Discussion Group at the New York Academy of Sciences.
The greater New York Metropolitan area has become a leading center for research on chromosome biology and function, as well as for research at the interface between chromosome integrity and onset and progression of malignancy. The connection between cancer and genome integrity is widely appreciated, and the concentration of excellence in this field is unparalleled anywhere in the world. The Genome Integrity meetings are designed to provide a forum for interactions between the many basic science and clinically-oriented research groups working on these issues. We feel that these interactions will not only facilitate synergy between labs, but also provide a context in which previously unappreciated complementarities will be revealed.
In that spirit, the talks will cover a broad range of areas, including, but not limited to the DNA damage response and cancer predisposition, DNA replication, transcription, chromatin modification, recombination, cell cycle control, telomeres, chromosome segregation, epigenetic states, as well as the emergence of new technologies relevant to research in genome integrity. Although a primary focus is upon basic mechanisms and processes, these areas are pertinent to cancer and myriad human disease states, and it is expected that this will be reflected in the substance of our discussions.
Genome Integrity Discussion Group meetings are organized under the leadership of John Petrini (Memorial Sloan Kettering Cancer Center), Susan Smith (NYU Langone Medical Center) and Lorraine Symington (Columbia University). This meeting will include a scientific symposium from 1:30 to 4:30 PM, followed by a networking reception from 4:30 to 5:30 PM.
|Nonmember (Student / Postdoc / Resident / Fellow)
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* Presentation titles and times are subject to change.
April 7, 2014
|Slicer and the Argonautes
Leemor Joshua-Tor, PhD, Cold Spring Harbor Laboratory
|DNA Damage-Dependent Regulation of Poly(ADP-ribosyl)ation Reactions
John M. Pascal, PhD, Thomas Jefferson University
|DNA Interrogation by the CRISPR RNA-guided Endonuclease Cas9
Sy Redding (Greene Lab, Columbia University)
|Identification and Biochemical Characterization of CMGE, a Minimal Eukaryotic Leading Strand Replisome
Lance Langston, PhD (O’Donnell Lab, The Rockefeller University)
|Regulation of Dual Incision and Repair Synthesis in Human Nucleotide Excision Repair
Orlando D. Schärer, PhD, Stony Brook University
|ATM and DNA-PKcs in DNA Repair – Beyond Phosphorylation
Shan Zha, MD, PhD, Columbia University
John Petrini, PhD
Memorial Sloan-Kettering Cancer Center
Susan Smith, PhD
NYU Langone Medical Center
Susan Smith, PhD, is a Professor in the Skirball Institute and Department of Pathology in the NYU School of Medicine. Prior to joining NYU in 1999 as an Assistant Professor, Dr. Smith trained as a postdoctoral fellow with Dr. Titia de Lange and Dr. Gunter Blobel at The Rockefeller University. She earned her PhD from Stony Brook University working with Dr. Bruce Stillman at Cold Spring Harbor Laboratory. Dr. Smith’s current research focuses on: regulation of telomere protein stability and function by post-translational modifications including poly(ADP-ribosyl)ation and ubiquitylation; mechanisms controlling establishment and resolution of sister chromatid cohesion at telomeres; impact of defective telomere cohesion in the human stem cell disease dyskeratosis congenita; and cell cycle regulation and non-telomeric functions of the PARP tankyrase 1, including its role at spindle poles and centrosomes. The long-term goal of her research is to understand the mechanisms that ensure genome integrity and cell survival. In addition to serving on the Skirball internal advisory committee, she is the Graduate Advisor for the Molecular Oncology and Tumor Immunology Graduate program at NYU and she served as a regular member of the NIH Molecular Genetics B Study Section.
Lorraine Symington, PhD
Columbia University Medical Center
DNA repair mechanisms are essential for the maintenance of genome integrity and defects in these processes are associated with cancer predisposition in humans. Research in the Symington laboratory is focused on three critical aspects of homology-directed double-strand break (DSB) repair: mechanism and regulation of DNA end processing; mechanisms of break-induced replication (BIR); and identification of nucleases involved in maturation of homologous recombination intermediates. The Symington laboratory has made important contributions to understanding how the ends of double-strand breaks are processed to generate 3’ single-stranded tails and the interplay between non-homologous end joining and the initiation of DNA end resection. These studies paved the way for research in vertebrate systems showing the mechanism of end resection is highly conserved. Their BIR studies were the first to demonstrate Rad51 dependence, that the initial strand invasion intermediate is unstable and for a conservative mode of DNA synthesis during BIR. They also identified Mus81-Mms4 as the main nuclease responsible for mitotic crossovers in yeast and proposed a novel role for the Rad1-Rad10 (XPF-ERCC1 in human) nuclease in cleaving recombination intermediates between ectopic sequences resulting in formation of a single Holliday junction intermediate.
