A Euglena gracilis zinc endonuclease.
A 26-kDa endonuclease has been purified to homogeneity from zinc-sufficient Euglena gracilis. The protein binds to single-stranded DNA with a higher affinity than to double-stranded DNA, but it exhibits nucleolytic activity toward both. Thus, it converts supercoiled plasmid pBR322 DNA into the linear form, a property characteristic of endonucleases, and it continues to act on the linearized DNA until it is completely degraded. It also hydrolyzes heat-denatured, single-stranded calf thymus DNA. Moreover, at amounts below 1 microgram, it enhances RNA synthesis by RNA polymerase II, a characteristic observed with other DNases. Its addition to an in vitro transcription assay increases RNA synthesis up to 3-fold. The nuclease requires two metal components to carry out its enzymatic activities. It hydrolyzes DNA only in the presence of millimolar amounts of magnesium or micromolar quantities of other activating metal ions, such as manganese, zinc, or cobalt. However, even when optimal concentrations of Mg2+ are present, micromolar amounts of the metal-chelating agents OP and HQSA completely inhibit pBR322 digestion. Transcription enhancement is also inhibited completely by both chelators at concentrations that do not affect the intrinsic polymerase II activity. By atomic absorption spectrometry, the enzyme contains 1 g-atom of Zn/mol, which is the likely target of chelator action. The nuclease protein can also be isolated from zinc-deficient E. gracilis, but remarkably it then contains 1 mol of Cu/g-atom and no zinc.(ABSTRACT TRUNCATED AT 250 WORDS)
Czupryn
M
M
Center for Biochemical and Biophysical Sciences and Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115.
Falchuk
K H
KH
Stankiewicz
A
A
Vallee
B L
BL
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002614:Chelating Agents / Q000494:pharmacology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004706:Endodeoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D005056:Euglena gracilis / Q000201:enzymology*; D007700:Kinetics; D008667:Metalloproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000032:analysis; D010618:Phenanthrolines / Q000494:pharmacology; D012319:RNA Polymerase II / Q000378:metabolism; D013054:Spectrophotometry, Atomic; D014158:Transcription, Genetic / Q000187:drug effects; D015032:Zinc / Q000032:analysis*
D000596:Amino Acids; D002614:Chelating Agents; D008667:Metalloproteins; D008670:Metals; D010618:Phenanthrolines; D012319:RNA Polymerase II; D004706:Endodeoxyribonucleases; D015032:Zinc; C025205:1,10-phenanthroline