INTRODUCTION- DNA replication is accomplished by a highly organized complex of proteins responsible for the coordinated synthesis of the leading and lagging strands of the replication fork. We are using a multienzyme system of bacteriophage T4 proteins to determine the nature of the proteins, enzymatic reactions, and protein-protein interactions that catalyze and control this intricate and essential process. In this system, the phage encoded DNA polymerase that synthesizes both strands is held in place by the gene 45 clamp, in turn loaded by the gene 44/62 clamp loader. Gene 41 helicase, which moves 5' to 3' on the lagging strand template, opens the duplex ahead of the leading strand polymerase and interacts with gene 61 primase to enable it to make the pentamer RNA primers that initiate each lagging strand fragment. T4 gene 59 protein preferentially binds forked DNA, accelerates the loading of the helicase, and binds the 32 protein that coats the single-stranded lagging strand template. 32 protein controls the cycle of lagging strand reactions by increasing the rates of primer synthesis, chain elongation by the lagging strand polymerase, and primer removal by T4 5' nuclease (RNaseH). HELICASE, HELICASE LOADER, AND 32 PROTEIN-In studies directed at understanding the function of the strong affinity between the 32 and 59 proteins at the replication fork, we showed that 32 protein is required for helicase-dependent leading strand DNA synthesis when the helicase is loaded by 59 protein. However, 32 protein is not required for leading strand synthesis when helicase is loaded, less efficiently, without 59 protein. Leading strand synthesis by wild type T4 polymerase is strongly inhibited when 59 protein is present without 32 protein. Since 59 protein can load the helicase on forks without 32 protein, our results are best explained by a model in which 59 helicase loader at the fork prevents the coupling of the leading strand polymerase and the helicase, unless the position of 59 protein is shifted by its association with 32 protein. In earlier studies on synthesis from the T4 uvsY origin, in collaboration with Ken Kreuzer, we found that 59 protein strongly blocked leading strand synthesis when 41 helicase was not present. Thus 59 protein helps to coordinate synthesis of the two strands at the fork by blocking leading strand synthesis until both the helicase and 32 protein are loaded. We have previously constructed a T4 phage with a complete deletion of gene 59, and shown that it is severely defective in DNA synthesis and produces a very low phage yield. We are currently using this mutant as the basis of a genetic screen to identify essential regions of 59 protein, by selecting for plasmid mutants that fail to restore replication of the T4 gene 59 deletion. In collaboration with Timothy Mueser (University of Toledo), we are continuing our efforts to get crystal structures of 59 protein and T4 RNaseH bound to DNA. INITIATION OF T4 DNA REPLICATION- The initial DNA synthesis after T4 phage infection of E. coli apparently originates from defined origins of replication, while continued DNA replication is dependent on homologous recombination. Both origin-dependent and recombination-dependent T4 DNA replication utilize an overlapping set of viral proteins, complicating the analysis of origin usage. Several possible origins have previously been identified by a variety of techniques, yet it is not known if these elements function in concert or individually. Moreover, the genetic requirements of origin activity have not been clearly defined. We are currently mapping the origins of T4 DNA replication in an attempt to understand the molecular processes required for the initiation of viral DNA synthesis. Our mapping approach uses an array of discrete T4 DNAs, amplified from evenly spaced regions within the viral genome. This array is then probed with T4 DNA produced early during infection, alllowing one to map this nascent DNA to discrete genomic loci and assign physical positions to the origins of T4 DNA synthesis. Using this approach we initially identified at least two major origins of T4 DNA replication that are active under our experimental conditions, oriE and oriF. Though both of these loci have been previously implicated in the initiation of T4 DNA synthesis, this is the first observation of activity from both origins during a single infection. We are also investigating the viral encoded requirements of origin function using our array and a panel of T4 mutants, deficient in a variety of replication associated gene products. STRUCTURE OF REPLICATION FORKS- In collaboration with the laboratory of Jack Griffith (University of North Carolina), we are using electron microscopy to probe the architecture of the DNA strands and T4 replication protein complex on rolling circle replication forks. In the ?trombone? model of Bruce Alberts, the nascent lagging strand fragment and the single-stranded DNA behind it are folded into a loop to allow contact between the leading and lagging strand polymerases. In confirmation of this model, we previously found that there is a single complex of the leading and lagging strand proteins at the fork, with a loop present on more than 50% of the molecules. However, in contrast to the original model, the DNA in the loops is completely double-stranded, with no visible extended single-stranded DNA. Instead, the protein-covered single-stranded DNA segments on the lagging strand are folded into highly compact structures, which may limit when primase recognition sites are accessible to the primase. We are currently using streptavidine-bound DNA ?pointers? to biotin-tagged replication proteins to determine the location of specific proteins in the replication complex, at different stages in the lagging strand replication cycle.

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Nossal, Nancy G; Makhov, Alexander M; Chastain 2nd, Paul D et al. (2007) Architecture of the bacteriophage T4 replication complex revealed with nanoscale biopointers. J Biol Chem 282:1098-108
Gangisetty, Omkaram; Jones, Charles E; Bhagwat, Medha et al. (2005) Maturation of bacteriophage T4 lagging strand fragments depends on interaction of T4 RNase H with T4 32 protein rather than the T4 gene 45 clamp. J Biol Chem 280:12876-87
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Jones, Charles E; Green, Erin M; Stephens, Julia A et al. (2004) Mutations of bacteriophage T4 59 helicase loader defective in binding fork DNA and in interactions with T4 32 single-stranded DNA-binding protein. J Biol Chem 279:25721-8
Jones, Charles E; Mueser, Timothy C; Nossal, Nancy G (2004) Bacteriophage T4 32 protein is required for helicase-dependent leading strand synthesis when the helicase is loaded by the T4 59 helicase-loading protein. J Biol Chem 279:12067-75
Chastain 2nd, Paul D; Makhov, Alexander M; Nossal, Nancy G et al. (2003) Architecture of the replication complex and DNA loops at the fork generated by the bacteriophage t4 proteins. J Biol Chem 278:21276-85
Nossal, N G; Dudas, K C; Kreuzer, K N (2001) Bacteriophage T4 proteins replicate plasmids with a preformed R loop at the T4 ori(uvsY) replication origin in vitro. Mol Cell 7:31-41
Bhagwat, M; Nossal, N G (2001) Bacteriophage T4 RNase H removes both RNA primers and adjacent DNA from the 5' end of lagging strand fragments. J Biol Chem 276:28516-24
Jones, C E; Mueser, T C; Dudas, K C et al. (2001) Bacteriophage T4 gene 41 helicase and gene 59 helicase-loading protein: a versatile couple with roles in replication and recombination. Proc Natl Acad Sci U S A 98:8312-8
Mueser, T C; Jones, C E; Nossal, N G et al. (2000) Bacteriophage T4 gene 59 helicase assembly protein binds replication fork DNA. The 1.45 A resolution crystal structure reveals a novel alpha-helical two-domain fold. J Mol Biol 296:597-612

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