Investigating local DNA conformational trapping dynamics during DNA polymerase holoenzyme assembly and exonuclease proof-reading activity.

Abstract

The sugar-phosphate backbones of DNA continuously undergo thermally-activated conformational fluctuations at equilibrium, which directly affect base-pair unstacking and hydrogen bond breaking. The influence of these motions on biomolecular function are particularly important at single-stranded (ss)—double-stranded (ds) DNA forks and primer-template (p/t) junctions. At these junctions, non-canonical structures that deviate from the Watson-Crick B-form DNA duplex may serve as recognition sites for protein binding, and the kinetics of these interactions likely play a central regulatory role for fundamental biological processes such as DNA replication and repair. The DNA polymerase/sliding clamp holoenzyme is the central protein complex that carries out the process of complementary nucleotide addition to nascent ssDNA template strands initiated at p/t junctions. In addition to playing a role in protein assembly, local DNA conformations resulting from nucleotide misincorporation regulate the exonuclease proof-reading activity of DNA polymerase. To study the details of these processive local DNA conformational dynamics, our group has developed a molecular probing method that relies on the rigid insertion of two fluorescent cyanine (Cy3) probes at opposite positions within the sugar-phosphate backbones of complementary DNA strands. Electrostatic coupling between probes results in delocalized optical properties that sensitively depend on relative probe orientations. Utilizing a series of complementary ensemble-based (i.e., fluorescence, absorbance, circular dichroism) and single-molecule spectroscopic methods, we study the local DNA backbone conformational dynamics as they relate to protein binding and activity. In this presentation, I will discuss recent studies of DNA polymerase holoenzyme assembly and exonuclease proofreading activity at p/t junctions. Specifically, results from microsecond-resolved polarization-sweep single-molecule spectroscopy (PS-SMS) demonstrate how protein-dependent conformational trapping of otherwise sparsely populated transient DNA local conformations is involved in processive holoenzyme formation.