Studies on T4-head maturation. 1. Purification and characterization of gene-49-controlled endonuclease.
An endonuclease has been purified more than 300-fold from Escherichia coli infected with bacteriophage T4. The enzyme degrades rapidly sedimenting (greater than 1000 S) DNA in vitro by introducing a limited number of breaks. The substrate is the replicative DNA isolated from cells infected with gene-49-defective phage [Kemper, B, and Janz, E. (1976) J. Virol. 18, 992-999]. Molecules of approximately a third the size of unit-length T4 DNA are exclusively found in a limit digest. The enzyme also reacts with single-stranded DNA from various sources. Heat-denatured T4 DNA is converted into acid-soluble oligonucleotides. Circular single-stranded M13 DNA is linearized by endonucleolytic cleavage causing a reduction of infectivity during transfection. The enzyme behaves like a typical late-gene product. Its activity is 100-fold reduced in cells infected with gene-55-defective phage (defect in expression of late functions). A 30-fold reduction in its specific activity was found in cells infected with gene-49-defective phage suggesting that gene 49 codes for the enzyme or controls its expression. The purified enzyme binds to native or denatured DNA from various sources. The protein has a molecular weight of 42000 as determined by gel filtration and sedimentation analysis. Optimal activity on rapidly sedimenting DNA is obtained at pH 8.6 in Tris/HCl buffer in the presence of 10 mM MgCl2. Some 75% of the activity can be obtained with 7 mM MnCl2. 5 mM CaCl2 has a stimulatory effect on the reaction with MgCl2 or MnCl2 each present at its individual optimal concentration. The enzyme does not require the addition of sulfhydryl reagent for full activity. The reaction can be inhibited by compounds like KCl, spermidine, p-hydroxymercuribenzoate or tRNA.
D055598:Chemical Phenomena; D002621:Chemistry; D004279:DNA, Viral / Q000302:isolation & purification*; D004720:Endonucleases / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008970:Molecular Weight; D009691:Nucleic Acid Denaturation; D013604:T-Phages / Q000235:genetics*; D013696:Temperature
D004279:DNA, Viral; D004720:Endonucleases