TY - JOUR
T1 - 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)
AU - Hartman, William R.
AU - Walters, D. Eric
AU - Hentosh, Patricia
N1 - Funding Information:
This work was supported by the National Institutes of Health Grant CA55414 (to P.H.) and a Sigma Xi Grants-in-Aid of Research (to W.R.H.). We thank Dr. Dennis Peffley for his constructive reading of the manuscript, and Ortho Biotech Products, L.P. for the cladribine.
PY - 2007/3/15
Y1 - 2007/3/15
N2 - 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 ∼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.
AB - 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 ∼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.
KW - Cancer drug
KW - Cladribine
KW - DNA minor groove
KW - Hairy cell leukemia
KW - Promoter sequence
KW - Protein conformation
KW - Protein:protein associations
KW - RNA polymerase II
KW - TATA-binding protein
KW - Transcription factors
UR - http://www.scopus.com/inward/record.url?scp=33847653338&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33847653338&partnerID=8YFLogxK
U2 - 10.1016/j.abb.2006.12.031
DO - 10.1016/j.abb.2006.12.031
M3 - Article
C2 - 17320040
AN - SCOPUS:33847653338
SN - 0003-9861
VL - 459
SP - 223
EP - 232
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 2
ER -