MOLECULAR INTERACTION IN T4 DNA REPLICATION COMPLEX

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The next stages of a continuing physical biochemical study of the structural and functional interactions of the protein and nucleic acid components of the bacteriophage T4 DNA replication system are outlined in this proposal. We will continue to study the mechanism of action of each functional subset of proteins of this in vitro replication system, focussing on how these subsets are assembled into the integrated DNA replication complex. These subsets include the central DNA polymerase (gene 43 protein), the polymerase accessory proteins complex (genes 44/62 and 45 proteins), and the helicase -primase complex ((genes 41 and 61 proteins), each of which also interacts with the T4 single-stranded DNA binding (gene 32) protein. During the next granting period we will pursue, in particular, the following specific aims. (i) We will continue our physical biochemical studies of the structure and function of the five-protein polymerase holoenzyme complex, focussing primarily on how the processivity ring (gp45) is loaded on to the primer-template junctions within the replication fork in a PuTP-driven "loading' reaction carried out by the gp44/62 sub-assembly of the polymerase accessory proteins, and how encounter with Okazaki fragments in lagging strand synthesis might trigger rapid release of the processivity ring, and thus the holoenzyme from the template; (ii) to elucidate the mechanisms whereby the gp41 hexamer functions as a helicase of double-stranded DNA (and as a unidirectional "translocase" on single- stranded DNA); (iii) to characterize the interactions between the helicase (gp41) and the primase (g61) components of the functional primosome of the T4 DNA replication, system, and to determine how these interactions modulate and regulate the helicase (and translocase) activities of gp41, as well as control the primary activity of gp61; (iv) to use low resolution (but "real time") structural information obtained by cryoelectron microscopy, and detailed molecular structural information obtained by x-ray crystallography to learn how these individual proteins and protein subassemblies operate and interact as molecules in discharging their physiological roles; and (v) to use the knowledge gathered in these studies to examine the integration of these subassemblies into a functional seven- protein (or eight-protein, if the helicase loading factor gp59 is also required) complex that is capable of elongating DNA at the physiological rate and with physiological processivity and fidelity. These studies now appear to be particularly directly health related, since considerable recent work has shown that both the processivity and the helicase components of the T4 system may be very analogous to the eukaryotic proteins that are involved in connecting DNA replication and repair to cell cycle control and tumorogenesis.