Electronic cameras for low-light microscopy.

This chapter introduces to electronic cameras, discusses the various parameters considered for evaluating their performance, and describes some of the key features of different camera formats. The chapter also presents the basic understanding of functioning of the electronic cameras and how these properties can be exploited to optimize image quality under low-light conditions. Although there are many types of cameras available for microscopy, the most reliable type is the charge-coupled device (CCD) camera, which remains preferred for high-performance systems. If time resolution and frame rate are of no concern, slow-scan CCDs certainly offer the best available performance, both in terms of the signal-to-noise ratio and their spatial resolution. Slow-scan cameras are thus the first choice for experiments using fixed specimens such as measurements using immune fluorescence and fluorescence in situ hybridization. However, if video rate imaging is required, one need not evaluate slow-scan CCD cameras. A very basic video CCD may suffice if samples are heavily labeled or are not perturbed by high intensity illumination. When video rate imaging is required for very dim specimens, the electron multiplying CCD camera is probably the most appropriate at this technological stage. Intensified CCDs provide a unique tool for applications in which high-speed gating is required. The variable integration time video cameras are very attractive options if one needs to acquire images at video rate acquisition, as well as with longer integration times for less bright samples. This flexibility can facilitate many diverse applications with highly varied light levels.

Single-molecule FRET analysis of DNA binding and bending by yeast HMGB protein Nhp6A.

High-mobility group B (HMGB) proteins bind duplex DNA without sequence specificity, facilitating the formation of compact nucleoprotein structures by increasing the apparent flexibility of DNA through the introduction of DNA kinks. It has remained unclear whether HMGB binding and DNA kinking are simultaneous and whether the induced kink is rigid (static) or flexible. The detailed molecular mechanism of HMGB-induced DNA 'softening' is explored here by single-molecule fluorescence resonance energy transfer studies of single yeast Nhp6A (yNhp6A) proteins binding to short DNA duplexes. We show that the local effect of yNhp6A protein binding to DNA is consistent with formation of a single static kink that is short lived (lifetimes of a few seconds) under physiological buffer conditions. Within the time resolution of our experiments, this static kink occurs at the instant the protein binds to the DNA, and the DNA straightens at the instant the protein dissociates from the DNA. Our observations support a model in which HMGB proteins soften DNA through random dynamic binding and dissociation, accompanied by DNA kinking and straightening, respectively.

Conformational trapping of mismatch recognition complex MSH2/MSH3 on repair-resistant DNA loops.

Insertion and deletion of small heteroduplex loops are common mutations in DNA, but why some loops are prone to mutation and others are efficiently repaired is unknown. Here we report that the mismatch recognition complex, MSH2/MSH3, discriminates between a repair-competent and a repair-resistant loop by sensing the conformational dynamics of their junctions. MSH2/MSH3 binds, bends, and dissociates from repair-competent loops to signal downstream repair. Repair-resistant Cytosine-Adenine-Guanine (CAG) loops adopt a unique DNA junction that traps nucleotide-bound MSH2/MSH3, and inhibits its dissociation from the DNA. We envision that junction dynamics is an active participant and a conformational regulator of repair signaling, and governs whether a loop is removed by MSH2/MSH3 or escapes to become a precursor for mutation.

Branch migration enzyme as a Brownian ratchet.

In recent years, it has been shown that helicases are able to perform functions beyond their traditional role in unwinding of double-stranded nucleic acids; yet the mechanistic aspects of these different activities are not clear. Our kinetic studies of Holliday junction branch migration catalysed by a ring-shaped helicase, T7 gp4, show that heterology of as little as a single base stalls catalysed branch migration. Using single-molecule analysis, one can locate the stall position to within a few base pairs of the heterology. Our data indicate that the presence of helicase alone promotes junction unfolding, which accelerates spontaneous branch migration, and individual time traces reveal complex trajectories consistent with random excursions of the branch point. Our results suggest that instead of actively unwinding base pairs as previously thought, the helicase exploits the spontaneous random walk of the junction and acts as a Brownian ratchet, which walks along duplex DNA while facilitating and biasing branch migration in a specific direction.

Dissecting metal ion-dependent folding and catalysis of a single DNAzyme.

