Effect of spermine on interaction of DNA polymerase α from the loach ( Misgurnus fossilis) eggs with DNA

Abstract

Polyamines (putrescine, spermidine and spermine) cause a marked increase in the activity of the loach Misgurnus fossilis DNA polymerase α 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 K m of the polymerase for activated DNA progressively increases from 14 to 1200 μM (nucleotide), if the concentration of spermine rises up to 2 mM, while V max 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 α-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 α 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-β- 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 α-polymerase on activated DNA.