Spectroscopic studies of position-specific DNA “breathing” fluctuations at replication forks and primer-template junctions

Abstract

Junctions between ssDNA and dsDNA sequences are important in many cellular processes, including DNA replication, transcription, recombination, and repair. Significant transient conformational fluctuations (''DNA breathing'') can occur at these ssDNA-dsDNA junctions. The involvement of such breathing in the mechanisms of macromolecular complexes that operate at these loci is not well understood, in part because these fluctuations have been difficult to measure in a position-specific manner. To address this issue we constructed forked or primer-template DNA constructs with 1 or 2 adjacent 2-aminopurine (2-AP) nucleotide residues (adenine analogues) placed at specific positions on both sides of the ssDNAdsDNA junction. Unlike canonical DNA bases, 2-AP absorbs, fluoresces, and displays CD spectra at wavelengths >300 nm, where other nucleic acid and protein components are transparent. We used CD and fluorescence spectra and acrylamide quenching of these probes to monitor the extent and nature of DNA breathing of A-T base pairs at specific positions around the ssDNA-dsDNA junction. As expected, spectroscopically measurable unwinding penetrates Ϸ2 bp into the duplex region of these junctions under physiological conditions for the constructs examined. Surprisingly, we found that 2-AP bases at ssDNA sites directly adjacent to ssDNA-dsDNA junctions are significantly more unstacked than those at more distant ssDNA positions. These local and transient DNA conformations on both sides of ssDNA-dsDNA junctions may serve as specific interaction targets for enzymes that manipulate DNA in the processes of gene expression.2-aminopurine ͉ circular dichroism ͉ DNA unwinding ͉ fluorescence ͉ forked DNA D ouble-stranded DNA molecules are stabilized by a network of interstrand hydrogen bonds between complementary A-T and G-C base pairs and by intrastrand base stacking. These DNA duplexes exist primarily in the Watson-Crick B-form conformation in aqueous solution at physiological temperature and salt concentration. Heating dsDNA induces a cooperative transition to singlestranded components; the midpoint of this transition is defined as the melting temperature (T m ), which depends on base composition, base sequence, and solvent conditions. dsDNA molecules also experience small thermal ''breathing'' fluctuations that transiently break hydrogen bonds and unstack bases at temperatures well below T m . These fluctuations may play a role in the initial exposure of specific ssDNA binding targets that are otherwise buried in the duplex DNA (promoters, recombination or repair sites, etc.) to various regulatory protein complexes. However, this ''interior DNA breathing'' involves a very small fraction (Ϸ10 Ϫ6 ) of the total DNA at any one time and consequently cannot be observed by normal spectroscopic techniques. Methods such as hydrogen exchange (1-3) or chemical probes that interact irreversibly with and trap ssDNA sequences (for example, formaldehyde or dimethyl sulfate (4) can be used to monitor this interior breathing by accumulating the si...

Local Conformations and Competitive Binding Affinities of Single- and Double-stranded Primer-Template DNA at the Polymerization and Editing Active Sites of DNA Polymerases

“…In contrast, the free 2mm construct (two mismatches at the primer terminus directly adjacent to the P/T junction) exhibited a dramatic increase in CD intensity at 330 nm. This observed increase in CD amplitude most likely reflects the constrained conformation of an unpaired 2-AP dimer probe located directly at the P/T junction that we have demonstrated previously (33) for bases located directly vicinal to a replication fork. The above results also suggest that although these probes were only partially (the 1mm construct) or not (the 3mm and 4mm constructs) base-paired with the template strand, the terminal 2-AP dimer retained sufficient B-form character to display exciton character, as observed also for ssP16-1.…”

“…The free construct exhibited a characteristic excitoncoupled low energy CD spectrum, suggesting that the probecontaining base pairs were in a B-form DNA conformation. We note that this exciton coupling appears much larger than that seen with the 0mm-A or P14-1 constructs, perhaps reflecting some "end fraying" in these latter constructs in which the 2-AP probes are closer to or at the P/T junction (33).…”

Fifty years of DNA “Breathing”: Reflections on old and new approaches

“…The details, at the equilibrium and steady state ’breathing’ level, are presented elsewhere 38,45 , 48 . Here we briefly summarize our findings and conclusions.…”

Coupling Translocation with Nucleic Acid Unwinding by NS3 Helicase

“…The preferential association may come from interactions of the duplex backbone with the helicase as suggested for PcrA 25,30 and may also be contributed by partial base unstacking at the junction. 47 The junction stabilization reduces the dissociation rate of the helicase during unwinding but does not affect the translocation velocity much. Nevertheless, both the base pair-unwinding and the junction-stabilization effects arise from the helicase interacting with the duplex.…”

“…Interestingly, recent experimental studies indicate that bases immediately adjacent to the junction are less stacked than ssDNA bases elsewhere. 47 Hence, it is also possible that the partial unstacking of the bases at the junction contributes to the preferential helicase-duplex association.…”