Probing the structure of RecA–DNA filaments. Advantages of a fluorescent guanine analog

Singleton, Scott F.; Roca, Alberto I.; Lee, Andrew M.; Xiao, Jie

doi:10.1016/j.tet.2006.10.092

Cited by 17 publications

(14 citation statements)

References 95 publications

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“…Furthermore, 3-MI has been evaluated for single-molecule detection purposes (Sanabia et al 2004). The other guanine analogue, 6-MI, has been used in recombination to study the mechanism of the RecA-mediated DNA strand exchange (Roca & Singleton, 2003; Xiao et al 2006) as well as in structural studies of the RecA-DNA filament (Singleton et al 2007). The adenine analogue 6-MAP, on the other hand, has been used in premelting transition studies of DNA A-tracts (Augustyn et al 2006) and as a probe for base flipping by DNA photolyase (Yang et al 2007).…”

Section: Fluorescent Base Analoguesmentioning

confidence: 99%

Fluorescent nucleic acid base analogues

Wilhelmsson

2010

Quart. Rev. Biophys.

260

220

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The use of fluorescent nucleic acid base analogues is becoming increasingly important in the fields of biology, biochemistry and biophysical chemistry as well as in the field of DNA nanotechnology. The advantage of being able to incorporate a fluorescent probe molecule close to the site of examination in the nucleic acid-containing system of interest with merely a minimal perturbation to the natural structure makes fluorescent base analogues highly attractive. In recent years, there has been a growing interest in developing novel candidates in this group of fluorophores for utilization in various investigations. This review describes the different classes of fluorophores that can be used for studying nucleic acid-containing systems, with an emphasis on choosing the right kind of probe for the system under investigation. It describes the characteristics of the large group of base analogues that has an emission that is sensitive to the surrounding microenvironment and gives examples of investigations in which this group of molecules has been used so far. Furthermore, the characterization and use of fluorescent base analogues that are virtually insensitive to changes in their microenvironment are described in detail. This group of base analogues can be used in several fluorescence investigations of nucleic acids, especially in fluorescence anisotropy and fluorescence resonance energy transfer (FRET) measurements. Finally, the development and characterization of the first nucleic base analogue FRET pair, tC(O)-tC(nitro), and its possible future uses are discussed.

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Section: Fluorescent Base Analoguesmentioning

confidence: 99%

Fluorescent nucleic acid base analogues

Wilhelmsson

2010

Quart. Rev. Biophys.

260

220

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“…Although RecA-like proteins bind DNA, they do not appear to bind short ssDNA tight enough. It is generally accepted that oligonucleotides shorter than 30 nucleotides do not fully activate the ATPase activity of RecA (Brenner et al, 1987;Bianco & Weinstock, 1996), which can be attributed to length-dependent affinity between RecA and ssDNA (Singleton et al, 2007). Similar length-dependence may also exist for dsDNA binding.…”

Section: Putative Advantage Of Atp Hydrolysis For Dna Strand Exchangementioning

confidence: 99%

Conservation of a conformational switch in RadA recombinase fromMethanococcus maripaludis

Yang

Luo

2009

Acta Crystallogr D Biol Crystallogr

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Archaeal RadAs are close homologues of eukaryal Rad51s ($40% sequence identity). These recombinases promote ATP hydrolysis and a hallmark strand-exchange reaction between homologous single-stranded and double-stranded DNA substrates. Pairing of the 3 0 -overhangs located at the damaged DNA with a homologous double-stranded DNA enables the re-synthesis of the damaged region using the homologous DNA as the template. In recent studies, conformational changes in the DNA-interacting regions of Methanococcus voltae RadA have been correlated with the presence of activity-stimulating potassium or calcium ions in the ATPase centre. The series of crystal structures of M. maripaludis RadA presented here further suggest the conservation of an allosteric switch in the ATPase centre which controls the conformational status of DNA-interacting loops. Structural comparison with the distant Escherichia coli RecA homologue supports the notion that the conserved Lys248 and Lys250 residues in RecA play a role similar to that of cations in RadA. The conservation of a cationic bridge between the DNAinteracting L2 region and the terminal phosphate of ATP, together with the apparent stability of the nucleoprotein filament, suggests a gap-displacement model which may explain the advantage of ATP hydrolysis for DNA-strand exchange.

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“…Although the fluorescence of the 5′-fluorescein-labeled 32mer serves as a sensitive indicator of RecA-DNA filament assembly, the mean signal for the positive control (500 μM ADP) was only 40% above signal for the background (absence of ADP). Hence, we elected to explore fluorescence anisotropy, which is known to distinguish the free and RecA-bound oligonucleotides [55,56] as an assay observable.…”

Section: Fluorescence Assay For Reca-dna Filament Assembly Using a Flmentioning

confidence: 99%

A complementary pair of rapid molecular screening assays for RecA activities

Lee¹,

Wigle²,

Singleton³

2007

Analytical Biochemistry

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The bacterial RecA protein has been implicated in the evolution of antibiotic resistance in pathogens, which is an escalating problem worldwide. The discovery of small molecules that can selectively modulate RecA's activities can be exploited to tease apart its roles in the de novo development and transmission of antibiotic resistance genes. Toward the goal of discovering small-molecule ligands that can prevent either the assembly of an active RecA-DNA filament or its subsequent ATP-dependent motor activities, we report the design and initial validation of a pair of rapid and robust screening assays suitable for the identification of inhibitors of RecA activities. One assay is based on established methods for monitoring ATPase enzyme activity and the second is a novel assay for RecA-DNA filament assembly using fluorescence polarization. Taken together, the assay results reveal complementary sets of agents that can either suppress selectively only the ATP-driven motor activities of the RecA-DNA filament or prevent assembly of active RecA-DNA filaments altogether. The screening assays can be readily configured for use in future automated high-throughput screening projects to discover potent inhibitors that may be developed into novel adjuvants for antibiotic chemotherapy that moderate the development and transmission of antibiotic resistance genes and increase the antibiotic therapeutic index.

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Probing the structure of RecA–DNA filaments. Advantages of a fluorescent guanine analog

Cited by 17 publications

References 95 publications

Fluorescent nucleic acid base analogues

Fluorescent nucleic acid base analogues

Conservation of a conformational switch in RadA recombinase fromMethanococcus maripaludis

A complementary pair of rapid molecular screening assays for RecA activities

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