-- Abstract In this renewal application we describe recent progress in our studies of the molecular mechanisms and controls that regulate DNA replication, and outline our plans for the next four years. With this application we (Professors Andrew Marcus and Peter von Hippel) propose to further formalize our on-going and increasingly close collaboration in these studies by applying for this grant renewal as `joint' Principal Investigators (PIs). In earlier work on this grant the von Hippel lab largely 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. As a result these studies provide good model systems to examine how human DNA replication and RNA transcription proceed at the fundamental level, and provide insights into what goes wrong at these levels in various forms of cancer and genetic diseases that often seem to involve minor kinetic or structural changes in the properties or control of these `macromolecular machines'. During the last reporting period we completed a number of studies on the above mechanistic questions, using reconstituted DNA replication or RNA transcription complexes that carry out their functions with essentially the same rates, fidelities and processivities as the in vivo versions of the same complexes. We proceeded largely by placing fluorescent base analogue probes, or internal cyanine dye probes and FRET pairs, at defined positions within the nucleic acid frameworks of the reconstituted complexes, and then used fluorescent and circular dichroism spectroscopy at wavelengths great than 300 nm (an optical range in which the rest of the protein and nucleic acid components of the complexes are transparent) to monitor biologically relevant conformational changes at and near the probe sites. By these means we obtained significant information about replication and transcription mechanisms under steady state or equilibrium conditions, and then followed up with very successful initial spectroscopy studies that showed that various versions of these same optical probe approaches can be used in more complex arrangements to permit two-dimensional fluorescence spectroscopic (2DFS) and single molecule Fluorescence Resonance Energy Transfer (smFRET) and Fluorescence Linear Dichroism (smFLD) measurements that can follow the kinetics of reactions within these complexes in `real time' with sec to msec resolution. As described in the present proposal, these approaches now permit us to obtain local structural and dynamic information on conformational changes that occur at defined and biologically- relevant base analogue and DNA backbone probe sites, as well as to map transition states of individual rate- limiting molecular steps within in vitro reconstituted models of relatively complete DNA replication complexes. Using these approaches we are beginning to reveal aspects of mechanisms and regulatory control systems that have previously been inaccessible to direct experimental measurement.
Protein-nucleic acid complexes work in living cells to faithfully replicate (copy) the DNA genome to form the genetic material of the daughter cells, to transcribe and process specific messenger RNA sequences to permit `expression' of the defined genes, and to direct the synthesis of specific required proteins at various stages of development. These molecular complexes, which drive and direct these central processes of cellular function, largely self-assemble from individual protein and nucleic acid components to form the so-called `macromolecular machines of gene expression'. We are continuing a program of research that uses the methods of physics and chemistry to understand the specific molecular processes that are involved in the function and regulation of these machines, in part because understanding these central processes in molecular detail will help in the development of specific tools to control diseases of anomalous gene expression and development, including various forms of cancer and related genetic diseases.
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