High stability binding of poly(ADPribose) polymerase-like thermozyme from S. solfataricus with circular DNA.
The poly(ADPribose) polymerase-like thermozyme from the hyperthermophilic archaeon S. solfataricus was found to bind DNA with high affinity and non-specifically. Binding was independent of base composition and length of the nucleic acid, and the protein showed a slight preference for the circular structure. By using pCMV-Neo-Bam plasmid as experimental model, the behaviour of the thermozyme upon binding with either circular or linear plasmid was analyzed. pCMV-Neo-Bam has a single HindIII site that allows to obtain the linear structure after digestion with the restriction enzyme. Intrinsic tryptophan-dependent fluorescence of poly(ADPribose) polymerase-like thermozyme noticeably changed upon addition of either circular or linear plasmid, showing the same binding affinity (K=2 x 10(9) M-1). However, experiments of protection against temperature and DNase I gave evidence that the thermozyme formed more stable complexes with the circular structure than with the linear pCMV-Neo-Bam. Increasing temperature at various DNA/protein ratios had a double effect to reduce the amount of circular DNA undergoing denaturation and to split the melting point towards higher temperatures. Nil or irrelevant effect was observed with the linear form. Similarly, DNase acted preferentially on the linear plasmid/protein complexes, producing an extensive digestion even at high protein/DNA ratios, whereas the circular plasmid was protected by the thermozyme in a dose-dependent manner. The complexes formed by archaeal poly(ADPribose) polymerase (PARPss) with the circular plasmid were visualized by bandshift experiments both with ethidium bromide staining and by labelling the circular plasmid with 32P. The stability of complexes was tested as a function of enzyme concentration and in the presence of a cold competitor and of 0.1% SDS. From the performed experiments, a number of 3-10 base pairs bound per molecule of enzyme was calculated, indicating a high frequency of binding. The presence of circular DNA was also able to increase by 80% the poly(ADPribose)polymerase-like activity, as compared to 25% activation induced by the linear pCMV-Neo-Bam.
Faraone-Mennella
Maria Rosaria
MR
Dipartimento di Chimica Biologica, Facolta' di Scienze, Universita' "Federico II", Via Mezzocannone, 16, 80134 Napoli, Italy. faraone@unina.it
De Luca
Paola
P
Giordano
Anna
A
Gambacorta
Agata
A
Nicolaus
Barbara
B
Farina
Benedetta
B
D001665:Binding Sites; D004270:DNA, Circular / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006358:Hot Temperature; D046911:Macromolecular Substances; D010957:Plasmids / Q000235:genetics; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D016944:Sulfolobus / Q000201:enzymology*
D004270:DNA, Circular; D046911:Macromolecular Substances; D011065:Poly(ADP-ribose) Polymerases; D003850:Deoxyribonuclease I