DNA binding and 3'-5' exonuclease activity in the murine alternatively-spliced p53 protein.
In this study we show that the naturally occurring C-terminally alternative spliced p53 (referred to as AS-p53) is active as a sequence-specific DNA binding protein as well as a 3'-5'-exonuclease in the presence of Mg2+ ions. The two activities are positively correlated as the sequence-specific DNA target is more efficiently degraded than a non-specific target. In contrast, a mutated AS-p53 protein that is deficient in DNA binding lacks exonuclease activity. The use of modified p53 binding sites, where the 3'-phosphate is replaced by a phosphorothioate group, enabled the inhibition of DNA degradation under the binding conditions. We demonstrate that AS-p53 interacts with its specific DNA target by two distinct binding modes: a high-affinity mode characterized by a low-mobility protein-DNA complex at the nanomolar range, and a low-affinity mode shown by a high-mobility complex at the micromolar range. Comparison of the data on the natural and the modified p53 binding sites suggests that the high-affinity mode is related to AS-p53 function as a transcription factor and that the low-affinity mode is associated with its exonuclease activity. The implications of these findings to a specific cellular role of AS-p53 are discussed.
Shakked
Zippora
Z
Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel. Z.Shakked@weizmann.ac.il
Yavnilovitch
Michael
M
Kalb Gilboa
A Joseph
AJ
Kessler
Naama
N
Wolkowicz
Roland
R
Rotter
Varda
V
Haran
Tali E
TE
D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D013379:Substrate Specificity; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism*
D016159:Tumor Suppressor Protein p53; D004247:DNA; D005090:Exodeoxyribonucleases