Chromosome replication is one of the most crucial events in the cell cycle of Escherichia coli, and it is regulated at the initiation step. The initiation occurs at a unique site on the chromosome termed oriC which must be activated by several interacting proteins. The initiation event also requires RNA and protein synthesis. The biochemical nature of the protein synthesis requirement is not understood. Under certain circumstances, E. coli exhibits altered modes of DNA replication. These are constitutive stable DNA replication (cSDR) seen in rnh mutants which are devoid of ribonuclease H and induced stable DNA replication (iSDR) seen during the SOS response. Unlike normal replication, both modes of replication can be repeatedly initiated without concomitant protein synthesis. cSDR is initiated from a new set of origins )termed oriKs) other than oriC, and requires RNA synthesis. On the other hand, iSDR is initiated at two origins (termed oriMs) that are different from oriKs and from oriC. The initiation at oriMs requires neither protein synthesis nor RNA synthesis. This project is focused on the characterization of these two alternative DNA replication systems which are normally repressed but can be activated by genetic stress such as damage to DNA and a mutation in the rnh gene. The experiments are proposed: 1. To clone and characterize several oriK sites which are active only in rnh mutants; 2. To characterize the DNA-damage inducible origins of replication, oriMs. 3. To develope an in vitro DNA replication system to study the mechanism of the activation of oriMs by SOS-inducing signals. 4. To characterize a new type of Sdr mutants (sdrB) with the expectation that the study will lead to an insight into the nature of the protein synthesis requirements for initiation of chromosome replication at oriC.
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