Protein metalloenzymes use various modes for functions for which metal-dependent global conformational change is required in some cases but not in others. In contrast, most ribozymes require a global folding that almost always precedes enzyme reactions. Herein we studied metal-dependent folding and cleavage activity of the 8-17 DNAzyme using single-molecule fluorescence resonance energy transfer. Addition of Zn2+ and Mg2+ induced folding of the DNAzyme into a more compact structure followed by a cleavage reaction, which suggests that the DNAzyme may require metal-dependent global folding for activation. In the presence of Pb2+, however, the cleavage reaction occurred without a precedent folding step, which suggests that the DNAzyme may be prearranged to accept Pb2+ for the activity. Neither ligation reaction of the cleaved substrates nor dynamic changes between folded and unfolded states was observed. These features may contribute to the unusually fast Pb2+-dependent reaction of the DNAzyme. These results suggest that DNAzymes can use all modes of activation that metalloproteins use.

Nonblinking and long-lasting single-molecule fluorescence imaging.

Photobleaching and blinking of fluorophores pose fundamental limitations on the information content of single-molecule fluorescence measurements. Photoinduced blinking of Cy5 has hampered many previous investigations using this popular fluorophore. Here we show that Trolox in combination with the enzymatic oxygen-scavenging system eliminates Cy5 blinking, dramatically reduces photobleaching and improves the signal linearity at high excitation rates, significantly extending the applicability of single-molecule fluorescence techniques.

Real-time observation of RecA filament dynamics with single monomer resolution.

RecA and its homologs help maintain genomic integrity through recombination. Using single-molecule fluorescence assays and hidden Markov modeling, we show the most direct evidence that a RecA filament grows and shrinks primarily one monomer at a time and only at the extremities. Both ends grow and shrink, contrary to expectation, but a higher binding rate at one end is responsible for directional filament growth. Quantitative rate determination also provides insights into how RecA might control DNA accessibility in vivo. We find that about five monomers are sufficient for filament nucleation. Although ordinarily single-stranded DNA binding protein (SSB) prevents filament nucleation, single RecA monomers can easily be added to an existing filament and displace SSB from DNA at the rate of filament extension. This supports the proposal for a passive role of RecA-loading machineries in SSB removal.

Unraveling helicase mechanisms one molecule at a time.

Recent years have seen an increasing number of biological applications of single molecule techniques, evolving from a proof of principle type to the more sophisticated studies. Here we compare the capabilities and limitations of different single molecule techniques in studying the activities of helicases. Helicases share a common catalytic activity but present a high variability in kinetic and phenomenological behavior, making their studies ideal in exemplifying the use of the new single molecule techniques to answer biological questions. Unexpected phenomena have also been observed from individual molecules suggesting extended or alternative functionality of helicases in vivo.

Repetitive shuttling of a motor protein on DNA.

Many helicases modulate recombination, an essential process that needs to be tightly controlled. Mutations in some human disease helicases cause increased recombination, genome instability and cancer. To elucidate the potential mode of action of these enzymes, here we developed a single-molecule fluorescence assay that can visualize DNA binding and translocation of Escherichia coli Rep, a superfamily 1 DNA helicase homologous to Saccharomyces cerevisiae Srs2. Individual Rep monomers were observed to move on single-stranded (ss)DNA in the 3' to 5' direction using ATP hydrolysis. Strikingly, on hitting a blockade, such as duplex DNA or streptavidin, the protein abruptly snapped back close to its initial position, followed by further cycles of translocation and snapback. This repetitive shuttling is likely to be caused by a blockade-induced protein conformational change that enhances DNA affinity for the protein's secondary DNA binding site, thereby resulting in a transient DNA loop. Repetitive shuttling was also observed on ssDNA bounded by a stalled replication fork and an Okazaki fragment analogue, and the presence of Rep delayed formation of a filament of recombination protein RecA on ssDNA. Thus, the binding of a single Rep monomer to a stalled replication fork can lead to repetitive shuttling along the single-stranded region, possibly keeping the DNA clear of toxic recombination intermediates.

Surfaces and orientations: much to FRET about?

Single molecule FRET (fluorescence resonance energy transfer) is a powerful technique for detecting real-time conformational changes and molecular interactions during biological reactions. In this Account, we examine different techniques of extending observation times via immobilization and illustrate how useful biological information can be obtained from single molecule FRET time trajectories with or without absolute distance information.

Probing single-stranded DNA conformational flexibility using fluorescence spectroscopy.

Single-stranded DNA (ssDNA) is an essential intermediate in various DNA metabolic processes and interacts with a large number of proteins. Due to its flexibility, the conformations of ssDNA in solution can only be described using statistical approaches, such as flexibly jointed or worm-like chain models. However, there is limited data available to assess such models quantitatively, especially for describing the flexibility of short ssDNA and RNA. To address this issue, we performed FRET studies of a series of oligodeoxythymidylates, (dT)N, over a wide range of salt concentrations and chain lengths (10 < or = N < or = 70 nucleotides), which provide systematic constraints for testing theoretical models. Unlike in mechanical studies where available ssDNA conformations are averaged out during the time it takes to perform measurements, fluorescence lifetimes may act here as an internal clock that influences fluorescence signals depending on how fast the ssDNA conformations fluctuate. A reasonably good agreement could be obtained between our data and the worm-like chain model provided that limited relaxations of the ssDNA conformations occur within the fluorescence lifetime of the donor. The persistence length thus estimated ranges from 1.5 nm in 2 M NaCl to 3 nm in 25 mM NaCl.

DNA-binding orientation and domain conformation of the E. coli rep helicase monomer bound to a partial duplex junction: single-molecule studies of fluorescently labeled enzymes.

The SF1 DNA helicases are multi-domain proteins that can unwind duplex DNA in reactions that are coupled to ATP binding and hydrolysis. Crystal structures of two such helicases, Escherichia coli Rep and Bacillus stearothermophilus PcrA, show that the 2B sub-domain of these proteins can be found in dramatically different orientations (closed versus open) with respect to the remainder of the protein, suggesting that the 2B domain is highly flexible. By systematically using fluorescence resonance energy transfer at the single-molecule level, we have determined both the orientation of an E.coli Rep monomer bound to a 3'-single-stranded-double-stranded (ss/ds) DNA junction in solution, as well as the relative orientation of its 2B sub-domain. To accomplish this, we developed a highly efficient procedure for site-specific fluorescence labeling of Rep and a bio-friendly immobilization scheme, which preserves its activities. Both ensemble and single-molecule experiments were carried out, although the single-molecule experiments proved to be essential here in providing quantitative distance information that could not be obtained by steady-state ensemble measurements. Using distance-constrained triangulation procedures we demonstrate that in solution the 2B sub-domain of a Rep monomer is primarily in the "closed" conformation when bound to a 3'-ss/ds DNA, similar to the orientation observed in the complex of PcrA bound to a 3'-ss/ds DNA. Previous biochemical studies have shown that a Rep monomer bound to such a 3'-ss/ds DNA substrate is unable to unwind the DNA and that a Rep oligomer is required for helicase activity. Therefore, the closed form of Rep bound to a partial duplex DNA appears to be an inhibited form of the enzyme.

Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase.

Helicases are motor proteins that couple conformational changes induced by ATP binding and hydrolysis with unwinding of duplex nucleic acid, and are involved in several human diseases. Some function as hexameric rings, but the functional form of non-hexameric helicases has been debated. Here we use a combination of a surface immobilization scheme and single-molecule fluorescence assays--which do not interfere with biological activity--to probe DNA unwinding by the Escherichia coli Rep helicase. Our studies indicate that a Rep monomer uses ATP hydrolysis to move toward the junction between single-stranded and double-stranded DNA but then displays conformational fluctuations that do not lead to DNA unwinding. DNA unwinding initiates only if a functional helicase is formed via additional protein binding. Partial dissociation of the functional complex during unwinding results in interruptions ('stalls') that lead either to duplex rewinding upon complete dissociation of the complex, or to re-initiation of unwinding upon re-formation of the functional helicase. These results suggest that the low unwinding processivity observed in vitro for Rep is due to the relative instability of the functional complex. We expect that these techniques will be useful for dynamic studies of other helicases and protein-DNA interactions.