B.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.
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
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:
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
are a class of chemical compounds that intercalate into DNA and are covalently linked to one strand (forming monoadducts) or both strands (forming crosslinks) of the DNA when photoactivated. Psoralens have been used in the treatment of a number of hyperproliferative diseases such as psoriasis and even cancer. Because they damage DNA, psoralens interfere with transcription by blocking elongation of RNA polymerases and with replication by blocking elongation of DNA polymerases. We are interested in examining how DNA polymerases are blocked by psoralen adducts, whether the psoralen adduct is binding the active site or other allosteric sites on the enzyme, or whether the psoralen adduct is altering the structure of DNA and thereby affecting DNA polymerase binding and synthesis. We feel these three related approaches will help us to better understand how DNA polymerases work and just as importantly how they make errors so that we can ultimately understand how mutations arise.
Miranda, M.P.L., and Islas, A.L., " Temperature profiling of template-switching activity by exonuclease-deficient thermophilic DNA polymerases" (manuscript in preparation).
Nosé, K.Y. and Islas, A.L. “Non-template-directed nucleotide addition by DNA polymerase: a kinetic analysis of inhibition and nearest neighbor effects.” FASEB Journal. Vol. 20: A481 (2006). Poster Abstract: Experimental Biology 2006 (ASBMB) San Francisco, CA.
Faucett, A.M. and Islas, A.L., “Reiterative template switching: the effect of single-strand homopolymeric DNA on non-template-directed nucleotide addition by DNA polymerase”, Biochem Biophys Res Comm 337:1030-1037 (2005).
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
Lynae R. Green 2002
Juan José Fung 2002
Joyce A. C. Viloria 2003
Kelly C. Vranas 2003
Elizabeth A. Scott 2004