The origin of rolling circle replication in filamentous coliphage consists of a core origin that is absolutely required and an adjacent replication enhancer sequence that increases in vivo replication 30 to 100-fold. The core origin binds the initiator protein (gpII) which either nicks or relaxes negatively superhelical replicative form DNA (RFI). Nicking at the origin, but not relaxation, leads to initiation of DNA replication. Our results indicate that the ratio of nicking to relaxation (nicking-closing) in vitro depends on the superhelical density of the substrate. We have studied the effect of a single amino acid substitution In gpII, which allows wild-type levels of replication in the absence of the enhancer, on origin nicking and binding. The enhancer-independent mutation yields more nicking and less relaxation of RFI, compared to the wild-type protein. The mutant gpII also shows a reduced requirement for superhelicity of the substrate in the nicking reaction. At the same time, the mutant gpII increases the co-operativity of protein-protein interactions in origin binding. We propose that the relaxation activity of gpII negatively regulates replication initiation, and that both increase in the negative superhelicity of the substrate and action of the replication enhancer may antagonize the relaxation activity.
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We thank Susumu Hirose for helpful suggestions, Norton Zinder and Peter Model for critical reading of the manuscript, and Nahoko Higashitani and Jodi Radassao for their help in experiments. This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan, and by grants from the Fujisawa Foundation, the Association for Propagation of the Knowledge of Genetics, Japan, the National Science Foundation and the National Institutes of Health. D.G. was supported by training grant AI07233 from the National Institutes of Health.