Carey Phelps

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

Significance Unique single-molecule fluorescence techniques were used to monitor DNA “breathing” at and near the junctions of model DNA replication forks on biologically relevant microsecond-to-millisecond time scales. Experiments performed in the absence and presence of helicase complexes addressed the role of these fluctuations in helicase function during DNA replication. These studies simultaneously monitored single-molecule Förster resonance energy transfer and single-molecule fluorescence linear dichroism of “internal” Cy3/Cy5 labels placed rigidly into the DNA backbones at positions near the fork junction. Our results showed significant breathing at the fork junction that was greatly augmented by the presence of weakly bound helicase, followed by still larger fluctuations and strand separation after full duplex DNA unwinding by the complete tightly bound and processive helicase complex. DNA “breathing” is a thermally driven process in which base-paired 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.

Using microsecond single-molecule FRET to determine the assembly pathways of T4 ssDNA binding protein onto model DNA replication forks

Significance A microsecond-resolved single-molecule FRET method was used to monitor the binding and unbinding of the ssDNA binding protein (gene product 32) of the T4 bacteriophage replication complex to biologically relevant primer-template DNA constructs. A unique multitime correlation function analysis was applied to the resulting sparse data, which permitted the investigation of the kinetics and mechanisms of noncooperative and cooperative protein binding, unbinding, and “sliding.” Our results indicate that noncooperatively bound monomer proteins dissociate on the timescale of tens of milliseconds, which is consistent with the known rate of nucleotide addition during DNA replication. The rapid dissociation of the monomer suggests that sliding is a much more likely mechanism for translocation of cooperatively bound clusters of indeterminate size. DNA replication is a core biological process that occurs in prokaryotic cells at high speeds (∼1 nucleotide residue added per millisecond) and with high fidelity (fewer than one misincorporation event per 107 nucleotide additions). The ssDNA binding protein [gene product 32 (gp32)] of the T4 bacteriophage is a central integrating component of the replication complex that must continuously bind to and unbind from transiently exposed template strands during DNA synthesis. We here report microsecond single-molecule FRET (smFRET) measurements on Cy3/Cy5-labeled primer-template (p/t) DNA constructs in the presence of gp32. These measurements probe the distance between Cy3/Cy5 fluorophores that label the ends of a short (15-nt) segment of ssDNA attached to a model p/t DNA construct and permit us to track the stochastic interconversion between various protein bound and unbound states. The length of the 15-nt ssDNA lattice is sufficient to accommodate up to two cooperatively bound gp32 proteins in either of two positions. We apply a unique multipoint time correlation function analysis to the microsecond-resolved smFRET data obtained to determine and compare the kinetics of various possible reaction pathways for the assembly of cooperatively bound gp32 protein onto ssDNA sequences located at the replication fork. The results of our analysis reveal the presence and translocation mechanisms of short-lived intermediate bound states that are likely to play a critical role in the assembly mechanisms of ssDNA binding proteins at replication forks and other ss duplex junctions.

Persistence of trions and quenching of excitons in optically induced two-dimensional electron gases in mixed-type GaAs/AlAs quantum wells

We report experimental studies of absorption spectra of two-dimensional electron gases (2DEGs) in a GaAs/AlAs mixed-type quantum well (MTQW) structure. The 2DEGs are induced and the corresponding Landau level filling factor is controlled through optical excitations of the narrow well in the MTQW. In an external magnetic field normal to the quantum well plane, the absorption spectra of the 2DEGs are characterized by both exciton and trion resonances. As the filling factor approaches 1, exciton resonances are quenched, while a strong trion resonance persists. These properties make 2DEGs in MTQWs a promising ensemble spin system for pursuing electromagnetically induced transparency and related applications in semiconductors.