Formation and cleavage of a DNA network during in vitro bacteriophage T7 DNA packaging: Light microscopy of DNA metabolism

To understand in vivo DNA metabolism, in vitro systems are developed that perform DNA metabolism, while maintaining in vivo (physiological) character. To determine the state of DNA during in vitro physiological metabolism, the present study develops procedures of fluorescence light microscopy for observation of stained DNA molecules during in vitro physiological metabolism in a crude extract of bacteriophage T7-infected cells. The extract inhibits illumination-induced breakage of DNA. The following DNA metabolism remains active for 2-3 min during microscopy: exonuclease-dependent end-to-end joining (concatemerization) of T7 DNA and subsequent cleavage of concatemers. When the T7 gene 3-encoded DNA debranching endonuclease is absent during in vitro T7 DNA concatemerization, DNA progressively partitions to form a continuous, mostly immobile (i.e., no detected Brownian motion) fibrous network that encloses the DNA-depleted solution; presumably because of reduced branching, a less extensive network forms when the gene 3-encoded debranching endonuclease is present. Most strands of the network consist of multiple DNA segments. After a time interval of 5-10 min, the DNA network undergoes cleavage that depends on the presence of both ATP, capsids, and the DNA packaging accessory proteins encoded by genes 18 and 19; multiple Cleavages eventually disrupt the continuity of the DNA network. The dependence of the observed cleavage on these factors is explained by the hypothesis that this cleavage is the first of two cleavages known to occur during the packaging of T7 DNA concatemers both in vivo and in vitro. The first cleavage is also known to initiate entry of DNA into a T7 capsid. The cleavage observed here is usually preceded by an approximately 10 s burst of oscillatory motion of the DNA network near the point of eventual cleavage. If the in vivo presence of a similar concatemer- containing DNA network is assumed, requirement for DNA packaging-associated release of DNA from this network is a possible explanation for the evolution of a T7 DNA packaging pathway that is initiated by cleavage of a concatemer.