Spectroscopic Studies of Position-specific DNA ‘Breathing’ Fluctuations at Replication Forks and Primer-Template Junctions

Abstract

The properties of single- (ss) and double-stranded (ds) DNA sequences at replication forks and primer-template DNA junctions are central to the function of the protein complexes that drive DNA replication, transcription, recombination and repair. Significant base-pair ‘breathing’ (or ‘fraying’) occurs at these ss-dsDNA junctions and the physiological consequences of this ‘breathing’ is not well understood, in part because it has been difficult to measure in a sequence-specific manner in biological systems. We have used single and dimer pairs of 2-aminopurine (2-AP) residues, site-specifically placed at various positions on both sides of the ss-dsDNA junctions of DNA constructs, as spectroscopic probes of this breathing. Replacing adenosine with 2-AP in DNA has minimal biological or physical consequences, and these moieties absorb, fluoresce and display CD spectra at wavelengths >300 nm where other nucleic acid and protein components are transparent. The optical properties of these constructs have been used to measure the position-specific extent and nature of the fluctuations of forked and P/T DNA junctions. We find that spectroscopically measurable melting penetrates ∼2 bps into the interior of the duplex region of these junctions under physiological conditions. In addition 2-AP bases in ssDNA loci directly adjacent to these junctions display significantly more unstacked character than do 2-AP probes located within long ssDNA sequences. Quenching of 2-AP fluorescence with acrylamide has independently confirmed these results. These local and transient DNA conformations have possible biological significance as interaction targets for DNA-manipulating enzymes, and we are using these breathing properties of DNA in isolation as a platform to study helicase activity on duplex DNA and ultimately the coupling of these helicases to the other components of the ‘macromolecular machines of gene expression’.