Degradation of cytosine-containing bacterial and bacteriophage DNA after infection of Escherichia coli B with bacteriophage T4D wild type and with mutants defective in genes 46, 47 and 56

Abstract

The time-course of degradation of bacterial DNA after infection of Escherichia coli B with bacteriophage T4 has been followed by means of neutral and alkaline sucrose density-gradient centrifugation. This degradation is found to occur in two stages. The first stage involves limited endonucleolytic cleavage of the DNA molecule, causing a gradual decrease in average molecular weight to a minimum of about 10 6. There are a few single-strand breaks per double-strand fragment early in this stage but not later, indicating that both strands are broken at or near the same time. This first stage of the degradation still proceeds normally when genes 46 and/or 47 are defective. A second nuclease system is necessary to degrade these large DNA fragments to acid-soluble form. This second stage of the degradation proceeds rapidly once a fragment is attacked; little or no acid-insoluble material below a molecular weight of about 10 6 is found. Mutations in either gene 46 or 47 completely block this stage, leaving all of the bacterial DNA as fragments with a molecular weight of about 10 6. The implications of these findings are discussed with respect to known nucleases, the phage-directed inhibition of host nucleic acid synthesis, and genetic recombination. Evidence is presented that the degradation of newly synthesized T4 DNA which occurs in infected cells lacking the phage-directed dCTPase occurs in two similar stages; in such cells cytosine is substituted extensively for the hydroxymethylcytosine normally found in T4 phage DNA. We have not determined whether the first stage of the degradation utilizes the same enzymes or factors on both host and cytosine-containing phage DNA, nor whether these enzymes are primarily of bacterial origin or represent new, phage-direeted functions. However, our evidence does establish that T4 genes 46 and 47 are essential to the second stage of degradation of both types of DNA. Although the cytosine-containing DNA produced by a triple mutant defective in genes 56 (dCTPase), 46 and 47 is not rendered acid-soluble by the cell, it is fragmented to a molecular weight of about 10 7; this is a sufficient explanation, though not the only one, for the failure of this mutant to produce phage particles. Possible explanations for the apparent cytosine specificity in the degradation of DNA are discussed; an attractive possibility is that the first enzyme to act on both host and phage DNA may be an endonuclease that attacks only at cytosinerich clusters.