Leemor Joshua-Tor, PhD
Cold Spring Harbor Laboratory
Leemor Joshua-Tor, Ph.D. is a Howard Hughes Medical Institute Investigator and Professor at Cold Spring Harbor Laboratory. She uses a combination of biochemistry, molecular biology and biophysics to uncover how cells work at a molecular level. One way she does this is using a method called x-ray crystallography to deduce the structures of proteins, DNA and RNA molecules and determine how they interact, which leads to a better understanding of how these components work to control important cellular events. She is perhaps best known for her groundbreaking work revealing the inner workings of the gene-silencing mechanisms of RNA interference (RNAi), a process that regulates many important cellular processes and is emerging as a powerful took in biotechnology and medicine. She is also known for her studies on the molecular motors that are essential for DNA replication.
Dr. Joshua-Tor was born in Israel and received a B.Sc. in chemistry from Tel-Aviv University and a Ph.D. in chemistry from the Weizmann Institute of Science in Rehovot. She was awarded the presitgious Jane Coffin Childs postdoctoral fellowship for her continued training at the California Institute of Technology prior to joining the CSHL faculty. At CSHL, she was the Director of the Undergraduate Summer Research Program and then the Dean of the Watson School of Biological Sciences, CSHL’s graduate school. She is widely recognized as one of the top scientists in her area, and has been recognized by several awards, including the Dorothy Crowfoot Hodgkin Award from the Protein Society and the Beckman Young Investigator Award. She was recently elected as a Fellow of the American Association for the Advancement of Science. In addition to her research activities, Dr. Joshua-Tor serves on the editorial boards of several international scientific journals and on several advisory committees at the National Institutes of Health.
John Pascal, PhD
Thomas Jefferson University
John M. Pascal, Ph.D., is an Associate Professor of Biochemistry and Molecular Biology and a member of the Kimmel Cancer Center at Thomas Jefferson University, Philadelphia, USA. He studied the structural biology of DNA replication and repair as a postdoctoral fellow at Harvard Medical School, and earned his Ph.D. at the University of Texas, Austin. His research currently focuses on proteins involved in maintaining genome stability.
Orlando Scharer, PhD
Stony Brook University
Orlando D. Schärer grew up in Zürich, Switzerland, where he received his MSc in Chemistry from the ETH Zürich. He obtained a PhD in chemistry with Gregory Verdine at Harvard University and conducted postdoctoral studies with Roland Kanaar and Jan Hoeijmakers at Erasmus University in Rotterdam in the Netherlands. He started his independent research group at the University of Zürich, Switzerland, before moving to Stony Brook University, NY in 2005, where he is currently professor of pharmacological sciences and chemistry. His research combines organic chemistry, biochemistry and cell biology to study mechanism of mammalian DNA repair pathways and the relationships of these pathways to genetic disorders, carcinogenesis and antitumor therapy.
Shan Zha, MD, PhD
The Zha lab is interested in the molecular mechanism of DNA double stand break repair and its implications in the development of normal lymphocytes and lymphoid malignancies. The goal of the lab is to use the knowledge in DNA repair understand the etiology of primary immunodeficiency and oncogenic translocations and ultimately facilitate better diagnosis and treatment for leukemia and lymphomas. Normal lymphocyte utilizes programmed DNA breaks as obligated intermediates during the assembly and subsequent modifications of the antigen receptor gene product. While these recombination events are critical for the diversity, specificity and longevity of the adaptive immune responses, mistakes during this process drive oncogenic translocations characteristic for human lymphoid malignancies. While these programmed breaks are initiated by lymphocyte specific genes, the DNA repair and completion of these recombination events rely on ubiquitously expressed DNA damage responsive genes and the non-homologous end-joining pathways. Dr. Zha finished her post-doctoral training in the laboratory of Dr. Frederic Alt and established her own group in Columbia University in 2010. In the past ten years, Dr. Zha and her lab have used mouse genetic approach to understand the role of DNA repair gene deficiencies in primary immunodeficiency and lymphomas. In this process, they have generated mouse models carrying disease causing mutations in DNA damage response genes (e.g. ATM, H2AX) and in members of the non-homologous end-joining pathway (e.g. XLF, DNA-PKcs). Using these tools, they identified extensive crosstalk and redundancies in DNA repair pathways. A subset of the animal models developed spontaneous lymphomas sharing molecular features with human lymphoid malignances. The Zha lab has characterized the translocation patterns and mechanism and identified novel tumor suppressor genes for acute lymphoblastic leukemias.
Slicer and the Argonautes
Argonautes are the central protein component in small RNA silencing pathways. Of the four human Argonautes (hAgo1-4) only hAgo2 is an active slicer. We have determined structures of the catalytically active hAgo2 as well as the catalytically inactive hAgo1, both bound to discrete miRNAs. The structures are strikingly similar. A conserved catalytic tetrad within the PIWI domain of hAgo2 is required for its slicing activity. Completion of the tetrad combined with a mutation on a loop adjacent to the active site of hAgo1 results in slicer activity that is substantially enhanced by swapping in the N domain of hAgo2. hAgo3, with an intact tetrad, becomes an active slicer by swapping the N domain of hAgo2, without additional mutations. Intriguingly, the elements that make Argonaute an active slicer involve a sophisticated interplay between the active site and more distant regions of the enzyme.
