Protein binding to DNA containing UV photoproducts and its effect on DNA repair

Binding of transcription factors to their cognitive sequences containing cyclobutane pyrimidine dimers (CPDs) was studied using transcription factor IIIA (TFIIIA) and high mobility group A1 (HMGA1) as model systems. Homogeneously damaged oligonucleotides containing a single cis-syn cyclobutane thymine dimer (CTD) in the internal control region (ICR) of the 5S rRNA gene (5S rDNA) were prepared, and the binding to TFIIIA was assayed by measurement of dissociation constants (K_d), dissociation rates (k _off) and protein-DNA contacts. We found that a single CTD in the ICR does not significantly affect the K_d of TFIIIA. In contrast, CTDs at positions +55 and +72 (from the transcription start site) in the ICR enhance k_off of TFIIIA from the complex, and reduce the protein-DNA contacts of zinc finger(s) and 5S rDNA. Furthermore, CTDs at +55 and +72 are more efficiently removed from the complex than CTDs at other sites in the ICR by Xenopus oocyte nuclear extracts. This suggests that the fast repair of CTDs may be due to changes in the binding of individual zinc fingers of the TFIIIA-ICR complex. Alternatively, the close correlation of dissociation and repair profiles indicates that release of DNA molecules from the TFIIIA-5S rDNA complex may be an important factor in repair of CTDs within the ICR. In addition, a CPD-interference assay demonstrates that CPDs within the ICR enhance dissociation of the TFIIIA-5S rDNA complex and also decrease DNA-binding affinity of TFIIIA.; The effect of UV photoproducts on HMGA1 binding was examined using UV-irradiated DNA fragments consisting of the positive regulatory region II of the interleukin-2 receptor α-chain. In gel mobility shift analysis, there are no significant changes in HMGA1 binding due to UV photoproducts. However, UV photofootprints of HMGA1 within an 18mer poly T sequence (T-tract) show that binding of HMGA1 may induce a structural change in the T-tract. Furthermore, HMGA1 proteins markedly inhibit repair of CPDs in the HMGA1-DNA complex in vitro.