We are continuing our study of the E. Coli bacteriophage T4 model system for duplex DNA replication in which efficient DNA replication in vitro is achieved with purified proteins encoded by T4 phage: T4 DNA polymerase (gene 43), gene 32 DNA helix-destabilizing protein, the gene 44/62 and gene 45 polymerase accessory proteins, the genes 41, 61, and 59 primase-helicase, RNase H, and DNA ligase. We are collaborating with Tim Meuser and Craig Hyde, NIAMS, to determine the structure of the T4 DNA replication proteins by X-ray diffraction. The T4 gene 59 helicase assembly protein plays an important role in DNA replication and recombination by accelerating the loading of the gene 41 helicase at replication origins and forks and on recombination intermediates. It binds both the T4 gene 41 helicase and gene 32 single-stranded DNA binding proteins. We have shown that the helicase assembly protein has a much higher affinity for forked DNA than for either single or double-stranded DNA, and are using DNA footprinting to define the length of each type of DNA bound by 59 protein. The crystal structure of the 59 helicase assembly protein has recently been solved by our collaborators Tim Meuser and Craig Hyde. There is a single short beta strand in an otherwise completely helical protein. We have begun site-directed mutagenesis to test our model of proposed binding sites on 59 protein for the helicase, the single-stranded DNA binding protein, and for the single and double-stranded regions of forked DNA. T4 RNaseH is a 5' to 3' exonuclease that is a member of the RAD2 family of eukaryotic and prokaryotic replication and repair nucleases. We have shown that this nuclease removes the pentanucleotide RNA primers and 10-50 nucleotides of adjacent DNA from each discontinuous lagging strand fragment on the DNA replication fork in vitro. Removing the first DNA added to each primer is likely to improve replication accuracy. The extent of DNA removal by the nuclease is regulated by its interaction with the gene 32 single-stranded DNA binding protein, which binds directly to T4 RNaseH, even in the absence of DNA, and converts it into a moderately processive exonuclease that removes 10-50 nucleotides each time it binds to the DNA duplex. A C-terminal helical region of the nuclease and the N-terminal domain of 32 protein are each required for this interaction. On nicked and gapped model substrates for DNA repair, DNA degradation is stimulated by loading the gene 45 polymerase clamp protein behind the nuclease. A short (11 amino acid) disordered region at the N-terminus of T4 RNaseH is essential for its interaction with the clamp protein. In collaboration with Kathleen Dudas and Ken Kreuzer (Duke University) we have established an in vitro system for the extension of R-loops on plasmids with phage T4 DNA replication origins. Full length synthesis is dependent on T4 DNA topoisomerase as well as the polymerase, clamp, and clamp loader; the primase, helicase, and helicase assembly protein; and 32 protein.
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