REQUIREMENT FOR THE POLYMERIZATION AND 5'-]3' EXONUCLEASE ACTIVITIES OF DNA-POLYMERASE-I IN INITIATION OF DNA-REPLICATION AT ORIK SITES IN THE ABSENCE OF RECA IN ESCHERICHIA-COLI RNHA MUTANTS

In previous studies, we found that the requirement for RecA protein in constitutive stable DNA replication (cSDR) can be bypassed by derepression of the LexA regulon and that DNA polymerase I (DNA PolI) is essential for this Rip (RecA-independent process) pathway of cSDR (Y. Cao, R. R. Rowland, and T. Kogoma, J. Bacteriol. 175:7247-7253, 1993). In this study, the role of DNA PolI in the Rip pathway was further examined. By using F' plasmids carrying different parts of the polA gene, a series of complementation tests was carried out to investigate the requirement for the three enzymatic activities, polymerization, 3'-->5' exonuclease, and 5'-->3' exonuclease activities, of DNA PolI. The result indicated that both the 5'-->3' exonuclease and polymerization activities of DNA Poll are essential for bypassing the requirement for RecA in cSDR but that the 3'-->5' exonuclease activity can be dispensed with. Complementation experiments with rat DNA Polbeta also supported the hypothesis that a nick translation activity is probably involved in cSDR in the absence of RecA. An analysis of DNA synthesis suggested that DNA PolI is involved in the initiation but not the elongation stage of cSDR. Moreover, the dnaE293(Ts) mutation was shown to render the bypass replication temperature sensitive despite the presence of active DNA PolI, suggesting that DNA PolIII is responsible for the elongation stage of the Rip pathway. A model which describes the possible roles of RecA in cSDR and the possible function of DNA Poll in the Rip pathway is proposed.