Research Interest/Area of Expertise
Biochemistry, molecular biology, chemical carcinogenesis, DNA replicaton, enzyme mechanism
Understanding how carcinogenic DNA adducts compromise accurate DNA replication is an important goal in cancer research. These lesions can occur by endogenous agents in the cell or by exposure to exogenous agents such as the toxins found in cigarette smoke. The DNA damage caused by these agents can result in mutations by reducing the accuracy of DNA replication across from or near the damaged site. A central goal of the studies in our lab is to determine the molecular mechanism that allows a DNA polymerase to incorporate a nucleotide across from and past a bulky adduct in a DNA template, which we anticipate will lead to a better understanding of how the damage leads to mutations.
It is well established that during synthesis DNA polymerases incorporate a nucleotide through a multi-step mechanism that involves a conformational change from an open binary to a catalytically active closed ternary complex. We have developed a method to detect the formation of this closed complex which forms only in presence of the next correct nucleotide. Any deviation from correct Watson/Crick (W/C) geometry for the resulting base-pair was shown to have a destabilizing effect on the complex. The presence of a bulky carcinogenic adduct, such as an N-acetyl-2-aminofluorene-dG adduct (dG-AAF), resulted in two interesting effects. First, surprisingly, the polymerase binds much more tightly to the modified primer-template than to an unmodified one. Second, the presence of the adduct completely inhibited the conformational change, explaining why this adduct acts as a strong block to DNA synthesis. We have recently obtained a crystal structure of a polymerase bound to an AAF-modified primer-template (shown in the figure) and find that this structure provides an explanation for the biochemical effects of the adduct. We found that the adduct caused the O helix to tilt forward and block the site were the nucleotide must bind prior to incorporation. We have recently begun to determine how specific amino acid substitutions within the polymerase active site contribute to the properties that affect polymerase mechanism and fidelity. We have shown that the substitution of a serine for a tyrosine at the base of the O helix result in a significant increase in the relative ability to incorporate the correct nucleotide dC opposite the dG-AAF adduct. Moreover, we are able to detect a conformational change for the mutant polymerase when it is bound to an AAF-modified template, suggesting that the increased room provided by removing the bulky tyrosine allows for nucleotide binding in the active site. Finally, our most recent studies involve using FRET at the single molecule level to determine how these types of adducts effect the rate of polymerase binding and synthesis on DNA templates.
Education – Degrees, Licenses, Certifications
- B.A. Chemistry Rutgers College (1972)
- Ph.D. Organic Chemistry (1976), Rutgers University
- NIH Postdoctoral Fellowship, Harvard Medical School (1976-1980)
Joshua P. Gill and Louis J. Romano (2005) "Mechanism for AAF-induced Frameshift Mutagenesis by Escherichia coli DNA Polymerase I (Klenow Fragment)," Biochemistry 44, 15387-15395.
Dutta, S., Li, Y., Johnson, D., Dzantiev, L., Richardson, C. C., Romano, L. J., and Ellenberger, T. (2004) "Crystal Structures of N-2-acetylaminofluorene and N-2-aminofluorene in Complex With T7 DNA Polymerase Reveal Mechanisms of Replication Blockage and Mutagenesis," Proc. Natl. Acad. Sci. U.S.A. 101, 16186-16191.
Lone, S., Romano, L. J. (2003) "Mechanistic Insights into Replication Across from Bulky DNA Adducts: A Mutant Polymerase I Allows an N-Acetyl-2-aminofluorene Adduct to be Accommodated During DNA Synthesis", Biochemistry 42, 3826-3834.
Alekseyev, Y.; Romano, L. J. (2002) "Effect of Benzo[a]pyrene Adduct Stereochemistry on Downstream DNA Replication In Vitro: Evidence for Different Adduct Conformations Within the Active Site of DNA Polymerase I (Klenow Fragment)", Biochemistry 41, 4467-4479.
Dzantiev, L.; Romano, L. J. (2000) "Differential effects of N-Acetyl-2-aminofluorene and N-2-Aminofluorene Adducts on the Conformational Change of DNA Polymerase I (Klenow Fragment)", Biochemistry 39, 5139-5145.