MITOGENIC FACTOR SECRETED BY STREPTOCOCCUS-PYOGENES IS A HEAT-STABLE NUCLEASE REQUIRING HIS(122) FOR ACTIVITY

The gene encoding a mitogenic factor, termed Mr, was cloned from Strep tococcus pyogenes and the recombinant MF was overexpressed in Escheric hia coli. Both the natural and recombinant MF had heat-resistant nucle ase activity. The nuclease activity of MF was characterized using the recombinant protein. MF showed endonuclease activity, digesting ssDNA, dsDNA and tRNA. The optimal pH for the DNase activity of MF was 9.5. The DNase activity was enhanced approximately tenfold by the simultane ous presence of two divalent cations, Mg2+ and Ca2+, compared to eithe r alone and was inhibited by EDTA or NaCl. The heat stability of MF wa s biphasic; the DNase activity was heat-stable from 0 to 50 degrees C and over 80 degrees C but very unstable at around 60 degrees C. DNA di gested by MF possessed 5'-phosphorylated and 3'-hydroxylated termini, identical to those obtained by digestion of DNA by pancreatic deoxyrib onuclease I. A mutant clone revealed that His(122) was a residue essen tial to the nuclease activity.