Studies on deoxyribonucleases from Haemophilus influenzae on DNA · agarose affinity chromatography

Abstract

In a first part of this report, purification and characterization of several nucleases from lysates of Haemophilus influenzae are described. The enzymes bind to DNA · agarose columns and are removed by elution with phosphate buffer. Among the considered enzymes, the exonucleases 1 and 3, an 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 · 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 the isolated enzymes with a biological method.

Finally, with enzymatic extracts of mutant strain Rd com⁻ 56, a strain which integrates shorter than normal pieces of DNA and which is suspected to possess an “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.