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J. Bacteriol., 01 1998, 83-89, Vol 180, No. 1
Copyright © 1998, American Society for Microbiology

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 [In Process Citation]

MG Kramer, SA Khan and M Espinosa
Centro de Investigaciones Biologicas, CSIC, Madrid, Spain.

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.

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