Dynamic Interactions between DNA and the T4 Single-Stranded Binding Protein gp32: Multi-Dimensional Correlation Analysis of Microsecond Single-Molecule FRET and Linear Dichroism Fluctuations

Abstract

Protein-nucleic acid interactions are of central importance in genome-regulatory processes. The DNA replication system of the T4 bacteriophage is an excellent model system to study DNA replication in higher organisms, since the T4-coded replication machinery utilizes many of the same components. Here we present studies of the kinetics of binding of gp32, the single-stranded (ss) DNA binding protein of the T4 system, whose roles include the functional integration of the other components of the T4 replication complex as well as protecting the transiently-exposed single-stranded DNA template sequences from DNA nucleases and melting out unfavorable secondary structures potentially formed by the lagging strand during DNA replication. We have performed single-molecule measurements of internally-labelled Cy3/Cy5 labeled primer-template DNA constructs in the presence of gp32 by simultaneously monitoring single-molecule Forster resonance energy transfer (smFRET) and single-molecule fluorescence-detected linear-dichroism (smFLD) on the microsecond time scale. smFRET measurements probe the distance between the fluorophores, while smFLD measurements are sensitive to local orientations of the Cy3-labeled sugar-phosphate backbones of the DNA construct. Multiple transient FRET states are observed when adding gp32 protein to tethered model replication fork DNA constructs, permitting us to track the dynamics of millisecond interconversions between various configurations of the gp32 / ssDNA system. The distribution of FRET states changes as a function of gp32 concentration, suggesting that we may be monitoring fluctuations that reflect changes associated with the progression from isolated to cooperative gp32 binding. We apply a multi-dimensional correlation function analysis to our microsecond-resolved smFRET and smLD data to reveal statistically relevant mechanistic information about the reaction pathways of the ssDNA-gp32 system at the replication fork.