loechler@bu.edu

Biochemistry Cancer Biology Microbial Physiology and Genetics Genetics

Professor of Biology
Ph.D., Brandeis University, 1979

Molecular biology; mutagenesis and carcinogenesis, computational chemistry

BACKGROUND: CARCINOGENS, MUTAGENS AND BENZO[A]PYRENE

Carcinogens are cancer-causing substances, and are generally active because they cause mutations in the genetic substance, DNA. This makes sense, because tumor cells are derived from normal cells via mutations in certain key genes that are involved in cellular growth control.

Benzo[a]pyrene (B[a]P) is a potent mutagen and carcinogen, and is an example of the class of substances called "polycyclic aromatic hydrocarbons" (PAHs), which are found ubiquitously in the environment as the result of incomplete combustion. For example, PAHs are found in car exhaust, power plant emissions, cigarette smoke and charred foods. In many cases, B[a]P is the most important component of soot based on its prevalence and its potency as a mutagen and carcinogen. B[a]P has also proven to be a good model for the action of PAHs in general.

The goal of our research is to understand the fundamental mechanism by which potent carcinogens, such as B[a]P, induce mutations that turn normal cells into tumor cells.

B[a]P is metabolized in cells to its corresponding diol epoxide [(+)- anti -B[a]PDE], which we showed can induce all classes of mutations, though base substitutions at G:C prevail, of which ~60% are G->T. (+)- anti -B[a]PDE principally reacts at the N2-position of guanine to give the DNA adduct [+ta]-B[a]P-N2-dG (+BP).   +BP can induce each of these mutations, though G->T dominates in many sequence contexts.  

CURRENT WORK ON LESION-BYPASS DNA POLYMERASES AND BENZO[A]PYRENE MUTAGENESIS

We are investigating what DNA polymerases (DNAPs) are involved in mutagenic and non-mutagenic bypass of +BP and why? Human cells have at least fifteen DNAPs with four being involved in bypassing DNA adducts (DNAPs eta, kappa, iota and zeta), while E. coli has five DNAPs with three being involved in adduct bypass (DNAPs II, IV and V).   Lesion-bypass DNAPs are involved in both non-mutagenic and mutagenic bypass of DNA adducts/lesions, and we wish to understand the mechanistic differences.

Regarding the non-mutagenic pathway, we (and others) have shown that DNAP IV inserts dCTP correctly opposite [+ta]-B[a]P-N2-dG. DNAP V is also involved in the non-mutagenic pathway, as it does the next step after adduct-G:C formation ("extension"). In the G->T mutagenic pathway, DNAP V inserts dATP opposite [+ta]-B[a]P-N2-dG (and does extension).

Data in the literature show that human DNAP kappa and E. coli DNAP IV are functional orthologs and have probably evolved to insert the correct dNTP opposite adducts like [+ta]-B[a]P-N2-dG. Human DNAP eta and E. coli DNAP V are also functional orthologs and have evolved to insert the correct dNTP opposite DNA damaged by UV light (e.g., thymine dimers). Thus, it appears that G->T mutations occur when the wrong DNAP (i.e., V or eta) inserts opposite +BP.

Currently, we are trying to understand why DNAPs IV and V function so differently, in spite of both being in the Y-Family (as are DNAPs eta and kappa).   Because no X-ray structures existed, we built models of DNAPs IV, V, eta and kappa, taking a homology modeling approach.

Based on what appeared to be a functionally important structural difference, we changed one amino acid in DNAP IV that made it work almost as well as DNAP V in the extension step with +BP.   Taking this fundamental approach (i.e., determining what amino acid changes are minimally required to make DNAP IV function like DNAP V--and vice versa), we are determining the key structural features that define how these Y-Family DNAPs work.   For example, we have a hypothesis for what amino acid changes are needed to allow DNAP V to insert dCTP opposite +BP; i.e., to allow it to function like DNAP IV.   We are also trying to crystallize both DNAPs IV and V for X-ray structural studies.

Coordinates for E. coli DNAPs IV and V, and human DNAPs eta and kappa based on our molecular dynamics studies are available upon request (loechler@bu.edu).

Chandani, S. and Loechler, E.L. (submitted) Y-Family DNA Polymerases in Different Mechanistic Classes Have Structural Differences in Two Regions ("Roof"/"Chimney") That Affect Which of Two Different dNTP Shapes Is Used During Insertion.

