Bacteriophage P22 in vitro DNA packaging monitored by agarose gel electrophoresis: rate of DNA entry into capsids

Abstract

Bacteriophage P22, like other double-stranded DNA bacteriophages, packages DNA in a preassembled, DNA-free procapsid. The P22 procapsid and P22 bacteriophage have been electrophoretically characterized; the procapsid has a negative average electrical surface charge density (sigma) higher in magnitude than the negative sigma of the mature bacteriophage. Dextrans, sucrose, and maltose were shown to have a dramatic stimulatory effect on the in vitro packaging of DNA by the P22 procapsid. However, sedoheptulose, smaller sugars, and smaller polyols did not stimulate in vitro P22 DNA packaging. These and other data suggest that an osmotic pressure difference across some particle, probably a capsid, stimulates P22 DNA packaging. After in vitro packaging was optimized by including dextran 40 in extracts, the entry kinetics of DNA into P22 capsids were measured. Packaged DNA was detected by: (i) DNA-specific staining of intact capsids after fractionation by agarose gel electrophoresis and (ii) agarose gel electrophoresis of DNase-resistant DNA after release of DNase-resistant DNA from capsids. It was found that the first DNA was packaged by 1.5 min after the start of incubation. The data further suggest that either P22 capsids with DNA partially packaged in vitro are too unstable to be detected by the above procedures or entry of DNA into the capsid occurs in less than 0.25 min.