| Summary: | A DNA-binding protein, PHI, was partially purified from extracts of Hela cells by high-speed centrifugation, and chromatography on DEAE-cellulose, phosphocellulose and UV-irradiated DNA-cellulose columns. It eluted from the phosphocellulose column with 0.375 M potassium phosphate and from the UV-irradiated DNA-cellulose column between 0.5 M and 1 M NaCl. PHI binds preferentially to supercoiled PM2 DNA treated with ultraviolet light (UV-DNA) or N-acetoxy-N-acetyl-2-aminofluorene (AAAF-DNA) as compared to native supercoiled PM2 DNA. The binding is noncooperative.
A filter-binding assay utilizing GF/C glass fibre filters was used to detect PHI during the purification steps. Characterisation of PIII-DNA complex by glycerol gradient centrifugation indicates that the retention of the complex by the filters does not involve DNA precipitation, aggregration, or a conformational change of the DNA which results in a detectable change in the sedimentation coefficient of the DNA. The binding of PHI to DNA is reversible.
PHI is a protein as indicated by its sensitivity to proteinase K. The sedimentation coefficient of the protein estimated by glycerol gradient centrifugation is 2.0-2.5 S corresponding to a molecular weight of about 20-25,000 if the protein is spherical.
The binding between UV- or AAAF-DNA and PHI is optimal at around 100-200 mM NaCl and is relatively independent of temperature and pH. MgCl₂ and MnCl₂ at concentrations between 1 mM and 7 mM do not markedly affect the binding but it is inhibited by sucrose, ATP and caffeine.
Competition experiments indicate that PHI is a single protein which binds to AAAF-induced and UV-induced DNA binding sites with equal affinity. PHI also binds preferentially to supercoiled PM2 DNA treated with N-methyl-N'-nitro-nitrosoguanidine but has little or no preferential binding activity for methyl methanesulphonate-treated or depurinated PM2 DNA. It also possesses some binding activity to unit length, single-stranded PM2 DNA. Nicked or linear forms of PM2 DNA (damaged or untreated) are not efficient substrates for PHI, indicating a requirement of DNA supercoiling for the binding activity of PHI. The possible nature of the DNA-binding sites for PHI is discussed.
The biological significance of PHI remains to be determined. It does not possess significant glycosylase, endonuclease or exonuclease activities. The binding of PHI does not alter the susceptibility of UV-irradiated supercoiled PM2 DNA to the single-stranded endonuclease of Neurospora crassa. A DNA-binding protein similar to PHI was found to be present in extracts of a normal human fibroblast cell line and two xeroderma pigmentosum fibroblast cell lines (XP-cell lines). The concentration of this protein in the extracts of these cell lines was comparable to that of PHI in Hela cells. The two XP-cell lines were XP5EG and XP2NE. They belong to the A and D complementation groups of xeroderma pigmentosum, respectively. The cell line XP5EG appeared to be deficient in another DNA-binding protein, which eluted from the phosphocellulose column with 180-250 mM potassium phosphate.
The dissociation equilibrium constant for the binding reaction of PHI to the UV- or AAAF-induced binding sites on DNA is estimated to be 7 x 10⁻¹¹ M. The association rate constant and the dissociation rate constant are 4 x 10⁶ M⁻¹sec⁻¹ and 3 x 10⁻⁴sec⁻¹, respectively. There are at least 10⁵ molecules of PHI per Hela cell. === Medicine, Faculty of === Medical Genetics, Department of === Graduate
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