DNA synthesis in purified DNA-membrane complexes extracted from a Bacillus subtilis pol A mutant.

Abstract

The cell membrane of prokaryotes has been implicated as the site of DNA replication in the replicon model first proposed by Jacob, Brenner and Cuzin1. Ultrastructural, genetic and biochemical data have since been compiled suggesting an association between the cell membrane and replicating DNA (for review, see ref. 2). However, few studies have attempted to test this aspect of the replicon model directly by investigating the ability of DNA–membrane complexes to synthesize DNA in vitro. Such studies have been performed mainly with Pneumococcus and it has been shown that DNA–membrane complexes extracted by the M-band technique3 retain their capacity to synthesize DNA even after extensive purification4. However, mutants defective in various aspects of DNA replication are unavailable in Pneumococcus, and this is an important requirement for elucidating the physiological significance of the complex. Fortunately, they are available in Escherichia coli and Bacillus subtilis, and of the two organisms, much of the important work with the complex has been accomplished with B. subtilis5–12. These studies have indicated that the origin and terminus of replication are permanently associated with the cell membrane. Nonetheless, there have been no previous studies of the synthetic capacity of B. subtilis DNA–membrane complexes. We now offer evidence that two DNA–membrane sub-complexes extracted and purified from a pol A mutant of B. subtilis are capable of synthesizing DNA in vitro and that these activities are inhibited by hydroxyphenyl azouracil, a specific inhibitor of DNA polymerase III activity in B. subtilis.