Effect of spermine on interaction of DNA polymerase alpha from the loach (Misgurnus fossilis) eggs with DNA.
Polyamines (putrescine, spermidine and spermine) cause a marked increase in the activity of the loach Misgurnus fossilis DNA polymerase alpha on activated (gapped) DNA. The stimulatory effect increases in the order: putrescine, spermidine, spermine. Kinetic analysis shows that spermine does not change the affinity of the polymerase for dTTP, but it decreases the enzyme affinity for DNA. The apparent Km of the polymerase for activated DNA progressively increases from 14 to 1200 microM (nucleotide), if the concentration of spermine rises up to 2 mM, while Vmax reaches a maximum at 0.5 mM spermine and then drops at higher polyamine concentrations. Native calf thymus DNA and especially single-stranded DNA from phage M13 appear to be inhibitors of alpha-polymerase activity on gapped DNA. Dixon plots suggest simple competitive inhibition of the polymerase activity by single- or double-stranded DNA and absence of cooperativity in the interaction of the polymerase with DNA. Hill-plot analysis is compatible with the interpretation that there is only one DNA binding site on each DNA polymerase alpha molecule. Spermine, even at low concentrations, decreases sharply the affinity of the enzyme for double-stranded DNA, while the enzyme affinity for single-stranded DNA changes insignificantly. Another result of spermine action is the destabilization of the polymerase-DNA complex. The ratio of the 'static affinity' of the enzyme to its 'kinetic affinity' decreases 2.2-fold in the presence of 0.5 mM spermine. As a result, the sensitivity of DNA synthesis to 3'-deoxy-3'-aminothymidine 5'-triphosphate and to 1-beta-D-arabinofuranosylcytidine 5'-triphosphate decreases in the presence of the polyamine. Both spermine effects, the decrease in the 'nonproductive binding' of the polymerase to double-stranded regions in DNA and the destabilization of the polymerase-DNA complex, presumably account for the increase in the activity of the loach alpha-polymerase on activated DNA.
D000818:Animals; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D004257:DNA Polymerase II / Q000378:metabolism*; D005260:Female; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D015636:Magnesium Chloride; D009865:Oocytes / Q000201:enzymology*; D011700:Putrescine / Q000494:pharmacology; D013095:Spermidine / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology*; D013942:Thymine Nucleotides / Q000494:pharmacology
D013942:Thymine Nucleotides; D015636:Magnesium Chloride; D013096:Spermine; C043038:3'-deoxy-3'-aminothymidine-5'triphosphate; D004247:DNA; D004257:DNA Polymerase II; D008274:Magnesium; D013095:Spermidine; D011700:Putrescine