A single-strand specific endonuclease activity copurifies with overexpressed T5 D15 exonuclease

• Published in: Nucleic acids research Vol. 19; no. 15; pp. 4127 - 4132
• Main Authors: SAYERS, J. R, ECKSTEIN, F
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 11-08-1991
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Base Sequence; Biological and medical sciences; DNA - metabolism; DNA, Circular - metabolism; DNA, Single-Stranded - metabolism; DNA-Directed DNA Polymerase - metabolism; Endonucleases - antagonists & inhibitors; Endonucleases - isolation & purification; Endonucleases - metabolism; Enzymes and enzyme inhibitors; Escherichia coli; Escherichia coli - metabolism; Exodeoxyribonucleases - isolation & purification; Exodeoxyribonucleases - metabolism; Fundamental and applied biological sciences. Psychology; Hydrolases; Magnesium - metabolism; Molecular Sequence Data; T-Phages - enzymology; Transfection - genetics; Viral Plaque Assay; Virus; Exonuclease; Enzymatic activity; Enzyme; Siphoviridae; Homology; Single stranded DNA; Phage T5; Endonuclease; Aminoacid sequence; Phage
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/19.15.4127

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.