Structure and Relations of Proteins and Nucleic Acids

– Abstract In this MIRA application we describe recent progress and general plans for the next five years of our studies that focus largely on understanding the molecular mechanisms of DNA ‘breathing’ fluctuations and their roles in the assembly, function and control of the macromolecular complexes that direct DNA replication in bacteriophage T4. This work is presently funded by our R01 grant from NIGMS (GM15792), which has one more year to run, and which we do not plan to renew. Our proposed MIRA project in this area represents a continuation of our present tightly knit collaborative research program between the groups of Professors Andrew H. Marcus and Peter H. von Hippel at the University of Oregon. In earlier collaborative work, the Marcus and von Hippel labs have focused on solution studies of the replication complex of bacteriophage T4, which involves essentially the same molecular mechanisms for ‘driving’ and regulating these central life processes as do those of ‘higher organisms,’ including humans. The T4 replication system thus serves as a good model to determine how human DNA replication proceeds at the functional level, and these studies can help provide insights into what might go wrong in various genetic diseases, which are often caused by minor quantitative changes in the composition and molecular mechanisms of the ‘macromolecular machines’ of genome expression. In recent years the Marcus and von Hippel groups (and collaborators) have developed a series of powerful single-molecule fluorescence and ensemble spectroscopic methods that can be used to determine the local conformations, conformational disorder and microsecond-resolved ‘breathing fluctuations’ of DNA constructs that have been site-specifically labeled with fluorescent optical probes. These DNA constructs contain fluorescent base analogue or cyanine dye probes located at defined biologically relevant positions within model DNA frameworks. We use fluorescence, circular dichroism (CD) and two-dimensional fluorescence spectroscopy (2DFS) methods to monitor biologically relevant changes in local conformation and conformational disorder at and near the probe-labeled sites. By this means we obtain significant information about average local DNA base and backbone conformations and conformational disorder in bulk solution. We then use these same optical probe-labeling strategies in single-molecule Förster Resonance Energy Transfer (smFRET) and Polarization- Sweep Single-Molecule Fluorescence (PS-SMF) experiments to monitor microsecond-resolved DNA ‘breathing’ fluctuations, including within protein-DNA complexes. As described in this MIRA proposal, these approaches now permit us to obtain structural and dynamic information about local conformational changes that occur at defined and biologically relevant DNA backbone and base analogue probe sites, as well as to determine free energy surfaces (and define transition states) of individual rate-limiting molecular steps within such reconstituted models of regulatory genomic control systems. Project Number: 1R35GM161360-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Andrew Marcus | Institution: UNIVERSITY OF OREGON, EUGENE, OR | Award Amount: $403,836 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award B Study Section[MRAB] View on NIH RePORTER: https://reporter.nih.gov/project-details/11259163