The molecular mechanisms involved in DNA replication are being studied biochemically. The system used is the in vitro initiation of replication of double-stranded Lambda dv plasmid DNA. This reaction requires two phage initiation proteins, O and P gene products, many host replication proteins, including the products of dnaB, dnaG, polC, dnaN, dnaJ, dnaK, dnaZ, dnaX, dnaY, gyrA, gyrB, and lig, RNA transcription in the region of the origin of replication and a specific site, ori, on the lambda DNA. I have been isolating the proteins required for this reaction with the goal of reconstituting lambda replication with all purified proteins. Towards this end, I have already prepared Lambda O , Lambda P, dnab, dnaG, pol III holoenzyme (containing polC, dnaN, dnaX, and dnaZ polypeptide) and DNA gyrase. I have developed in vitro complementation assays for dnaJ and dnaK proteins and have designed purification procedures for their isolation. I am currently characterizing a weak ATPase activity associated with dnaK and DNA binding activity associated with dnaJ.
