Dr. Angel L. IslasAssociate Professor & Director of Biotechnology ProgramEducational BackgroundB.S. – Biochemistry, University of California, Davis Ph.D. – Department of Biological Sciences, Stanford University Postdoctoral Fellow – Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford University Postdoctoral Fellow – Laboratory of Radiobiology and Environmental Health, University of California, San Francisco. Teaching Biology 9: Cancer Biology 24: Cell Biology/Genetics Biology 174: Cell Biology Biology 175: Molecular Biology Biology 177: Biotechnology Lab II: Gene Expression & Protein Purification
*Undergraduate Research Brochure Research
Our research program examines the molecular events that occur when DNA polymerases, the enzymes responsible for duplicating DNA, encounter damaged DNA. Our research can be divided into three major themes:
Template Switching The main question of our lab is: what do DNA polymerases do when they encounter DNA strand breaks? The two strands of DNA in all living cells can be broken as a result of exposure to ionizing radiation, certain anti-cancer drugs, or spontaneously through normal metabolic processes. DNA double-strand breaks are very harmful to human cells – some estimates suggest one DNA double-strand break in a human cell is sufficient to lead to cell death. DNA polymerases are dependent on an unbroken DNA template for synthesis of new DNA; it uses the sequence of the template strand to create a new complementary copy of DNA. Therefore, one would expect the enzyme to simply stop or fall off when it reaches a strand break. But we have seen a surprising phenomenon: occasionally some DNA polymerases can synthesize DNA past a break in a template strand – in essence switching from one strand to another. The biological implications to DNA double-strand break repair and rejoining could be important. It suggests that DNA polymerases may play a role in rejoining a broken DNA template during the course of replication. And it may have important implications for mutagenesis and cancer. We focus on quantifying this template-switching activity by DNA polymerases and on determining its underlying mechanism. Using a variety of experimental approaches from protein biochemistry to molecular biology, our research seeks to understand how DNA polymerases recognize broken template strands, what proteins influence template switching by DNA polymerases, and how DNA structure affects the efficiency of template switching.
Non-template-directed DNA synthesis
Related to template switching is the observation that at the ends of DNA very often some DNA polymerases will add extra nucleotides leaving a 3´ overhang. These extra nucleotides are not added in a template-directed manner; in other words they are not coded in the DNA. Rather, they are added in an almost random fashion depending on the DNA polymerase involved. Some DNA polymerases specialize in this kind of DNA synthesis. For example, terminal deoxynucleotidyl transferase (TdT) will add non-template directed nucleotides almost exclusively and in fact these nucleotides are in part responsible for the diversity of antibodies and T-cell receptors generated during V(D)J recombination. While TdT is usually only expressed in lymphoid tissue, a number of other DNA polymerases ubiquitously expressed in all tissues can also add nucleotides to the ends of DNA. Estimates are that around 10 double strand breaks occur per cell per day in humans and well over 1000 single strand breaks occur per cell per day; meaning that there are plenty of opportunities for DNA polymerases to encounter breaks and add nucleotides. In fact, as many as 10% of double strand breaks in mammalian cells show nucleotide insertions consistent with non-template directed DNA synthesis. This means that the potential for mutagenesis as the result of non-template directed synthesis is extremely high. Our research focuses on how DNA polymerases add nucleotides in non-template directed manner, how this kind of synthesis differs from normal template-directed synthesis, and what proteins are responsible for regulating and/or inhibiting this potentially mutagenic activity.
Psoralen Crosslinks and Monoadducts Psoralens Publications García, P.B., Robledo, N.L.,and Islas, A.L., “In vitro DNA synthesis at DNA ends: A kinetic analysis of non-template-directed synthesis and template switching by DNA polymerase." Manuscript in Preparation. Islas, A.L., Fairley, C.F., and Morgan, W. F., “DNA Synthesis on Discontinuous Templates by Human DNA Polymerases: Implications for Non-homologous DNA Recombination.”, Nucleic Acids Research 26(16):3729-38 (1998). Islas, A.L. and Hanawalt, P.C. “DNA Repair in the MYC and FMS Proto-oncogenes in Ultraviolet Light-irradiated Human HL60 Promyelocytic Cells During Differentiation.”, Cancer Research 55(2):336-41 (1995). Lieber, M. R., Chang, C.-P., Gallo, M., Gauss, G., Gerstein, R., and Islas, A., “The Mechanism of V(D)J Recombination: Site-Specificity, Reaction Fidelity and Immunologic Diversity.”, Seminars in Immunology 6:304-315 (1994). Islas, A.L., Baker, F.J. and Hanawalt, P.C., “Transcription-coupled Repair of Psoralen Crosslinks But Not Monoadducts in Chinese Hamster Ovary Cells.”, Biochemistry 33:10794-10799 (1994). Islas, A.L., Vos, J.-M., and Hanawalt, P.C. “Differential Introduction and Repair of Psoralen Photoadducts to DNA in Specific Human Genes.”, Cancer Research 51:2867-2873 (1991). Islas, A.L., Vos, J.-M. and Hanawalt, P.C. “Processing of Psoralen Adducts in Defined Human Sequences.”, Journal of Cellular Biochemistry Supplement 12A:290 (1988).
Former Members of the Islas Lab
Nicole L. Robledo, Research Associate Allison M. Faucett, Student Patty B. García, Student Aaesha I. Sheikh, Student Maria P. Miranda, Student
Diana M. Maldonado 2001 |
