TATA binding protein discriminates between different lesions on DNA, resulting in a transcription decrease

Abstract

DNA damage recognition by basal transcription factors follows different mechanisms. Using transcription-competition, nitrocellulose filter binding, and DNase I footprinting assays, we show that, although the general transcription factor TFIIH is able to target any kind of lesion which can be repaired by the nucleotide excision repair pathway, TATA binding protein (TBP)-TFIID is more selective in damage recognition. Only genotoxic agents which are able to induce kinked DNA structures similar to the one for the TATA box in its TBP complex are recognized. Indeed, DNase I footprinting patterns reveal that TBP protects equally 4 nucleotides upstream and 6 nucleotides downstream from the A-T (at position -29 of the noncoding strand) of the adenovirus major late promoter and from the G-G of a cisplatin-induced 1,2-d(GpG) cross-link. Together, our results may partially explain differences in transcription inhibition rates following DNA damage.

FIG. 1

Chemical structure of mono- and bifunctional drugs reacting with DNA.

FIG. 2

Inhibition of in vitro transcription from AdMLP by the presence of damaged DNA. (A) Transcription of AdMLP (50 ng) was performed with 30 μg of HeLa WCE or with RTS fractions in the presence of increasing amounts (25, 50, and 100 ng) of plasmid pHM untreated (NT) (lane 2) or treated with CDDP (lanes 3 to 5), TDDP (lanes 6 to 8), Dach (lanes 9 to 11), or Dien (lanes 12 to 14) (100 lesions/plasmid). Lane 1, absence of DNA competitor. (B) (Left panel) Densitometric quantification of the above autoradiogram (309-nt band) as well as additional experiments (at least three, when standard deviation is indicated), presented as the percentage of transcription from AdMLP as a function of DNA competitor. Lanes correspond to those in panel A. (Right panel) Quantification of experiments performed as described for panel A, with DNA competitors previously treated with AAF (50 and 100 ng), MMS (25 and 50 ng), or EtAz (100 and 200 ng) (lanes 17 to 22, respectively). Transcription in the absence of competitor DNA equals 100%. (C) Transcription of AdMLP was performed with WCE in the presence of 50 ng of different damaged DNA competitors containing 100 (lanes 3, 5, 7, and 9) or 200 (lanes 4, 6, 8, and 10) lesions per molecule as indicated at the top of the figure. Lane 1 represents transcription without DNA competitor; lane 2 (+) contains 50 ng of undamaged pHM plasmid. (D) Recovery of transcription inhibited in an RTS by CDDP (lanes 1 to 8)-, Dach (lanes 9 to 12)-, and AAF (lanes 13 to 16)-damaged plasmid (containing 100 lesions/plasmid for CDDP and Dach and 15 to 20 lesions/plasmid for AAF; the amount of each competitor is indicated in the figure in nanograms). TBP and TFIIB added to restore transcription were as described in reference .

FIG. 3

Analysis of damaged-DNA-associated proteins. Aliquots of HeLa WCE were incubated with damaged pHM plasmids adsorbed in microwells under conditions described in Materials and Methods. After a 2-h incubation, the protein fraction bound to DNA was recovered and analyzed by Western blotting. (A) One hundred nanograms of pHM was either NT or treated with CDDP, TDDP, Dach, or Dien to an rb value of ∼100 Pt atoms/plasmid (lanes 2 to 5 and 9); EtAz (2.4 μM, leading to ∼30 adducts/plasmid [lane 10]); or MMS (15 mM, leading to ∼90 methylations/plasmid [lane 11]). Lane 6, as a positive control, 200 ng of a TATA box-containing plasmid (pUC309, 3.4 kb) was adsorbed in the well. Lane 7, 50 μg of WCE was analyzed in parallel. The presence of the p62 subunit of TFIIH, TBP, and TFIIEα was analyzed with 3C9, 3G3, and 2A1 monoclonal antibodies, respectively. (B) pHM was either NT; was platinated with CDDP, TDDP, Dach, or Dien to rb values of ∼50, ∼100, and ∼200 Pt atoms/plasmid; or was methylated with MMS (5, 10, and 15 mM, leading to ∼30, 60, and 90 methylations/plasmid, respectively).

FIG. 4

Preferential binding of TBP to damaged DNA. CDDP-, TDDP-, Dach-, or Dien-treated (100 lesions/plasmid); EtAz-treated (30 lesions/plasmid); or AAF-treated (15 to 20 lesions/plasmid) DNA or untreated (NT) DNA was labelled with [α-³²P]dATP. NT or damaged DNA was then incubated with increasing amounts of TBP and tested for retention on nitrocellulose filters. Graphs represent the percentages of DNA retained on the filters as a function of the amount of TBP. One hundred percent represents the total counts obtained when 1 μl of each DNA probe (input) was spotted onto Whatman paper and simultaneously exposed with the nitrocellulose filter.

FIG. 5

Footprinting of TBP onto DNA containing either a TATA box or CDDP damage. (A) TBP footprint on the TATA box could be competed by adding increasing amounts (10, 20, and 30 ng) of either undamaged (NT) or CDDP-, TDDP-, Dach-, or Dien-damaged DNA (containing 100 lesions per molecule) or TATA box-containing fragment (AdMLP). Lanes 1 and 22, naked DNA; lanes 2 and 21, no DNA competitor. (B) Footprint of TBP onto a 1,2-d(GpG) cisplatin lesion (T) compared with NT DNA. (C and D) Footprint of TBP onto a 1,2-d(GpG) cisplatin lesion (T) compared with a TATA box-containing fragment in the presence or absence of TFIIB as indicated. The TATA box is boxed and the 1,2-d(GpG) cisplatin lesion position is shaded on the left of the figure, and TBP footprint protection is determined upon sequencing.