Lagging-strand replication from the ssoA origin of plasmid pMV158 in Streptococcus pneumoniae: in vivo and in vitro influences of mutations in two conserved ssoA regions.
The streptococcal plasmid pMV158 replicates by the rolling-circle mechanism. One feature of this replication mechanism is the generation of single-stranded DNA intermediates which are converted to double-stranded molecules. Lagging-strand synthesis initiates from the plasmid single-stranded origin, sso. We have used the pMV158-derivative plasmid pLS1 (containing the ssoA type of lagging-strand origin) and a set of pLS1 derivatives with mutations in two conserved regions of the ssoA (the recombination site B [RS(B)] and a conserved 6-nucleotide sequence [CS-6]) to identify sequences important for plasmid lagging-strand replication in Streptococcus pneumoniae. Cells containing plasmids with mutations in the RS(B) accumulated 30-fold more single-stranded DNA than cells containing plasmids with mutations in the CS-6 sequence. Specificity of lagging-strand synthesis was tested by the development of a new in vitro replication system with pneumococcal cell extracts. Four major initiation sites of lagging-strand DNA synthesis were observed. The specificity of initiation was maintained in plasmids with mutations in the CS-6 region. Mutations in the RS(B) region, on the other hand, resulted in the loss of specific initiation of lagging-strand synthesis and also severely reduced the efficiency of replication.
Kramer
M G
MG
Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
Khan
S A
SA
Espinosa
M
M
D001483:Base Sequence; D017124:Conserved Sequence / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D018741:Replication Origin / Q000235:genetics*; D013296:Streptococcus pneumoniae / Q000235:genetics*
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded