title: Interaction of the ADP-ribosylating enzyme from the hyperthermophilic archaeon S. solfataricus with DNA and ss-oligo deoxy ribonucleotides.
abstract: The DNA-binding ability of the poly-ADPribose polymerase-like enzyme from the extremely thermophilic archaeon Sulfolobus solfataricus was determined in the presence of genomic DNA or single stranded oligodeoxyribonucleotides. The thermozyme protected homologous DNA against thermal denaturation by lowering the amount of melted DNA and increasing melting temperature. The archaeal protein induced structural changes of the nucleic acid by modifying the dichroic spectra towards a shape typical of condensing DNA. However, enzyme activity was slightly increased by DNA. Competition assays demonstrated that the protein interacted also with heterologous DNA. In order to characterize further the DNA binding properties of the archaeal enzyme, various ss-oligodeoxyribonucleotides of different base composition, lengths (12-mer to 24-mer) and structure (linear and circular) were used for fluorescence titration measurements. Intrinsic fluorescence of the archaeal protein due to tryptophan (excitation at 295 nm) was measured in the presence of each oligomer at 60 degrees C. Changes of tryptophan fluorescence were induced by all compounds in the same range of base number per enzyme molecule, but independently from the structural features of oligonucleotides, although the protein exhibited a slight preference for those adenine-rich and circular. The binding affinities were comparable for all oligomers, with intrinsic association constants of the same order of magnitude (K=10(6) M(-1)) in 0.01 M Na-phosphate buffer, pH 8.0, and accounted for a "non-specific" binding protein. Circular dichroism analysis showed that at 60 degrees C the native protein was better organized in a secondary structure than at 20 degrees C. Upon addition of oligonucleotides, enzyme structure was further stabilized and changed towards a beta-conformation. This effect was more marked with the circular oligomer. The analysed oligodeoxyribonucleotides slightly enhanced enzyme activity with the maximal increase of 50% as compared to the control. No activation was observed with the circular oligomer.
authors.affiliation: Dipartimento di Chimica Biologica, Facolta' di Scienze, Universita' "Federico II," via Mezzocannone, Napoli, Italy. faraone@unina.it
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: Faraone-Mennella
authors.forename: Gennaro
authors.initials: G
authors.lastname: Piccialli
authors.forename: Paola
authors.initials: P
authors.lastname: De Luca
authors.forename: Sabrina
authors.initials: S
authors.lastname: Castellano
authors.forename: Anna
authors.initials: A
authors.lastname: Giordano
authors.forename: Daniela
authors.initials: D
authors.lastname: Rigano
authors.forename: Lorenzo
authors.initials: L
authors.lastname: De Napoli
authors.forename: Benedetta
authors.initials: B
authors.lastname: Farina
mesh_terms: D019843:Archaeal Proteins / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D019641:DNA, Archaeal / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D016944:Sulfolobus / Q000201:enzymology* / Q000378:metabolism; D017186:Titrimetry
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004277:DNA, Single-Stranded; D011065:Poly(ADP-ribose) Polymerases