The proposed work will continue our laboratory's effort to detail the events that occur during the initiation of lagging-strand DNA synthesis and the subsequent maintenance of the priming apparatus on the DNA. Two regions of pBR322 DNA have been shown to act as effector DNAs for the singlestrand DNA-dependent ATPase activity of replication factor Y (Zipursky and Marians, PNAS, 77, 6521-6525 (1980)), a pre-priming E. coli replication protein which directs the assembly of the primosome. We have demonstrated that the factor Y effector sites of pBR322 DNA can act as origins of complementary strand synthesis when cloned into single-strand phage vectors (Zipursky and Marians, PNAS, 78, 6111-6115(1981). The proposed work can be divided into two major sections. We will first study the interaction of factor Y with its effector sites on a molecular level. Through the use of DNA footprinting, methylation enhancement and in vitro mutant construction we will determine precisely those bases within a factor Y effector site which are required for origin function. This work will define the mechanism of interaction between factor Y and its effector DNAs and define the requirements for a region of DNA to function as a lagging-strand origin of replication. The second section of the proposed work is biochemical in nature. Reconstitution of ss(c) greater than RF DNA synthesis with purified proteins using the recombinant Y-site-fl phage DNAs as templates will allow us to determine which proteins are involved, the direction of primosome movement, and the role of the ATP hydrolyzing proteins (factor Y and the dnaB protein) in unwinding duplex DNA during replication.
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