Anomalous DNA binding by E2 regulatory protein driven by spacer sequence TATA.
We have investigated the anomalously weak binding of human papillomavirus (HPV) regulatory protein E2 to a DNA target containing the spacer sequence TATA. Experiments in magnesium (Mg(2+)) and calcium (Ca(2+)) ion buffers revealed a marked reduction in cutting by DNase I at the CpG sequence in the protein-binding site 3' to the TATA spacer sequence, Studies of the cation dependence of DNA-E2 affinities showed that upon E2 binding the TATA sequence releases approximately twice as many Mg(2+) ions as the average of the other spacer sequences. Binding experiments for TATA spacer relative to ATAT showed that in potassium ion (K(+)) the E2 affinity of the two sequences is nearly equal, but the relative dissociation constant (K(d)) for TATA increases in the order K(+ )< Na(+ )< Ca(2+ )< Mg(2+). Except for Mg(2+), K(d) for TATA relative to ATAT is independent of ion concentration, whereas for Mg(2+) the affinity for TATA drops sharply as ion concentration increases. Thus, ions of increasing positive charge density increasingly distort the E2 binding site, weakening the affinity for protein. In the case of Mg(2+), additional ions are bound to TATA that require displacement for protein binding. We suggest that the TATA sequence may bias the DNA structure towards a conformation that binds the protein relatively weakly.
Xi
Zhiqun
Z
Department of Chemistry, Yale University, New Haven, CT 06511, USA.
Zhang
Yongli
Y
Hegde
Rashmi S
RS
Shakked
Zippora
Z
Crothers
Donald M
DM
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D009690:Nucleic Acid Conformation; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D011485:Protein Binding
D004268:DNA-Binding Proteins; C054855:E2 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D004247:DNA; D003850:Deoxyribonuclease I; D008274:Magnesium