Purification and characterization of normal and mutant forms of T4 endonuclease V.
Endonuclease V of bacteriophage T4 has been purified to physical homogeneity from T4D-infected Escherichia coli 1100. The enzyme, whose molecular weight is 16,000, possessed two distinct catalytic activities, a pyrimidine dimer-DNA glycosylase and an apurinic/apyrimidinic endonuclease. They acted on UV-irradiated poly(dA) . poly(dT) in a sequential manner; the glycosylase cleaved the N-glycosyl bond between the 5'-pyrimidine of a dimer and the corresponding sugar and then the endonuclease hydrolyzed a phosphodiester bond on the 3'-side of the apyrimidinic site. The 5'-termini thus generated were phosphorylated by T4 polynucleotide kinase only after they had been subjected to direct photoreversal and then treated with alkaline phosphatase. By using two phage mutants, uvs-5 and uvs-13, it was shown that occurrence of an amber mutation in the denV gene caused a simultaneous loss of the two activities. Suppression of the mutation of uvs-5 rendered both activities partially active. When the mutation of uvs-13 was suppressed, a mutant form of enzyme that possessed only a glycosylase activity was produced. This suggests that there are two distinct domains in a single enzyme, each of which corresponds to one of the activities.
D043224:Deoxyribonuclease (Pyrimidine Dimer); D003851:Deoxyribonucleases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004706:Endodeoxyribonucleases; D004720:Endonucleases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008970:Molecular Weight; D009154:Mutation; D009994:Osmolar Concentration; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D014466:Ultraviolet Rays; D014764:Viral Proteins
D014764:Viral Proteins; D003851:Deoxyribonucleases; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer)