MULTIPLE ROLES FOR DNA-POLYMERASE-I IN ESTABLISHMENT AND REPLICATION OF THE PROMISCUOUS PLASMID PLS1

The polymerase activity of DNA polymerase I is important for the estab lishment of the pLS1 replicon by reconstitutive assembly in Streptococ cus pneumoniae after uptake of exogenous pLS1 plasmid DNA. In polA mut ants lacking the polymerase domain, such establishment was reduced at least 10-fold in frequency. Chromosomally facilitated establishment of pLS1-based plasmids carrying DNA homologous to the host chromosome wa s not so affected. However, both types of plasmid transfer gave mostly small colonies on initial selection, which was indicative of a defect in replication and filling of the plasmid pool. Once established, the pLS1-based plasmids replicated in polA mutants, but they showed segre gational instability. This defect was not observed in strains with the wild-type enzyme or in an S. pneumoniae strain that encodes the polym erase and exonuclease domains of the enzyme on separate fragments. The role of DNA polymerase I in stably maintaining the plasmids depends o n its polymerizing function in three separate steps of rolling-circle replication, as indicated by the accumulation of different replication intermediate forms in polA mutants. Furthermore, examination of the s egregational stability of the pLS1 replicon in an Escherichia coil mut ant system indicated that both the polymerase and the 5'-to-3' exonucl ease activities of DNA polymerase I function in plasmid replication.