Single-molecule FRET and linear dichroism studies of DNA breathing and helicase binding at replication fork junctions

Abstract

DNA "breathing" is a thermally driven process in which basepaired DNA sequences transiently adopt local conformations that depart from their most stable structures. Polymerases and other proteins of genome expression require access to single-stranded DNA coding templates located in the double-stranded DNA "interior," and it is likely that fluctuations of the sugar-phosphate backbones of dsDNA that result in mechanistically useful local base pair opening reactions can be exploited by such DNA regulatory proteins. Such motions are difficult to observe in bulk measurements, both because they are infrequent and because they often occur on microsecond time scales that are not easy to access experimentally. We report single-molecule fluorescence experiments with polarized light, in which tens-of-microseconds rotational motions of internally labeled iCy3/iCy5 donor-acceptor Förster resonance energy transfer fluorophore pairs that have been rigidly inserted into the backbones of replication fork constructs are simultaneously detected using single-molecule Förster resonance energy transfer and single-molecule fluorescence-detected linear dichroism signals. Our results reveal significant local motions in the ∼100-μs range, a reasonable time scale for DNA breathing fluctuations of potential relevance for DNA-protein interactions. Moreover, we show that both the magnitudes and the relaxation times of these backbone breathing fluctuations are significantly perturbed by interactions of the fork construct with a nonprocessive, weakly binding bacteriophage T4-coded helicase hexamer initiation complex, suggesting that these motions may play a fundamental role in the initial binding, assembly, and function of the processive helicase-primase (primosome) component of the bacteriophage T4-coded DNA replication complex.smFRET | single-molecule linear dichroism | thermal fluctuations | T4 primosome helicase | DNA helicase D NA "breathing" is defined as the transient opening, due to thermal fluctuations, of nucleic acid base pairs at experimental temperatures below the melting temperature of dsDNA duplex. Such fluctuations are thought to be important to the function of replication, transcription, recombination, and repair systems, which depend on the ability of the relevant protein complexes to gain access to ssDNA templates that are located in the dsDNA "interior" (1-3). Furthermore, the ability of the genome to spontaneously expose partial template sequences is a central feature of protein-DNA binding models in which open conformations serve as substrates that can be "trapped" by a functional protein complex. McConnell and von Hippel (4) suggested that breathing of duplex DNA might be structurally decomposed into a set of distinct breathing elements, each involving characteristic fluctuations of the sugar-phosphate backbone and the nucleobases. For example, a possible motion might involve compression along the double-helix axis (perhaps coupled with twisting or bending), leading to the disruption of WatsonCrick hydrogen bonds without...

Topological impact of noncanonical DNA structures on Klenow fragment of DNA polymerase

“…Based on the nearest neighbor model, stability of a duplex region of a hairpin is determined by the sequence and number of stacked base pairs (36,37). Terminal base pairs "breathe" and are easily dissociated (38). Therefore, KF may induce unzipping of a hairpin in a mechanism that does not depend on the overall free energy of the hairpin structure (Fig.…”

Watching DNA breath one molecule at a time

“…Nevertheless, it is now more and more appreciated that physical properties, such as DNA bending (4,5) and DNA breathing (6)(7)(8), play important roles in gene expression by affecting binding and assembly of molecular machineries on DNA. In PNAS, Phelps et al introduce to us a unique single-molecule approach to study DNA breathing dynamics and investigate the role of DNA breathing in protein assembly on DNA (9).…”

Breathing dynamics based parameter sensitivity analysis of hetero-polymeric DNA

“…It is also important to mention that in most natural DNA, the opening probability is much higher due to torsional stress 7,8 . The breathing dynamics of bubble a) Electronic mail: rmetzler@uni-potsdam.de b) Electronic mail: skbanik@jcbose.ac.in c) Electronic mail: pinakc@rediffmail.com: Corresponding author plays a crucial role in the functioning of DNA [9][10][11][12][13][14][15][16][17] . Fundamental biological processes like replication and transcription largely rely on the local denaturation.…”

“…Using single molecule florescence correlation spectroscopy, breathing dynamics has been monitored and multistep relaxation kinetics with characteristic time scale has been accounted from the study 21,22 . Experimental studies have also been performed to explore correlations between the dynamics of hetero-polymeric DNA with the biological activities of nucleic acid enzymes 17 . Theoretical models of the dynamics have also been established based on the DNA free energy landscape [23][24][25][26][27] .…”