Project Summary – Abstract In this renewal application we describe recent progress in our ongoing studies of DNA-protein interactions, which have focused largely during the last grant cycle on understanding the molecular mechanisms involved in the assembly, function and control of the macromolecular complexes that direct DNA replication in bacteriophage T4, and outline our plans for the next five years. For many years Peter von Hippel served as the sole PI of this grant, but now this program is a tightly knit joint collaboration between the research groups of Professors Andrew Marcus and Peter von Hippel at the University of Oregon, who serve jointly as co-PIs of this research project. In earlier work supported by this grant, the von Hippel lab focused on solution studies of the replication complex of bacteriophage T4 and the transcription complex of E. coli. We note that these systems involve essentially the same molecular mechanisms for `driving' and regulating these central life processes as do those of `higher organisms,' including humans. These studies thus provide good model systems to examine how human DNA replication proceeds at the functional level, and can help provide insights into what might go wrong in various genetic diseases, which are often caused by minor quantitative changes in the properties of the `macromolecular machines' of genome expression. During the previous reporting period we completed a number of studies on the above mechanistic questions, using reconstituted DNA replication assemblies that carry out their functions in vitro with essentially the same rates, fidelities and processivities as do the in vivo versions of these complexes. We proceeded by placing fluorescent base analogue probes, or cyanine dyes, at defined positions within the DNA frameworks of the reconstituted complexes, and then used fluorescence and circular dichroism spectroscopy to monitor biologically relevant conformational changes at and near the probe labeling sites. In this way, we obtained significant information about replication mechanisms under steady-state or equilibrium conditions, and then carried forward this work by demonstrating that these same optical probe-labeling strategies can be used in more complex experiments to permit two-dimensional fluorescence spectroscopy (2DFS), single-molecule Förster Resonance Energy Transfer (smFRET) and Fluorescence-detected Linear Dichroism (smFLD), which provide structural interpretations and follow the kinetics of reactions within these complexes in `real time' with µsec to msec resolution. As described in the present proposal, these approaches now permit us to obtain structural and dynamic information about local conformational changes that occur at defined and biologically-relevant base analogue and DNA backbone probe sites, as well as to determine free energy surfaces (and define transition states) of individual rate-limiting molecular steps within reconstituted models of relatively complete DN...