Chromosome Dynamics and Gene Therapy
DNA Replication, Recombination and Repair
Session: Aberrant DNA Repair, Genomic Instability and Cancer
• The Role of Rev3L in Genome Maintenance, Richard D. Wood, University of Texas M. D. Anderson Cancer Center
• Aberrant Base Excision Repair and Cancer, Joann B. Sweasy, Yale University
• Delineating Drivers of Large-scale DNA Sequence Rearrangements in Vivo, Bevin P. Engelward, Massachusetts Institute of Technology
Session: Site-specific Recombination in Chromosome Dynamics and Gene Therapy
• Structure and Function of Serine Integrases, Gregory D. Van Duyne, University of Pennsylvania
• Mobile Group II Introns: Site-specific Retroelements with Programmable DNA Target Specificity in Bacteria and Eukaryotes, Alan Lambowitz, University of Texas at Austin
• Gene Therapy Targeted by Meganucleases, Nancy Maizels, University of Washington School of Medicine
Session: Replication of Noncanonical DNA Sequences and Genomic Instability
• Coordination of the Leading and Lagging Strand During DNA Replication, Smita Patel, Robert Wood Johnson Medical School
• DNA Structure, Genetic Instability and Cancer, Karen M. Vasquez, University of Texas M. D. Anderson Cancer Center
• XPD-Dependent Induction of Apoptosis of Cells with DNA Containing Helical Repeats, Faye Rogers, Yale University
Session: Retroelements in
Genome Plasticity and Cancer
• Yeast Ty1 Retrotransposons and Genome Fragility, Joan Curcio, Wadsworth Center, New York State Department of Health
• Group II Introns Collaborate with their Host to Promote Genome Plasticity by Retrotransposition, Marlene Belfort, Wadsworth Center, New York State Department of Health
• Xenotropic Murine Leukemia Virus-related Virus and Prostate Cancer, Robert H. Silverman, Lerner Research Institute, Cleveland Clinic
The second session, titled “Site-specific Recombination in Chromosome Dynamics and Gene Therapy,” describes DNA transactions designed to repair genetic defects. Interestingly, in all cases, the agent that targets DNA to mediate recombination is derived from a naturally occurring mobile genetic element. First, Gregory D. Van Duyne (University of Pennsylvania) will describe the structure and function of a serine integrase. These integrases have great potential for use in a variety of transgenic and gene therapy applications. His group is working to develop a structural basis for understanding how these site-specific recombinases achieve what is effectively an irreversible integration reaction without the use of accessory proteins and auxiliary DNA sequences. Small angle x-ray and neutron scattering, combined with single crystal X-ray diffraction, have provided some important insights into this recombinase family.
In contrast, Alan Lambowitz (University of Texas at Austin) will describe group II introns as gene-targeting vehicles. Mobile group II introns, ribozymes that insert site-specifically into DNA, have been developed into gene targeting vectors (“targetrons”) with the unique feature of readily programmable DNA target specificity. Targetrons are used widely for gene targeting in diverse bacteria, and recent work is focusing on adapting targetrons to function efficiently in eukaryotes.
Eukaryotic gene therapy also is being attempted by Nancy Maizels and colleagues (University of Washington School of Medicine), using homing endonucleases called meganucleases. Meganuclease-targeted gene correction is an especially powerful strategy for gene therapy, and, like the two aforementioned gene targeting agents, it uses molecules and mechanisms optimized over billions of years of evolution to correct deleterious mutations in human cells.
Processing Non-B Form DNA
The third session is titled “Replication of Non-Canonical DNA Sequences and Genomic Instability.” Smita Patel (Robert Wood Johnson Medical School) will focus on the enzymatic mechanisms for coordinating leading and lagging strand DNA synthesis. The antiparallel nature of the double-stranded DNA and the 5’-3’ directionality of the polymerase enzyme pose unique problems in copying the two strands in the same time span. Several mechanisms have been identified that allow the lagging polymerase to keep up with the leading polymerase. The replication enzymes stay physically associated, and, as a consequence, the displaced DNA strand rolls out into a priming loop. The synergistic actions of the replication enzymes allow the two strands of the DNA to be copied in the same time span.
Naturally occurring DNA repeat sequences can form noncanonical DNA structures such as H-DNA and Z-DNA, which are abundant in mammalian genomes. Karen M. Vasquez (University of Texas M. D. Anderson Cancer Center) will discuss her work showing that both H-DNA and Z-DNA structures are intrinsically mutagenic in mammalian cells. Her findings suggest that both H-DNA and Z-DNA, which have been reported to correlate with chromosomal breakpoints in human tumors, are sources of genetic instability and demonstrate that naturally occurring DNA sequences are mutagenic in mammalian cells and may contribute to evolution and disease.
Faye Rogers (Yale University) also will describe her work on naturally occurring H-DNA in human cells. To counteract the potentially devastating effects of altered helical structures on genomic integrity, an intricate balance between DNA repair and apoptosis is critical. Rogers has found that the TFIIH factor XPD is implicated in triggering apoptosis in response to excessive H-DNA induced damage. The maintenance of this mechanism may be of central importance for avoiding induction of mutations and progression to cancer.
RNA and Genome Plasticity
The fourth and last session switches to “Retroelements in Genome Plasticity and Cancer.” The three talks in this session involve retroelements in organisms as diverse as bacteria, yeast and humans. Joan Curcio (Wadsworth Center, New York State Department of Health) will describe Ty1, a retrovirus-like transposon in Saccharomyces cerevisiae. Ty1 is associated with chromosome fragile sites and plays remarkably versatile roles in promoting chromosomal rearrangements and generating novel gene sequences. Chimeric cDNA molecules created by Ty1 reverse transcriptase function as molecular bridges, healing chromosome breaks and reordering the genome in the process. Her talk will examine how retrotransposition creates chromosomal sites that are prone to breakage and how DNA damage signaling pathways modulate the synthesis of cDNA molecules that straddle broken ends to form rearranged chromosomes.