In collaboration with UO Prof. Peter von Hippel, our groups are working to develop an improved understanding of how biomolecular assemblies drive the synthesis of DNA and RNA. We are interested in the mechanisms of how conformation fluctuations at specific sites within a nucleic acid framework affects the assembly of ‘macromolecular machines,’ such as the DNA replication complex, and how these fluctuations drive the functional activity of these complexes. An important focus is to understand the local conformations and conformational fluctuations near single-stranded (ss) — double-stranded (ds) DNA forks and junctions, which are key sites for the assembly of proteins that manipulate DNA.

A schematic multi-dimensional free energy surface that governs the coupled assembly steps of the T4 helicase loading protein (gp59) to the replication fork junction, and the subsequent cooperative assembly of the ssb proteins (gp32) to the exposed template strands. Gp59 helicase loading protein (purple) binds weakly to the ss-ds junction of a replication fork, favoring disassociation from the DNA. Single strands near the fork junction are internally labeled with Cy3 dyes (red ellipsoids). Introduction of ssb proteins (yellow) leads to the cooperative assembly of the DNA-gp59-(gp32)n nucleoprotein complex. The hypothetical free energy surface describing the sugar-phosphate backbone fluctuations near the fork junction is shown representing each of the three conformational states: unbound DNA fork substrate that can fluctuate between “closed” and “open” backbone conformations at the fork junction (red curve), weakly bound DNA-gp59 helicase loading protein complex (blue curve), and strongly bound DNA-gp59-(gp32)n complex (green curve). Figure adapted from von Hippel et al. Biopolymers, 99, 923-954 (2013).

To determine the conformations of nucleic acids, and to monitor their time dependent behavior, we develop and apply both novel and well-established spectroscopic methods, which we use to study nucleic acid constructs that are site-specifically labeled with fluorescent optical probes. We carry out these experiments both at the ensemble and single-molecule levels.

The methods we develop for this purpose include i) microsecond-resolved single-molecule Förster resonance energy transfer (FRET) experiments on DNA constructs labeled with donor-acceptor cyanine dyes to study the conformation fluctuations of single-stranded DNA, and ii) circular dichroism (CD) and two-dimensional fluorescence spectroscopy (2DFS) of cyanine dimer probes, which are site-specifically positioned relative to the DNA fork junction. In the latter experiments, we use model DNA fork constructs in which the exciton-coupled cyanine dimers are rigidly attached within the sugar-phosphate backbones on opposing strands of the duplex DNA to monitor local backbone conformation fluctuations, both in the presence and absence of DNA associating proteins. To study the behavior of the DNA bases, we use DNA constructs in which fluorescent base analogs are substituted for canonical bases at specific sites. The above probe labeling strategies allow us to study the effects of local DNA backbone and base-stacking conformation fluctuations on the mechanisms of assembly and function of protein-DNA complexes.

We are studying the functional mechanisms of the T4 bacteriophage replication complex. The T4 replisome is a model system that is useful for understanding DNA replication in higher organisms since it contains the essential ‘core’ protein sub-assemblies necessary for replication, but it does not include the additional regulatory proteins present in the systems of higher organisms. The three protein sub-assemblies of the T4 replisome are i) a helicase complex that unwinds the DNA genome to expose the parental ssDNA templates; ii) the DNA polymerases that that use the ssDNA templates to synthesize complementary DNA daughter strands; and iii) the replication clamp-clamp loader complex that loads and unloads sliding clamps to and from the functioning polymerases to control the processivity of DNA synthesis.

Schematic views of the T4 bacteriophage DNA replication complex with and without its protein components. (A) An exploded two-dimensional view of the functional sub-assemblies of the DNA replication complex, specifically the polymerases, the helicase and the clamp-clamp loader sub-assemblies, together with representative bound ssDNA binding (ssb) proteins and cooperatively bound clusters. (B) The same schematic view of the DNA replication complex, but shown here with the proteins removed. This visualization highlights the replication fork and the primer-template (p/t) junctions within the overall replication complex to which replication proteins can bind. From Lee et al., Nucleic Acids Research, 44, 10691-10710 (2016).

During its activity, the replisome forms a quasi-stable complex with the genomic DNA at a replication fork junction, synthesizing new daughter strands at an average rate of 1 nucleotide per millisecond. SsDNA binding proteins (or ssbs) bind and unbind to the transiently exposed ssDNA templates, possibly to provide protection from nuclease activity and to help configure the templates into optimal conformations for replication by the DNA polymerases. The ssbs interact with many components of the replisome and repair machinery, and they may additionally serve to couple the functional activities of the three sub-assemblies.

By applying novel spectroscopic methods and probe-labeling strategies, we are gaining new insights about protein-DNA recognition, assembly and function. Our ensemble measurements of local DNA backbone and base-stacking conformations provide us with molecular level information about the structures and free energies of the biochemical intermediates along a reaction coordinate, while our single-molecule fluctuation measurements provide us with information about the transition states and free energy barriers that mediate the forward and backward transitions between reaction intermediates.