Bacteriophage T4 endonucleases II and IV, oppositely affected by dCMP hydroxymethylase activity, have different roles in the degradation and in the RNA polymerase-dependent replication of T4 cytosine-containing DNA.
Bacteriophage T4 mutants defective in gene 56 (dCTPase) synthesize DNA where cytosine (Cyt) partially or completely replaces hydroxymethylcytosine (HmCyt). This Cyt-DNA is degraded in vivo by T4 endonucleases II and IV, and by the exonuclease coded or controlled by genes 46 and 47.-Our results demonstrate that T4 endonuclease II is the principal enzyme initiating degradation of T4 Cyt-DNA. The activity of endonuclease IV, but not that of endonuclease II, was stimulated in the presence of a wild-type dCMP hydroxymethylase, also when no HmCyt was incorporated into phage DNA, suggesting the possibility of direct endonuclease IV-dCMP hydroxymethylase interactions. Endonuclease II activity, on the other hand, was almost completely inhibited in the presence of very small amounts of HmCyt (3-9% of total Cyt + HmCyt) in the DNA. Possible mechanisms for this inhibition are discussed.-The E. coli RNA polymerase modified by the products of T4 genes 33 and 55 was capable of initiating DNA synthesis on a Cyt-DNA template, although it probably cannot do so on an HmCyt template. In the presence of an active endonuclease IV, Cyt-DNA synthesis was arrested 10-30 min after infection, probably due to damage to the template. Cyt-DNA synthesis dependent on the unmodified (33-55-) RNA polymerase was less sensitive to endonuclease IV action.
D003596:Cytosine; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D019877:Hydroxymethyl and Formyl Transferases; D007700:Kinetics; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*; D014166:Transferases / Q000378:metabolism*
D004279:DNA, Viral; D029968:Escherichia coli Proteins; D003596:Cytosine; D014166:Transferases; D019877:Hydroxymethyl and Formyl Transferases; C100226:deoxycytidylate hydroxymethyltransferase; D012321:DNA-Directed RNA Polymerases; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase