Two membrane sites for DNA synthesis in Pneumococcus

Summary

A DNA membrane fraction extracted from pneumococci can be separated into two subfractions with respect to macromolecular composition and DNA synthesis by centrifugation in a 30–60% w/v neutral sucrose gradient. Each fraction can be rebanded in a sucrose gradient or centrifuged to equilibrium in a CsCl density gradient without altering the ability of the fractions to synthesize DNA. The fast sedimenting (heavy) fraction contains 45% of the DNA, and the bulk of the phospholipid, protein, and RNA. The light fraction contains 50% of the DNA, and lower, but significant amounts of phospholipid, RNA, and protein. Both fractions contain a DNA replication complex consisting of a number of enzymes involved in synthesizing DNA or DNA precursors, as well as RNA polymerase activity. However, the specific activity of DNA polymerase in the light fraction is much greater than that in the heavy fraction. In addition, the following results suggest that the former is concerned primarily with replication of the genome while the latter has characteristics of a repair function for the genome. (1) newly synthesized DNA can be detected within 30 s in the light fraction but not until 4 min in the heavy fraction. (2) an RNA-DNA single-stranded hybrid can be demonstrated during initial stages of DNA synthesis in the light, but not heavy fraction. (3) extensive semiconservative DNA replication occurs in the light fraction, whereas little such replication is detected in the heavy fraction. (4) DNA polymerase activity in the light fraction has several of the characteristics of a polymerase identified by others as being concerned with normal DNA replication, such as inhibition by N-ethylmaleimide, and relatively high rates of chain elongation (4.9×10⁴ nucleotides/min). In contrast, DNA polymerase activity in the heavy fraction has characteristic properties associated with DNA polymerase I, a possible repair enzyme. These include higher activity for a d(A-T)n template than that detected in the light fraction, no effect of N-ethylmaleimide, and relatively low rates of chain elongation (9×10³ nucleotides/min).