Mechanism of action of deoxyribonuclease II from human lymphoblasts.
Deoxyribonuclease II has been purified through five fractionation steps from the human lymphoblast cell line K562. Isolation included DEAE-cellulose and heparin-agarose chromatography followed by fractionation on Mono-S, Mono-Q and Superose-12 FPLC columns. In an extension of previous studies, deoxyribonuclease II was found to introduce a much higher proportion of single-strand nicks relative to double-strand breaks into supercoiled DNA than has been reported for linear DNA. Application of DNA sequencing techniques has further revealed a unique resistance of 3' termini to hydrolysis by this enzyme. Deoxyribonuclease II cleaves at every available site along the duplexed portion of a paired oligonucleotide substrate with the exception of the last four nucleotides. Consistent with previous results, this deoxyribonuclease II is active at low pH in the absence of Mg2+ and is not inhibited by EDTA, but complete inhibition is observed with 100 microM Fe3+. Likewise we confirmed the presence of 3'-phosphoryl termini on the DNA cleavage products since they failed to function as primers for DNA synthesis catalyzed by Escherichia coli DNA polymerase I.
Harosh
I
I
Department of Genetics, University of California, Davis.
Binninger
D M
DM
Harris
P V
PV
Mezzina
M
M
Boyd
J B
JB
D001483:Base Sequence; D002460:Cell Line; D002853:Chromatography, Liquid; D004247:DNA / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008214:Lymphocytes / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity
D004278:DNA, Superhelical; D004247:DNA; D004706:Endodeoxyribonucleases; C019329:deoxyribonuclease II