| Abstract
| - Recently, it was reported that TATA-binding protein (TBP) enhances (6-4) photoproductformation in a TATA box under UVC irradiation [Aboussekhra and Thoma (1999) EMBO J. 18, 433−443]. The conclusions of that study were based on an indirect enzymatic assay that was not specific for(6-4) photoproducts. Herein we report the use of a recently developed coupled enzymatic digestion/massspectrometry assay [Wang et al. (1999) Chem. Res. Toxicol.12, 1077−1082] to identify unambiguouslyand quantify the photoproducts formed in a TATA box-containing dodecamer duplex sequence in thepresence or absence of TBP binding. Exposure of the adenovirus major late promoter TATA box to ahigh dose of UVC irradiation in the absence of the C-terminal domain of yeast TBP leads to predominantformation of the cis-syn dimer within the T4 tract, whereas exposure in the presence of TBP leads toalmost exclusive formation of the (6-4) photoproduct. In contrast, the (6-4) product is not detected athigh doses of UVB irradiation in the absence of TBP but is detected in the presence of TBP, although thecis-syn product predominates. When the products of UVB irradiation were subsequently exposed to ahigh dose of UVC irradiation in the presence of TBP, the (6-4) photoproduct again becomes nearly theexclusive photoproduct, indicating that the cis-syn dimer is being reversed to TT by UVC light. Bothcis-syn and (6-4) photoproducts are formed in approximately equal amounts upon irradiation with smalldoses of UVC in the presence of TBP, but the fraction of (6-4) photoproduct increases with dose. Throughthe use of a TATA box containing a site-specifically deuterated thymine, it was found that (6-4)photoproducts formed most selectively at the second and third positions of the T4 tract upon either UVBor UVC irradiation in the presence of TBP. By using the same substrate, it was found that UVC-inducedTA* formation was inhibited by TBP binding and that TA* formation was greatest at the 5‘ end of theTATA sequence.
|
