Presence of the anti-leukemic nucleotide analog, 2-chloro-2'-deoxyadenosine-5'-monophosphate, in a promoter sequence alters DNA binding of TATA-binding protein (TBP).
2-Chlorodeoxyadenosine (CldAdo, Cladribine), a nucleoside analog used in the treatment of hairy cell leukemia, is phosphorylated and incorporated into DNA, but is not an absolute chain terminator. We hypothesized that the presence of a chlorine molecule projecting into the DNA minor groove would affect DNA:protein-binding interactions. Here, we investigated recognition of and binding to double-stranded CldAMP-substituted TATA promoter sequences by human TATA-binding protein (TBP) using mobility shift assays. Depending on the site, CldAMP in place of dAMP within a TATA sequence decreased in vitro TBP binding by approximately 30% to 55% compared to control sites. When bound to a CldAMP-substituted TATA box, however, the TBP complex was more resistant to polyanions, suggesting enhanced stability. Limited exposure of the TBP:DNA complex to proteases indicated that TBP conformation was altered on CldAMP-substituted DNA compared to control. Further, binding of transcription factor IIB to TBP was diminished on analog-containing TATA sequences. These results suggest normal TBP-binding interactions--specifically recognition, stability, and conformation-are disrupted by CldAMP insertion into eukaryotic promoter sequences.
Hartman
William R
WR
Department of Pharmacology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA.
Walters
D Eric
DE
Hentosh
Patricia
P
D001483:Base Sequence; D001665:Binding Sites; D017338:Cladribine / Q000378:metabolism*; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D035181:TATA-Box Binding Protein / Q000235:genetics / Q000378:metabolism*
D035181:TATA-Box Binding Protein; D017338:Cladribine; D004247:DNA