We are attempting to use the individually purified protein components of the T4 bacteriophage DNA replication apparatus to reconstitute the entire replication process in a completely defined in vitro system. While the replication fork movement process have been faithfully reconstituted, and many of the mechanistic details of this process are well understood, the process that initiates replication forks at T4 replication origins has not yet been successfully reproduced. The E. coli host RNA polymerase plays a role in the latter process, along with the T4 ATP dependnet type II DNA topoisomerase and other suspected but unknown bacteriophage proteins. Using several different assays, we will attempt to define the missing components, as required to obtain origin-dependent replication fork initiation in vitro. We will also use a novel cloning strategy to define the exact DAN sequences that constitute a primary T4 replication origin. Several experiments will also be pursued that are aimed at understanding the detailed structure of the T4 multienzyme complex that moves a replication fork, including its interactions with DNA and the details of the process that recycles the lagging strand DNA polymerase molecule for successive rounds of Okazaki fragment synthesis. Finally, using the purified T4 uvsX and uvsY proteins, we will attempt to reconstitute the process that initiates replication forks from genetic recombination intermediates at late times of T4 infection. All of these studies are aimed at increasing our fundamental knowledge of the basic genetic mechanisms common to all cells, and they are important to an understanding of cell behavior in both normal and diseased states.
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