Incorporation of nucleoside triphosphates opposite stereoisomeric R and S N3-(2-hydroxy-3-buten-2-yl)-2'-deoxyuridine adducts by the Sulfolobus solfataricus DNA polymerase dpo4

TOXI 69

Wen Xu, wen.xu@vanderbilt.edu, Department of Chemistry, Vanderbilt University, Nashville, TN 37235, Richard P. Hodge, Sealy Center for Environmental Health and Medicine, Department of Pharmacology and Toxicology, The University of Texas Medical Branch at Galveston, Galveston, TX 77555, R. Stephen Lloyd,  rslloyd@utmb.edu, Sealy Center for Moelecular Sciences, UTMB-Galveston, Galveston, TX 775655, Martin Egli, martin.egli@vanderbilt.edu, Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, TN 37232-0146, and Michael P. Stone, michael.p.stone@vanderbilt.edu, Department of Chemistry, Center in Molecular Toxicology and the Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, TN 37235.
The R and S N3-(2-hydroxy-3-buten-2-yl)-2'-deoxyuridine DNA adducts are derived from the reaction of butadiene monoepoxide with deoxycytosine, followed by deamination. Ternary complexes of the Sulfolobus solfataricus DNA Polymerase Dpo4, a template-primer site-specifically modified with either the R or S N3-(2-hydroxy-3-buten-2-yl)-2'-dU adduct, and incoming d(d)ATP, d(d)CTP, or d(d)GTP, were examined using crystallography. Surprisingly, for each of the complexes, the 5' overhang of the template formed a hairpin such that the 5'-terminal template T was paired with the butadiene-modified template dU. This suggested a preference for Dpo4 to insert TTP opposite either the R or S N3-(2-hydroxy-3-buten-2-yl)-2'-dU adducts, consistent with the observation of C to A transversions in previous site-specific mutagenesis studies. In each of the ternary complexes, the incoming d(d)NTP was inserted into the position above the thymine. Supported by NIH grants ES-05355 (R.S.L.) and ES-05509 (M.P.S. and M.E.).

 

Poster Session and Awards
6:00 PM-10:00 PM, Tuesday, August 21, 2007 BCEC -- 204 A/B, Poster

Division of Chemical Toxicology

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007