Coauthors: Christopher R. Faehnle1,3, Elad Elkayam1,2,3, Astrid D. Haase2,3, Gregory J. Hannon2,3
1W. M. Keck Structural Biology Laboratory
2Howard Hughes Medical Institute
3Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
DNA Damage-Dependent Regulation of Poly(ADP-ribosyl)ation Reactions
John M. Pascal, PhD, Thomas Jefferson University, Philadelphia, Pennsylvania
Poly(ADP-ribose) polymerases (PARPs) use NAD+ to create the posttranslational modification poly(ADP-ribose), or PAR, to regulate a variety of cellular functions. The PAR synthesis activities of PARPs 1, 2, and 3 are potently stimulated through interaction with DNA damage, and the acute production of PAR is a hallmark of the cellular response to DNA damage. We have been working toward a molecular level understanding of the mechanism of DNA damage-dependent PARP activation. Our most recent structural and biochemical work have demonstrated how PARP-1 recognizes DNA damage, and how DNA damage organizes the modular domain architecture of PARP-1 into a conformation that activates the catalytic domain. The activated conformation of PARP-1 domains suggests a mechanism for in cis modification, and can explain PARP-1 preference for automodification over heteromodification. Moving forward, we are further studying the mechanism of PARP-1 activation using a range of techniques, including kinetic analysis and direct measurements of protein dynamics associated with activation. We are also leveraging our structural data to guide the design of mechanism-based PARP inhibitors that are functionally specific, e.g. DNA damage repair versus transcriptional regulation. PARPs 2 and 3 have fewer regulatory domains than PARP-1, but they are still regulated through interaction with DNA damage. Through structural, biochemical, and functional analyses we are establishing the similarities and differences among the DNA-damage dependent PARPs. These studies will improve our understanding of the specific cellular pathways that are regulated by PARPs, and will potentially contribute to the development of PARP inhibitors with improved specificity to be used as chemical probes and novel therapeutics.
Regulation of Dual Incision and Repair Synthesis in Human Nucleotide Excision Repair
Orlando D. Schärer, Department of Pharmacological Sciences & Department of Chemistry, Stony Brook University
Numerous structure-specific DNA endonucleases are required for genome maintenance in humans. While these enzymes have essential roles in repairing aberrant DNA structures, inadvertent incision of DNA could have deleterious consequences. Important questions are therefore how endonucleases are recruited to their specific DNA repair pathways and how the formation of long-lived nicks and gaps in DNA following incision are avoided. This talk will focus on how the activity nuclease activities are coordinated in nucleotide excision repair. Following damage recognition and preincision complex formation, the two incisions happen near simultaneously 5′ to lesion by ERCC1-XPF and 3′ to the lesion by XPG. Our studies revealed that the order and timing of the two incisions is tightly coordinated with the 5′ incision occurring first, followed by partial repair synthesis, incision by XPG and completion of the process. Furthermore, we found that the activity of the XPG endonuclease is tightly controlled and requires an interaction with ubiquitin and PCNA. Our results suggest that XPG is a latent endonuclease, the catalytic activity of which is controlled by multiple mechanisms. We will discuss the importance of these results in the context of UV-induced DNA damage signaling.
Coauthors: Yan Su, Lidija Staresincic, and Adebanke F. Fagbemi, Department of Pharmacological Sciences & Department of Chemistry, Stony Brook University; Bevin P. Engelward and Yang Su, Department of Biological Engineering, Massachusetts Institute of Technology.
ATM and DNA-PKcs in DNA Repair – Beyond Phosphorylation
Shan Zha, MD, PhD, Columbia University
ATM and DNA-PK are PI3 kinase related protein kinases that are recruited to the DNA ends and activated upon DNA double stand breaks by their specific accessory proteins. Activated DNA-PK and ATM phosphorylate overlapping substrates involved in DNA repair and checkpoint control to orchestra the DNA damage responses. In addition to other substrates, both ATM and DNA-PKcs are phosphorylated by themselves and also cross phosphorylated by each other. However the exact function of their auto and trans-phosphorylation are not well understood. To address these question, we knockin the kinase dead (KD) mutations into endogenous ATM and the catalytic subunit of DNA-PK (DNA-PKcs) genes in mouse germline. In contrast to the absence of protein expression in ATM null or DNA-PKcs null mice, ATMKD/KD and DNA-PKcsKD/KD mice express normal levels of ATM or DNA-PKcs protein lacking kinase activity.; While ATM null and DNA-PKcs null mice develop normally, both ATMKD/KD and DNA-PKcsKD/KD mice died during embryonic development with severe genomic instabilities. Further characterization of ATMKD/KD and DNA-PKcsKD/KD cells and mice revealed critical and specific “structural” function of ATM and DNA-PKcs protein at the DNA ends that is modulated by their auto-phosphorylation and cross-phosphorylation status.
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