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Projects RESEARCH PROJECT 1 Biochemical Consequences of Tamoxifen-DNA Adducts 2004 Merck Scholar Sarah Smith Despite tamoxifen’s profound effects in controlling the growth of breast cancer, tamoxifen treatment has been associated with an increased incidence of endometrial cancer. The basis for this carcinogenicity is believed to be caused by the formation of covalent adducts that arise from the reaction of metabolically activated tamoxifen derivatives and deoxyguanine of DNA. The resulting four diastereoisomers of a-(N2-deoxyguanosyl)tamoxifen have demonstrated different mutagenic properties.
The objective of our research is to understand how the different
stereoisomeric adducts affect lesion bypass by mutation-inducing
polymerases. We will determine if and how the error-prone polymerases, DNA
polymerase IV and V of Escherichia coli, and the mammalian paralog of
these enzymes, human DNA polymerase iota, replicate past these different
isomers of tamoxifen-DNA adducts within a single sequence context. This
research will correlate structural stability of the adducts with lesion
bypass activity by different polymerases. RESEARCH PROJECT 2 Drought
and Salinity Responses In C4 Grasses The Panicoideae and Chloridoideae grass subfamilies together comprise the great majority of the grass flora of tropical and subtropical regions of the world. These subfamilies differ in their distributions across natural precipitation and salinity gradients, with species of the Chloridoideae found in drier and more saline environments than those of the Panicoideae. As little is known of physiological differences between the two subfamilies that might account for these distributional differences, we will therefore compare the physiology of a selected group of species from these subfamilies grown under several different conditions of imposed drought and soil salinity. Plant tolerance of these factors will be assessed by examining several aspects of plant functioning known to be affected by drought and salinity stress, including biomass accumulation, membrane integrity, leaf chlorophyll content, stomatal conductance of water vapor, and rates of leaf photosynthesis and transpiration. Detailed examination of leaf chemistry will allow us to examine a variety of specific mechanisms of drought and salinity tolerance in these plants. One mechanism that enables some species of plants to tolerate soil salinity is the secretion of excess Na+ and Cl- from specialized salt glands onto leaf surfaces. We will compare secretion rates among species by washing leaf surfaces and analyzing the rinsate using potentiometry. We will also assess the concentrations of several key ions (e.g., Na+, Cl-, Ca+2, and K+) within the leaf tissue using ion chromatography. Species that are drought and salinity tolerant are often able to adjust the osmolarity of their cellular fluids by accumulating physiologically compatible solutes, such as proline and glycine betaine in grasses. We will therefore measure the concentration of these solutes in expressed leaf tissue sap using high performance liquid chromatography. RESEARCH PROJECT 3 Effects
of DNA-Reactive a-Hydroxytamoxifen on Endometrial Cell Lines The antiestrogen tamoxifen is widely used for the treatment of breast cancer; however, its use increases the risk of endometrial cancer. Tamoxifen could exert its agonist activity through binding to the estrogen receptor (ER) or, once metabolized, through the formation of covalent DNA adducts. The objective of this research is to determine if cellular proliferation is induced by a DNA-reactive metabolite, a-hydroxy-tamoxifen (a-OH TAM), and if it is mediated through its ability to bind to the ER resulting in an activated transcription factor and/or by forming DNA adducts. We plan to screen a panel of ER-responsive genes in cultured cells beginning with insulin growth factor 1, growth hormone, Estrogen receptor (ER) and progesterone receptor because of their demonstrated up-regulation in response to estrogen agonists. [1]To determine if a-OH TAM has agonistic activity, we will measure endometrial cell proliferation, ERa and ERb protein levels using a Western blot analysis, and mRNA levels of the estrogen regulated genes using Northern blot analysis. To address how a-OH TAM regulates the expression of estrogen responsive genes, we will characterize the important determinants required for transcriptional activation using an in vitro system from a nuclear extract depleted of ER. These studies will reveal if transcriptional activity requires the ER, and if it does, if DNA binding by the ligand-ER complex is necessary. RESEARCH PROJECT 4 Induction of SOS Mutagenesis under Starvation Conditions Martín Gonzalez (Biology) & Gulnar Rawji (Chemistry) Merck scholar 2004: Bhavik Kumar The E. coli SOS response to DNA damage is controlled by two proteins, LexA and RecA. Following exposure to DNA damaging agents, the replicative polymerase DNA pol III stalls at DNA lesions creating regions of single stranded DNA. This stimulates the RecA protein to undergo a change from its normal physiological state to an activated one referred to as RecA*. Preliminary studies suggest that RecA* is similarly stimulated by induction of inorganic polyphosphate (polyP) synthesis. PolyP has been shown to be involved in processes ranging from gene expression to protein degradation. This project focuses on the role of polyP in stimulating the activation of RecA to RecA*. It stems from the broader research interests of Gonzalez on the regulation of SOS mutagenesis and Rawji on the investigation of phosphate esters using NMR spectroscopy. We postulate that polyP mimics the negatively charged phosphate backbone of single-stranded DNA in attracting RecA. In this study, the genes required for polyP production will be deleted so that polyP levels can be controlled. Some questions we hope to answer in this investigation are: How and where on RecA does polyP bind? Does polyP chain length differentially affect activation? Once bound, what sequence of events leads to activation of RecA? We propose a dual track, complementary investigation of how polyP activates RecA. The in vivo studies will investigate DNA polymerase V activity in polyP deficient strains. Moreover, NMR spectroscopy (1H and 31P) will be used to probe the RecA structure in its unbound state in order to follow the conformational changes resulting from binding with polyP. [1] Denis Stygar, Natalia Muravitskaya, Britt Eriksson, Hĺkan Eriksson and Lena Sahlin, Reproductive Biology and Endocrinology 1, 2-8 (2003)
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