The molecular mechanisms involved in DNA replication are being studied biochemically. The system used is the in vitro replication of E. coli phage Lambda plasmid DNA. This reaction requires two phage initiation proteins, O and P gene products, many host proteins including DnaB, DnaG primase, pol III holonenzyme, DnaJ, DnaK, DNA gyrase, DNA ligase and Ssb, RNA transcription in the region of the origin of replication, and a specific site, ori Lambda, on the DNA. I have been isolating the proteins required for this reaction with the long range goal of reconstituting the Lambda replication pathway with all purified proteins. Currently, I am purifying DnaJ and DnaK proteins by in vitro complementation assays and characterizing them biochemically. DnaK has weak ATPase activity, DnaJ is a DNA binding protein and both form protein complexes with Lambda P protein. I am participating in a collaborative electron microscopic study of nucleoprotein structures formed by Lambda O, Lambda P, DnaB, Ssb, DnaJ and DnaK proteins at the origin of Lambda replication. I am also characterizing the DNA binding domain of the Lambda O protein.
