Andrew Marcus

 

Protein-nucleic acid interactions are of central importance in genome-regulatory processes. The DNA replication system of the T4 bacteriophage of E. coli is an excellent model system to study DNA replication in higher organisms, since the T4-coded replication machinery utilizes many of the same functional sub-assemblies. In this talk, I will present studies of the kinetics of binding of gp32, the single-stranded DNA binding (ssb) protein of the T4 system, whose roles include the functional integration of the other components of the T4 replication complex, the protection of transiently-exposed single-stranded DNA template sequences from DNA nucleases, and the elimination of unfavorable secondary structures formed on the lagging strand during DNA replication. We have performed single-molecule fluorescence measurements of Cy3/Cy5 labeled primer-template (p/t) DNA constructs in the presence of gp32 by monitoring single-molecule Förster resonance energy transfer (smFRET) on the microsecond time scale. smFRET measurements probe the distance between Cy3/Cy5 fluorophores that are incorporated into the sugar-phosphate backbones of the DNA constructs. Multiple transient FRET states are observed when adding gp32 protein to tethered model replication fork DNA constructs, permitting us to track the sub-millisecond inter-conversions between various configurations of the gp32 / ssDNA system. We apply a multi-point time correlation function analysis to our microsecond-resolved smFRET data to obtain information about the assembly pathways of the ssDNA-gp32 system at the replication fork. Our results provide a broadened understanding of the assembly dynamics of cooperatively bound clusters of gp32 molecules on the ssDNA ‘arms’ of model replication constructs as a function of ssDNA length, strand polarity, and protein interactions with the p/t DNA junction. This information, in turn, provides fundamental insight into the detailed molecular mechanisms that underlie and integrate the functions of DNA replication complexes.