A single-strand specific endonuclease activity copurifies with overexpressed T5 D15 exonuclease.
The T5 D15 exonuclease purified from an overproducing strain of E. coli was shown to possess a low level of endonucleolytic activity specific for single-stranded DNA when assayed with 1-10 mM Mg2+ as co-factor. Endonuclease activity on double-stranded circular DNA could not be detected under these conditions. Nicked circular DNA was first gapped by the enzyme's exonucleolytic activity, creating a single-stranded region. This gapped substrate was then endonucleolytically cleaved and rapidly degraded. We show that a gapped and not a nicked substrate is required for this activity as previously suggested (Moyer, R. W. and Roth, C. T. 1977, J. Virol. 24, 177-193). The single-strand endonuclease activity could be selectively suppressed by using low concentrations of Mg2+ as co-factor (less than 1 mM), thus allowing nicked double-stranded circular DNA to be gapped to a single-stranded circular species. We also report on sequence similarities between the T5 exonuclease and several prokaryotic DNA polymerases.
Sayers
J R
JR
Max-Planck-Institut für Experimentelle Medizin, Abteilung Chemie, Göttingen, FRG.
Eckstein
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004720:Endonucleases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D013604:T-Phages / Q000201:enzymology*; D014162:Transfection / Q000235:genetics; D010948:Viral Plaque Assay
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D004720:Endonucleases; D005090:Exodeoxyribonucleases; D008274:Magnesium