Anti-oncogene product p53 binds DNA helicase.
An oncogene product, p53, interacts with a simian virus 40-encoded T-antigen, which is an initiation protein for the viral DNA replication and also works as DNA helicase during elongation. Here we examine the interaction of p53 with cellular DNA helicase. A recombinant human wild type p53 fused with glutathione S-transferase was immobilized on glutathione-agarose as a ligand for affinity column. Hela cell extract was applied to the p53 column and the adsorbed proteins were eluted with buffers containing salt, 50% ethylene glycol, and glutathione. The ethylene glycol fraction contained a number of p53 binding proteins, and this fraction showed a DNA helicase activity measured by the displacement of DNA fragment from partially duplexed M13 DNA. The DNA helicase translocated in a 5'-to-3' direction on the single-stranded DNA using ATP as an energy source. The glutathione fraction that contained the p53 glutathione S-transferase fused protein also showed the same activity. The corresponding fractions from a control column carrying glutathione S-transferase showed only a trace amount of activity of DNA helicase. Therefore, the binding may be specific. Furthermore, an anti-p53 antibody column retained a p53-DNA helicase complex when the crude extracts of human placenta and of osteosarcoma cells were applied. These results indicate that p53 physically interacts with DNA helicase in vitro as well as in vivo.
Laboratory of Cancer Cell Biology, Nagoya University School of Medicine, Japan.
T
T
Sakurai
M
M
Suzuki
T
T
Sawazaki
S
S
Ishii
S
S
Yoshida
D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004265:DNA Helicases / Q000302:isolation & purification / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D016158:Genes, p53; D005982:Glutathione Transferase / Q000302:isolation & purification / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D016159:Tumor Suppressor Protein p53 / Q000302:isolation & purification / Q000378:metabolism*
D004277:DNA, Single-Stranded; D003854:Deoxyribonucleotides; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D005982:Glutathione Transferase; D004265:DNA Helicases