Studies on the structure of intracellular bacteriophage T4 DNA

Abstract

We have studied DNA metabolism in cells infected with bacteriophage T4 mutants producing different temperature-sensitive proteins required for T4 DNA replication. In general, the structure of the normal, 600 to 1000 S intracellular DNA is maintained when DNA synthesis is blocked by shifting such mutant-infected cells to a non-permissive temperature. However, in cells infected with a phage producing a thermolabile gene 32 protein (T4 DNA-unwinding protein), we find that the intracellular DNA is completely converted to simple DNA pieces ranging from one-fourth to two genomes in length (39 S to 80 S) within two minutes after a shift to high temperature. This conversion appears to require the presence of an active T4 gene 49 product. We present evidence that these DNA pieces are generated from the normal intracellular DNA by cutting at single-stranded regions uncovered by thermal inactivation of gene 32 protein, and that these single-stranded regions are greatly reduced or missing in a gene 49-deficient infection. The entire conversion can be closely mimicked in vitro by incubation of the intracellular DNA in detergent-treated lysates with the single-strand specific endonuclease, S 1. Striking features are the apparent first-order kinetics with which DNA is removed by S 1 nuclease from the normal rapidly sedimenting conformation, and the absence of appreciable quantities of normally sedimenting, oligomeric DNA intermediates in this breakdown process. The data suggests a “brush-like” model for the intracellular T4 chromosome, with the DNA held at roughly genome-length intervals by some non-DNA core material resistant to both strong detergent and protease treatments.