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). MATURATION OF T4 LAGGING STRAND FRAGMENTS DEPENDS ON INTERACTION OF T4 RNASEH WITH T4 32 PROTEIN RATHER THAN THE T4 GENE 45 CLAMP- T4 RNase H, which has a 5' nuclease that degrades both RNA:DNA and DNA:DNA duplexes, as well as a flap endonuclease activity, is responsible for removing the RNA primers from lagging strand fragments. This 5' nuclease has strong structural and functional similarity to the FEN1 nuclease family. We have previously shown that it is the 5' nuclease, rather than the flap endonuclease, that removes the RNA primers and about 30b of adjacent DNA from each T4 lagging strand fragment. T4 RNaseH is stimulated at nicks by the T4 replication clamp, and by 32 protein, when there is ssDNA behind the nuclease. Using mutant T4 RNase H with deletions in the binding site for 32 protein or the clamp, we find that it is the interaction of T4 RNase H with 32 protein that most affects the maturation of lagging strand fragments in the T4 replication system in vitro, and T4 phage production in vivo. This indicates that RNaseH normally removes the primers while there is still 32 protein-covered ssDNA between the nuclease and the polymerase extending the adjacent fragment. The interaction between the clamp and the nuclease serves a backup function, allowing the nuclease to be loaded on nicked molecules formed if polymerase completes synthesis before the primer is removed. 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. We have developed a powerful technique to use DNA """"""""pointers"""""""" to biotin-tagged replication proteins to determine which proteins are present in the replication complex at different stages in the lagging strand replication cycle. We have shown that many of the protein complexes at the fork contain two polymerases, as expected if the leading and lagging strand polymerases are coordinated. We find that the gene 59 helicase loading protein remains on the fork after loading the helicase, and is present on molecules with extensive replication. 59 protein is located predominantly at the fork, rather than with the 32-protein covering the ssDNA on the lagging strand. Biotin-tagged helicase is present on a large fraction of actively replicating molecules. However, when replication was limited to 60 or 400b of leading strand synthesis, by withholding a required 4th dNTP on different nicked circular templates, biotin-tagged polymerase remained in the complex, but there was much less biotin-helicase. This is consistent with previous biochemical experiments showing a tight coupling between a rapidly moving leading strand polymerase and the helicase. INITIATION OF PHAGE T4 REPLICATION IN VIVO- Phage T4 has a linear genome of 169 kb. The ends of its genome are both circularly permuted and terminally redundant, so that the end of one molecule can invade another by homologous recombination . Previous studies by others suggested the T4 replication begins at poorly defined origins, and then switches to recombination-dependent replication, which is responsible for most T4 DNA synthesis, at later times. Our recent experiments indicate that the switch to recombination-dependent replication is determined by the number of phage genomes per infected cell (multiplicity of infection, or MOI), rather than the length of time after infection. At an MOI of 0.5, replication is not dependent on the uvsX recombination protein ( serves the same function as E. coli recA), until 12 minutes after infection, while it is uvsX-dependent from the start of replication at an MOI of 5. We are using an array of discrete T4 DNAs, amplified from evenly spaced regions across the viral genome, to determine the region or regions of the T4 genome where most replication is initiated under normal infection conditions. Our studies show several active regions of T4 replication, with the region surrounding oriE being the most active. The oriE region was characterized by Gisela Mosig, who noted that it contained open reading frames for two short proteins, repEA and repEB. We find that truncation mutants in repEA or repEB, obtained from the Mosig laboratory, each reduce the relative abundance of early T4 DNA from the oriE region, and alter the pattern of actively replicating regions.
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