Seo, K.-Y., Yin, J., Chandani, S., Lee, C.H., Nagalingam, A. and Loechler, E.L. (Submitted) Amino Acid Architecture That Influences dNTP Insertion in Y-Family DNAP V of E. coli .

Chandani, S., Lee, C.H.   and Loechler, E.L. (2007) Molecular Modeling Benzo[a]pyrene N2-dG Adducts in Two Partially Overlapping Active Sites of the Y-Family DNA Polymerase Dpo4. Journal of Molecular Graphics and Modelling . 25 , 658 - 670.

Kalam, M.A., Haraguchi, K., Alimchandani, S., Loechler, E.L., Moriya, M., Greenberg, M.M. and Basu, A.K. 2006. Comparative mutagenesis of Fapy.G and 8-oxo-G in mammalian cells. Nucleic Acids Research 34, 2305 - 2315.

Seo, K.-Y., Nagalingam, A, Shadi Miri, Jun Yin, Alexander Kolbanovskiy, Anant Shastry, and Loechler, E.L. 2006. Mirror Image Stereoisomers of the Major Benzo[a]pyrene N2-dG Adduct Are Bypassed by Different Lesion-Bypass DNA Polymerases in E. coli DNA Repair 5, 515 - 527.

Chandani, S., Lee, C.H. and Loechler, E.L. 2005. Free energy perturbation methods to study structure and energetics of DNA adducts: Results for the major N2-dG adduct of benzo[a]pyrene in two conformations and different sequence contexts. Chemical Research in Toxicology. 18, 1108-1123.

Seo, K.-Y., Nagalingam, A. Tiffany, M. and Loechler, E.L. 2005. Mutagenesis studies on four stereoisomeric N2-dG benzo[a]pyrene adducts in the identical 5'-CGC sequence used in NMR studies: Although adduct conformation differs, mutagenesis outcome does not as G->T mutations dominate in each case. Mutagenesis 20, 441 - 448.

Seo, K.-Y., Nagalingam, A, and Loechler, E.L. 2005. Mutagenesis studies of the major benzo[a]pyrene N2-dG adduct in a 5'-TG vs. 5'-UG sequence: loss of the methyl group causes only a modest decrease in the [G->T/G->A] ratio. Mutagenesis 20, 105-110.

Yin, J., Seo, K.-Y. and Loechler, E.L. 2004. A role for DNA polymerase V in G->T mutagenesis from the major benzo[a]pyrene N2-dG adduct when studied in a 5'-TGT sequence in Escherichia coli. DNA Repair 3, 323-334.

Lee CH, Loechler EL. 2003. Molecular modeling of the major benzo[a]pyrene N(2)-dG adduct in cases where mutagenesis results are known in double stranded DNA. Mutat Res. Aug 28;529(1-2):59-76.

Lee CH, Chandani S, Loechler EL. 2002. Molecular modeling of four stereoisomers of the major B[a]PDE adduct (at N(2)-dG) in five cases where the structure is known from NMR studies: molecular modeling is consistent with NMR results. Chem Res Toxicol. Nov;15(11):1429-44.

Seo KY, Jelinsky SA, Loechler EL. 2000. Factors that influence the mutagenic patterns of DNA adducts from chemical carcinogens. Mutat Res. Oct;463(3):215-46.

Kozack R, Seo KY, Jelinsky SA, Loechler EL. 2000. Toward an understanding of the role of DNA adduct conformation in defining mutagenic mechanism based on studies of the major adduct (formed at N(2)-dG) of the potent environmental carcinogen, benzo[a]pyrene. Mutat Res. May 30;450(1-2):41-59.

Struck, R.F., Davis, R.L., Jr., Berardini, M.D. and Loechler, E.L. 2000. DNA guanine-guanine cross-linking sequence specificity of isophosphoramide mustard, the alkylating metabolite of the clinical antitumor agent ifosamide. Cancer Chemother. Pharmacol. 45, 59-62.

Berardini, M., Foster, P. and Loechler, E.L. 1999. DNA polymerase II (polB) is involved in a new DNA repair pathway for DNA Interstrand Cross-Links in Escherichia coli. J. Bact. 181, 2878 - 2882

Shukla, R., Geacintov, N. and Loechler, E.L. 1999. The major, N2-dG adduct of (+)-anti-B[a]PDE induces G->A mutations in a 5'-AGA-3' sequence context. Carcinogenesis 20, 261 - 268.

