Possible involvement of DNA-linked RNA in the initiation of Bacillus subtilis chromosome replication

Abstract

After thermal denaturation, an in vivo-labeled RNA was found in a temperature-sensitive initiation mutant of Bacillus subtilis (dna-37) associated with highmolecular-weight DNA. This RNA could be clearly distinguished from other RNA species by different techniques of separation, such as Sepharose 2B filtration, chromatography on nitrocellulose, and equilibrium centrifugation in density gradients. It was obtained even when HCHO was present during denaturation and chilling of nucleic acids and was still detected after a second denaturation as well as after incubation with proteinase K. Properties of the complex were not altered by prior treatment with RNase H. A control .experiment using two samples of the complex treated either with pancreatic DNase or with pancreatic RNase, denatured together and centrifuged in the same density gradient, showed that no artifactual associations occur between the DNA and the RNA components of the complex. These results demonstrate that the DNA and RNA in the complex are associated by neither hydrogen bonds nor proteins, but are indicative of a DNA-RNA covalent linkage. In addition, during synchronous replication after a previous period at a nonpermissive temperature, DNA-linked RNA synthesis took place at specific times which coincided with the appearance of rifampin resistance of the first and the second replication cycles. A possible involvement of this RNA in the initiation of chromosome replication is discussed.

Bacterial growth and division: genes, structures, forces, and clocks.

“…Both drug inhibition studies and the study of mutants have shown that RNA synthesis is needed for the initiation of DNA replication (87,127,259,260). DNA-RNA complexes have been found in both E. coli and B. subtilis that differ from the RNA found linked to Okazaki fragments during DNA replication.…”