Editorial

FRET tells us about proximities, distances, orientations and dynamic properties

In this short note we describe the conversion of the widely used fluorescence quenching azo-dyes DABCYL and HABA to fluorophores. The dyes were conjugated to the proteins RNase and human serum albumin (HSA) and subsequently reduced using sodium dithionite (Na₂S₂O₄), thus forming amine-containing fluorophores. Since this chemical reaction can be applied to any azo-containing quencher compound, a great variety of substances can be readily obtained synthetically. This approach provides a promising tool in the use of fluorescence-based investigations of biomolecular interactions.

The RAD51 DNA strand exchange protein plays an important role in maintaining the integrity of the human genome. It promotes homology-directed DNA repair by exchanging strands between the damaged and the intact DNA molecules. It also plays an important role in stabilizing distressed DNA replication forks. When overexpressed or misregulated, however, RAD51 contributes to “rogue,” genome destabilizing events that can lead to cancer, cell death, and to acquisition of chemotherapy resistance by cancerous cells. Human RAD51 is, therefore, an important and highly coveted anticancer drug target. Biochemical, biophysical, and structural studies of the human RAD51 and establishment of its structure–activity relationship require purification of large quantities of protein. In this chapter we describe a robust method for expression and purification of human RAD51 and the methods for assessing its activity based on the single-strand DNA-binding stoichiometry and its capacity to carry out the DNA strand exchange reaction.

The indodicarbocyanine fluorophores Cy3 and Cy5 are extensively used as donor-acceptor pairs in fluorescence resonance energy transfer experiments, especially those involving single molecules. When terminally attached to double-stranded nucleic acids via the 5′ phosphate group these fluorophores stack onto the ends of the molecule. Knowledge of the positions of the fluorophores is critical to the interpretation of fluorescence resonance energy transfer data. The positions have been demonstrated for double-stranded (ds) DNA using NMR spectroscopy. Here, we have used x-ray crystallography to analyze the location of Cy3 and Cy5 on dsRNA, using complexes of an RNA stem-loop bound to L5 protein determined at 2.4 Å resolution. This confirms the tendency of both fluorophores to stack on the free end of RNA, with the long axis of the fluorophores approximately parallel to that of the terminal basepair. However, the manner of interaction of both Cy3 and Cy5 with the terminus of the dsRNA is significantly different from that deduced for dsDNA using NMR. The fluorophores are stacked on the terminal basepair such that their indole nitrogen atoms lie on the major groove side, and thus their pendant methyl groups are on the minor groove side.

XPD-like helicases constitute a prominent DNA helicase family critical for many aspects of genome maintenance. These enzymes share a unique structural feature, an auxiliary domain stabilized by an iron-sulphur (FeS) cluster, and a 5′–3′ polarity of DNA translocation and duplex unwinding. Biochemical analyses alongside two single-molecule approaches, total internal reflection fluorescence microscopy and high-resolution optical tweezers, have shown how the unique structural features of XPD helicase and its specific patterns of substrate interactions tune the helicase for its specific cellular function and shape its molecular mechanism. The FeS domain forms a duplex separation wedge and contributes to an extended DNA binding site. Interactions within this site position the helicase in an orientation to unwind the duplex, control the helicase rate, and verify the integrity of the translocating strand. Consistent with its cellular role, processivity of XPD is limited and is defined by an idiosyncratic stepping kinetics. DNA duplex separation occurs in single base pair steps punctuated by frequent backward steps and conformational rearrangements of the protein–DNA complex. As such, the helicase in isolation mainly stabilizes spontaneous base pair opening and exhibits a limited ability to unwind stable DNA duplexes. The presence of a cognate ssDNA binding protein converts XPD into a vigorous helicase by destabilizing the upstream dsDNA as well as by trapping the unwound strands. Remarkably, the two proteins can co-exist on the same DNA strand without competing for binding. The current model of the XPD unwinding mechanism will be discussed along with possible modifications to this mechanism by the helicase interacting partners and unique features of such bio-medically important XPD-like helicases as FANCJ (BACH1), RTEL1 and CHLR1 (DDX11).

Fluorescence resonance energy transfer (FRET) is an important source of long-range distance information in macromolecules. However, extracting maximum information requires knowledge of fluorophore, donor and acceptor, positions on the macromolecule. We previously determined the structure of the indocarbocyanine fluorophores Cy3 and Cy5 attached to DNA via three-carbon atom tethers, showing that they stacked onto the end of the helix in a manner similar to an additional basepair. Our recent FRET study has suggested that when they are attached via a longer 13-atom tether, these fluorophores are repositioned relative to the terminal basepair by a rotation of ∼30°, while remaining stacked. In this study, we have used NMR to extend our structural understanding to the commonly used fluorophore sulfoindocarbocyanine-3 (sCy3) attached to the 5′-terminus of the double-helical DNA via a 13-atom flexible tether (L13). We find that L13-sCy3 remains predominantly stacked onto the end of the duplex, but adopts a significantly different conformation, from that of either Cy3 or Cy5 attached by 3-atom tethers, with the long axes of the fluorophore and the terminal basepair approximately parallel. This result is in close agreement with our FRET data, supporting the contention that FRET data can be used to provide orientational information.

Cyanine fluorophores are commonly used in single-molecule FRET experiments with nucleic acids. We have previously shown that indocarbocyanine fluorophores attached to the 5′-termini of DNA and RNA via three-carbon atom linkers stack on the ends of the helix, orienting their transition moments. We now investigate the orientation of sulfoindocarbocyanine fluorophores tethered to the 5′-termini of DNA via 13-atom linkers. Fluorescence lifetime measurements of sulfoindocarbocyanine 3 attached to double-stranded DNA indicate that the fluorophore is extensively stacked onto the terminal basepair at 15°C, with properties that depend on the terminal sequence. In single molecules of duplex DNA, FRET efficiency between sulfoindocarbocyanine 3 and 5 attached in this manner is modulated with helix length, indicative of fluorophore orientation and consistent with stacked fluorophores that can undergo lateral motion. We conclude that terminal stacking is an intrinsic property of the cyanine fluorophores irrespective of the length of the tether and the presence or absence of sulfonyl groups. However, compared to short-tether indocarbocyanine, the mean rotational relationship between the two fluorophores is changed by ∼60° for the long-tether sulfoindocarbocyanine fluorophores. This is consistent with the transition moments becoming approximately aligned with the long axis of the terminal basepair for the long-linker species.