Purification and characterization of an endo-exonuclease from adult flies of Drosophila melanogaster.
An endo-exonuclease (designated nuclease III) has been purified to near homogeneity from adult flies of Drosophila melanogaster. The enzyme degrades single- and double-stranded DNA and RNA. It has a sedimentation co-efficient of 3.1S and a strokes radius of 27A The native form of the purified enzyme appears to be a monomer of 33,600 dalton. It has a pH optimum of 7-8.5 and requires Mg2+ or Mn2+ but not Ca2+ or Co2+ for its activity. The enzyme activity on double-stranded DNA was inhibited 50% by 30 mM NaCl, while its activity on single-stranded DNA required 100 mM NaCl for 50% inhibition. Under the latter conditions, its activity on double-stranded DNA was inhibited approximately 98%. The enzyme degrades DNA to complete acid soluble products which are a mixture of mono- and oligonucleotides with 5'-P and 3'-OH termini. Supercoiled DNA was converted by the enzyme to nicked and subsequently to linear forms in a stepwise fashion under the condition in which the enzyme works optimally on single-stranded DNA. The amino acid composition and amino acid sequencing of tryptic peptides from purified nuclease III is also reported.
Shuai
K
K
Department of Molecular Biology and Genetics, Albert Einstein College of Medicine, Bronx, New York, NY 10461.
Das Gupta
C K
CK
Hawley
R S
RS
Chase
J W
JW
Stone
K L
KL
Williams
K R
KR
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D004277:DNA, Single-Stranded; D004331:Drosophila melanogaster / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000302:isolation & purification / Q000378:metabolism*; D005092:Exonucleases / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012965:Sodium Chloride; D013379:Substrate Specificity; D014461:Ultracentrifugation
D000596:Amino Acids; D004277:DNA, Single-Stranded; D012965:Sodium Chloride; D004720:Endonucleases; D005092:Exonucleases; C020104:endoexonuclease