The DNA/membrane fraction of Pneumococcus contains a DNA replication complex

Abstract

A DNA/membrane fraction extracted from cell suspensions of pneumococci (containing polyadenylic acid (poly(A)) and DNA precursors) with sodium lauroyl sarcosinate (Sarkosyl) contains a DNA-replication complex. Almost all of the nascent DNA, 70% of the nascent RNA and protein and 85% of the nascent phospholipid were detected in this fraction. The initial rate of synthesis calculated from incorporation of precursors in vivo corresponds to the incorporation of 2.66 × 10 5 nucleotides/min/cell. The entire fraction formed a pellicle at the meniscus of CsCl density gradients. Some of the DNA and other macromolecules could be dissociated upon treatment with nucleases, pronase or phospholipase. The sedimentation velocity of nascent DNA in the complex was smaller (9 to 12 s) than that of bulk DNA (16 to 20 s). The DNA/membrane fraction was capable of synthesizing new DNA in vitro with no external DNA template added when either deoxyribonucleoside triphosphates, deoxyribonucleoside mono-phosphates or ribonuoleoside diphosphates were added together with appropriate cofactors. A procedure designed to purify the complex with respect to in vitro DNA synthesis not only increased the specific activity of triphosphate- and mono-phosphate-initiated DNA synthesis but produced a “minimal” complex containing 9% of the total cell DNA and 8% of the protein. Experiments with puromycin suggested that enzyme activities in the DNA/membrane fraction were not due to nascent polypeptide chains on polysomes, but rather to “complete” enzymes.