DNA Helicases in DNA Replication and Repair

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J. CampbellThe goal of this research is to understand the mechanism by which an origin of DNA replication is converted into a replication fork and how the proteins at the fork are organized to coordinate the synthesis of the leading and lagging strands. The underlying assumption, which, while plausible, remains to be proved, is that there is a superassembly of proteins at the replication fork and that it functions like a machine whose moving parts are proteins. What is less certain is whether there is a single, multiprotein assembly at origins that changes conformation in a concerted manner to effect each stage of fork progression or if there are a number of smaller machines each assigned a different specific task and only transiently associated with the fork. To attack such a complex problem requires dividing it into partial reactions. In the coming granting period, the DNA2 helicase and its role in initiation and elongation will be studied in depth. These fundamental biological experiments in yeast will be combined with experiments in the powerful Xenopus laevis egg extracts, that recapitulate both initiation and elongation in vitro under strict cell cycle control. The Xenopus in vitro replication system faithfully mimics in vivo events and has been very useful in working out the order of assembly of initiation proteins onto the sperm chromatin. An important feature of this system is that the egg extracts stockpile replication proteins and are capable of very rapid and extensive replication in vitro. Thus, they also provide the only existing in vitro replication system for studying eukaryotic chromosomal replication forks. Previously, the first DNA helicase essential for DNA replication in yeast was identified in this laboratory. In order to take advantage of both yeast molecular biology and genetics and Xenopus in vitro replication system, the Xenopus homolog of the yeast helicase, called Dna2, was cloned. Each stage of DNA replication in Xenopus extracts will now be analyzed in the presence and absence of Xdna2p. The assembly and disassembly of the replication apparatus during the cell cycle in the presence and absence of Xdna2p will be followed. Yeast mutants defective in DNA2 are sensitive to X-rays and MMS. Since the Xenopus extracts have been shown to be useful for studies of repair of X-ray induced damage, the Xenopus extracts will be used to study the molecular contribution of Xdna2. Studies of the cell cycle and regulated formation and activation of the replication fork have important implications for the understanding of regulation of cell proliferation. DNA replication cycles are also a target of regulation during development and differentiation. This research is expected to provide important insights into these processes.