Kozack, R., Shukla, R. and Loechler, E.L. 1999. A Hypothesis for What Conformation of the Major Adduct of (+)-anti-B[a]PDE (N2-dG) Causes G->T vs. G->A Mutations Based Upon a Correlation between Mutagenesis and Molecular Modeling Results. Carcinogenesis 20, 95 - 104.

Kozack, R. and Loechler, E.L. 1999. Molecular modeling of the major adduct of (+)-anti-B[a]PDE (N2-dG) in the eight conformations and the five DNA sequences most relevant to base substitution mutagenesis. Carcinogenesis 20, 85 - 94.

Shukla, R, Jelinsky, S., Liu T., Geacintov, N.E. and Loechler, E.L. 1997. How stereochemistry affects mutagenesis by N2-dG adducts of B[a]PDE: configuration of the adduct bond is more important than of the hydroxyl groups. Biochemistry 36, 13263 - 13269.

Shukla, R., Liu, Y., Geacintov, N. and Loechler, E.L. 1997. The major, N2-dG adduct of (+)-anti-B[a]PDE shows a dramatically different mutagenic specificity (predominantly, G->A) in a 5'-CGT-3' sequence context. Biochemistry 36, 10256 - 10261.

Kozack, R. and Loechler, E.L. 1997. Molecular modeling of the conformational complexity of (+)-anti-B[a]PDE-adducted DNA using simulated annealing. Carcinogenesis. 18, 1585 - 1593.

Hanrahan, C.J., Bacolod, M.D., Vyas, R.R., , Liu, T., Geacintov, N.E., Loechler, E.L. and Basu, A.K. 1997. Sequence specific mutagenesis of the major (+)-anti-benzo[a]pyrene diol epoxide-DNA adduct at a mutational hotspot in vitro and in Escherichia coli cells. Chemical Research in Toxicology 10, 369 - 377.

Berardini, M., Mackay, W. and Loechler, E.L. 1997. A Site-Specific Study of a Plasmid Containing Single Nitrogen Mustard Interstrand Cross-Link: Evidence for a Second, Recombination-Independent Pathway for the DNA Repair of Interstrand Cross-Links. Biochemistry. 36, 3506 - 3513.

Min, Z., Gill, R.G., Cortez, C., Harvey, R.G., Loechler, E.L. and DiGiovanni, J. 1996. Targeted A->T, and G->T mutations induced by site-specific deoxyadenosine and deoxyguanosine adducts, respectively, from the (+)-anti-diol-epoxide of dibenz[a,j]anthracene in M13mp7L2. Biochemistry. 35, 4128 - 4138.

Jelinsky, S. A., Mao, B., Geacintov, N.E. and Loechler, E.L. 1995. The major, N2-Gua adduct of the (+)-anti-benzo[a]pyrene diol epoxide is capable of inducing G->A and G->C, in addition to G->T,mutations.Biochemistry 34, 13545 - 13553.

Loechler, E.L. 1995. How Are Potent Bulky Carcinogens Able to Induce Such a Diverse Array of Mutations? Mol. Carcinogenesis 13, 213 - 219

REVIEWS

Loechler, E.L. 1996. Commentary: The role of adduct-site-specific mutagenesis in understanding how carcinogen DNA adducts cause mutations: Perspective, Prospects and Problems. Carcinogenesis 17, 895-902.

Kozack, R, Seo, K.-W., Jelinsky, S.A. and Loechler, E.L. 2000. Toward an understanding of the role of DNA adduct conformation in defining mutagenic mechanism based on studies of the major adduct (formed at N²-dG) of the potent environmental carcinogen, benzo[a]pyrene. Mutation Res. 450, 41 - 59.

Loechler, E.L., Henry, B. and Seo, K.-Y. 2001. Biological Responses to Chemical Carcinogens. IN: The Molecular Basis of Human Cancer (Coleman and Tsongalis), Humana Press, Totowa, NJ, pp. 203 - 222.

Loechler, E.L. 2002. Environmental Mutagens and Carcinogens. IN: Nature Encyclopedia of Life Sciences, Nature Publishing Group, New York, NY, http://www.els.net.

Questions and comments are always welcome.
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This document was last modified on February 7, 2008.