Purification and characterization of an endonuclease specific for apurinic sites in DNA from a permanently established mouse plasmacytoma cell line.
An endonuclease specific for apurinic sites in double stranded DNA has been purified 373-fold from the nuclei of mouse plasmacytoma cells (line MPC-11). The enzyme is free of any detectable amounts of aspecific nucleases. The enzyme does not act on methylated or OsO4-treated DNA. However, high doses of UV-light and gamma-rays render the DNA slightly susceptible to endonucleolytic attack, which is believed to be due to depurination of depyrimidination caused by the treatment. The molecular weight of the enzyme is determined to be 28,000 and its apparent Km of the purified enzyme is calculated to be 2.7 nM apurinic sites. The activity is not absolutely dependent upon the presence of Mg2+ in the assay mixture although metal chelating agents such as sodium citrate and EDTA abolish the activity completely. The nuclease was stimulated by moderate concentrations of potassium chloride optimizing at 50 mM, and higher concentrations inhibiting the activity. The pH optimun for the reaction was 9.5.
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000528:radiation effects; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D003851:Deoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D004720:Endonucleases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D009374:Neoplasms, Experimental / Q000201:enzymology; D010954:Plasmacytoma / Q000201:enzymology*; D013379:Substrate Specificity; D014466:Ultraviolet Rays
D004247:DNA; D003851:Deoxyribonucleases; D004720:Endonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase