LAGGING-STRAND REPLICATION FROM THE SSOA ORIGIN OF PLASMID PMV158 IN STREPTOCOCCUS-PNEUMONIAE - IN-VIVO AND IN-VITRO INFLUENCES OF MUTATIONS IN 2 CONSERVED SSOA REGIONS

The streptococcal plasmid pMV158 replicates by the rolling-circle mech anism. One feature of this replication mechanism is the generation of single-stranded DNA intermediates which are converted to double-strand ed molecules. Lagging-strand synthesis initiates from the plasmid sing le-stranded origin, sso. We have used the pMV158-derivative plasmid pL S1 (containing the ssoA type of lagging-strand origin) and a set of pL S1 derivatives with mutations in two conserved regions of the ssoA (th e recombination site B [RSB] and a conserved 6-nucleotide sequence [CS -6]) to identify sequences important for plasmid lagging-strand replic ation in Streptococcus pneumoniae. Cells containing plasmids with muta tions in the RSB accumulated 30-fold more single-stranded DNA than tel ls containing plasmids with mutations in the CS-6 sequence. Specificit y of lagging-strand synthesis was tested by the development of a new i n vitro replication system with pneumococcal cell extracts. Four major initiation sites of lagging-strand DNA synthesis were observed. The s pecificity of initiation was maintained in plasmids with mutations in the CS-6 region. Mutations in the RSB region, on the other hand, resul ted in the loss of specific initiation of lagging-strand synthesis and also severely reduced the efficiency of replication.