Studies on deoxyribonucleases from Haemophilus influenzae on DNA agarose affinity chromatography. Two-step purification of ATP-dependent deoxyribonuclease.
In a first part of this report, purification and characterization of several nucleased from lysates of Haemophilus influenzae are described. The enzymes bind to DNA with agarose columns and are removed by elution with phosphate buffer. Among the considered enzymes, the exonucleases 1 and 3, and endonuclease, a DNA polymerase and a restriction enzyme were recovered mixed by raising the phosphate concentration from 0.1 to 0.3 M, while the ATP-dependent DNAase recovered well purified, by raising the phosphate concentration to 0.45 M. After a rechromatography, on a second DNA with agarose column, of the peak of the ATP-dependent DNAase, the specific activity tested with 3H-labeled DNA was 125 units/mg of protein, representing a 300-fold purification of the original crude extract. In a second part, we have investigated the inactivation, at various pH, of transforming DNA of Haemophilus influenzae wild strain Rd with the different eluted fractions of the column, in order to determine the importance of contamination with other enzymatic activities, and also in order to confirm the nature of theisolated enzymes with a biological method. Finally, with enzymatic extracts of mutant strain Rd com minus 56, a strain which integrates shorter than normal pieces of DNA and which is suspected to possess and "activated specific endonuclease" able to recognize even small conformational modifications in paired structures, we tried to detect this activity on artificially constructed heteroduplex regions in DNA.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002021:Buffers; D002413:Cations, Divalent / Q000494:pharmacology; D002846:Chromatography, Affinity; D004254:DNA Nucleotidyltransferases / Q000032:analysis; D004269:DNA, Bacterial / Q000494:pharmacology; D004277:DNA, Single-Stranded; D003851:Deoxyribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D004720:Endonucleases / Q000032:analysis; D005092:Exonucleases / Q000032:analysis; D006193:Haemophilus influenzae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D009154:Mutation / Q000187:drug effects; D012685:Sepharose; D014170:Transformation, Genetic / Q000187:drug effects
D002021:Buffers; D002413:Cations, Divalent; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D000255:Adenosine Triphosphate; D012685:Sepharose; D004254:DNA Nucleotidyltransferases; D003851:Deoxyribonucleases; D004720:Endonucleases; D005092:Exonucleases