Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli.
The gag-pol precursor protein of the avian sarcoma-leukosis virus is processed into three known pol-encoded mature polypeptides; the 95- and 63-kilodalton (kDa) beta and alpha subunits, respectively, of reverse transcriptase and the 32-kDa pp32 protein. The pp32 protein possesses DNA endonuclease activity and is produced from the precursor by two proteolytic cleavage events, one of which removes 4.1 kDa of protein from the C terminus. A 36-kDa protein (p36pol) which retains this C-terminal segment is detectable in small quantities in virions. We have constructed Escherichia coli plasmid clones that express the C-terminal domains of pol corresponding to pp32 and p36. These proteins have been purified by column chromatographic methods to near homogeneity. No significant differences could be detected in the enzymatic properties of the bacterially produced p32pol and p36pol proteins. Both possess DNA endonuclease activity and, like the pp32 protein isolated from virions, can cleave near the junction of two tandem avian sarcoma-leukosis virus long terminal repeats in double-stranded supercoiled DNA substrates. In the presence of Mg2+, both p32pol and viral pp32 cleave either strand of DNA 2 nucleotides 5' to the junction.
Terry
R
R
Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey 07110.
Soltis
D A
DA
Katzman
M
M
Cobrinik
D
D
Leis
J
J
Skalka
A M
AM
D000595:Amino Acid Sequence; D001358:Avian Sarcoma Viruses / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D004278:DNA, Superhelical / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D012191:Retroviridae Proteins / Q000235:genetics* / Q000378:metabolism
D004278:DNA, Superhelical; D004279:DNA, Viral; D011994:Recombinant Proteins; D012191:Retroviridae Proteins; C040520:avian retrovirus proteins; D004259:DNA-Directed DNA Polymerase; D004706:Endodeoxyribonucleases