Analysis of the bacteriophage T4 DNA polymerase holoenzyme and Dda helicase

Two questions are addressed: (1) What is the role of the bacteriophage T4 Dda DNA helicase during T4 infection? (2) What is the mechansim that enables the T4 DNA polymerase holoenzyme to synthesize DNA processively on the leading strand of the replication fork for many minutes, while allowing an identical holoenzyme on the lagging strand to recycle from finishing one Okazaki fragment to beginning the next in a few seconds?; The Dda protein is a 5{dollar}spprime{dollar}-3{dollar}spprime{dollar} DNA helicase that has been found to stimulate DNA replication and recombination reactions in vitro. Herein, I report the overexpression, purification, sequence analysis of the Dda protein. In addition, the T4 insertion-substitution system was used to create a deletion in the T4 dda gene. The deletion phage showed a delay in DNA synthesis during early times of infection, suggesting that the Dda protein is involved in the initiation of origin-dependent DNA synthesis. The gene 59 protein has been shown to load the gene 41 helicase onto DNA in vitro and to be important for recombination-dependent DNA synthesis in vivo. No DNA was synthesized during infection by double dda59 mutants. This suggests that the Dda protein is important for starting DNA replication in vivo, but that the gene 59-41 complex can partially substitute for it in this role.; To address the second question, I developed assays to monitor the dissociation of the T4 DNA polymerase holoenzyme--both when it is stalled by nucleotide omission and when it is stalled at a helical region of the DNA template. The dissociation of the holoenzyme stalled by nucleotide omission is a first order decay process with a half-life of 2.5 min. This half-life resembles that expected for the holoenzyme processively synthesizing DNA on the leading strand of the replication fork. In contrast, when the holoenzyme is stalled at a helical region, it dissociates with a half-life of 1 sec. From this data, a duplex DNA sensor model is presented, which explains the dissimilar behavior of the identical holoenzymes on the leading and lagging strands of the replication fork.