Structure of DNA-RecA complexes studied by residue differential linear dichroism and fluorescence spectroscopy for a genetically engineered RecA protein

Hagmar, Per; Nordén, Bengt; Baty, Daniel; Chartier, Martine; Takahashi, Masayuki

doi:10.1016/0022-2836(92)91061-s

Cited by 30 publications

(27 citation statements)

References 21 publications

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“…These spectra of the tryptophan residues are obviously very similar to each other. Furthermore, the Trp-290 spectrum obtained here is very similar to that obtained by another modified RecA protein and method (20). These results support our strategy for determining SSLD spectra.…”

Section: Ld Spectra Of Reca Protein Complexes With Poly(da) and Ssldsupporting

confidence: 75%

“…For the case of tryptophan substitution in the RecA protein, this chromophore is responsible for about 25% of the total protein absorption at 278 nm. Therefore, a rough estimate of the orientation factor S, such as from small-angle neutron scattering measurements (20), is enough for normalizing the LD spectrum to sufficient accuracy. However, for the SSLD of RecA's tyrosine residues, a more precise normalization is required because each tyrosine residue contributes only some 7% of the total protein absorption.…”

Section: Discussionmentioning

confidence: 99%

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Arrangement of RecA protein in its active filament determined by polarized-light spectroscopy

Morimatsu

Takahashi

Nordén

2002

Proc. Natl. Acad. Sci. U.S.A.

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Linear dichroism (LD) polarized-light spectroscopy is used to determine the arrangement of RecA in its large filamentous complex with DNA, active in homologous recombination. Angular orientation data for two tryptophan and seven tyrosine residues, deduced from differential LD of wild-type RecA vs. mutants that were engineered to attenuate the UV absorption of selected residues, revealed a rotation by some 40°of the RecA subunits relative to the arrangement in crystal without DNA. In addition, conformational changes are observed for tyrosine residues assigned to be involved in DNA binding and in RecA-RecA contacts, thus potentially related to the global structure of the filament and its biological function. The presented spectroscopic approach, called ''Site-Specific Linear Dichroism'' (SSLD), may find forceful applications also to other biologically important fibrous complexes not amenable to x-ray crystallographic or NMR structural analysis. Many proteins, like RecA, actin, etc., and also prions, display biological activities related to their ability to assemble into large filamentous structures. Whereas such assemblies are generally not amenable to structure analysis by x-ray crystallography or NMR, linear dichroism (LD), i.e., anisotropy to absorption of polarized light, may often provide valuable structural information about their internal organization (1). The basic principle is that absorption is maximum when the light polarization is parallel to the transition moment, i.e., the molecular ''antenna'' responsible for the interaction with the radiation field. Measurement of LD, defined as the absorption differential between orthogonal polarizations, can thus provide information about the angles between the respective transition moments and the orientation direction of the sample; a prerequisite is that the latter is macroscopically oriented. Knowledge about how the transition moments are directed within the molecular framework of a chromophoric residue, for example, obtained from quantum chemical calculations or experiments on small molecules (2) then allows conclusions about the orientation of the particular molecular residue in the structure. LD measured on flow-oriented DNA solutions has been used for a long time to determine binding angles for small ligand molecules, a measurement that may generally quickly discriminate between different binding modes, such as groove binding or intercalation (1). Also, average tilt and roll angles of nucleobases, electrophoretic orientation and flexibility of DNA helices have been studied (3). In a few cases, detailed binding geometries have been possible to deduce, including small angular distortions in diastereomeric complexes (4). From such directional information (i.e., in principle, two angular coordinates for each chromophore), a three-dimensional structure should be possible to determine by assistance from molecular modeling. Here, we present a study in which the RecA protein of Escherichia coli has been systematically modified to allow determination by LD of the ...

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Section: Ld Spectra Of Reca Protein Complexes With Poly(da) and Ssldsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Arrangement of RecA protein in its active filament determined by polarized-light spectroscopy

Morimatsu

Takahashi

Nordén

2002

Proc. Natl. Acad. Sci. U.S.A.

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“…The orientation created a well-resolved SANS pattern, where the helical diffraction cross provided exact information about the helix pitch and, most importantly, yielded the flow orientation distribution, making it possible to translate the LD signals into average base-plane orientations (Hagmar et al 1992;Nordén et al 1992). Electron microscopy had revealed that the DNA was extended by approximately a factor of 1·5 (Stasiak & Di Capua, 1982;Stasiak et al 1981), but surprisingly the base orientation concluded from LD did not show any inclination of the base planes (neither for ss-nor for dsDNA) as would be expected for a continuously stretched and unwound DNA form (Hagmar et al 1992;Nordén et al 1992Nordén et al , 1998. This observation remained puzzling until 2002, when the application of systematically mutated aromatic residues in RecA allowed a threedimensional model to be constructed for the aqueous solution structures of RecA-dsDNA and RecA-ssDNA (Morimatsu et al 2002).…”

Section: Introductionmentioning

confidence: 99%

A stretched conformation of DNA with a biological role?

Bosaeus

Reymer

Beke‐Somfai

et al. 2017

Quart. Rev. Biophys.

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Abstract. We have discovered a well-defined extended conformation of double-stranded DNA, which we call Σ-DNA, using laser-tweezers force-spectroscopy experiments. At a transition force corresponding to free energy change ΔG = 1·57 ± 0·12 kcal (mol base pair) −1 60 or 122 base-pair long synthetic GC-rich sequences, when pulled by the 3′−3′ strands, undergo a sharp transition to the 1·52 ± 0·04 times longer Σ-DNA. Intriguingly, the same degree of extension is also found in DNA complexes with recombinase proteins, such as bacterial RecA and eukaryotic Rad51. Despite vital importance to all biological organisms for survival, genome maintenance and evolution, the recombination reaction is not yet understood at atomic level. We here propose that the structural distortion represented by Σ-DNA, which is thus physically inherent to the nucleic acid, is related to how recombination proteins mediate recognition of sequence homology and execute strand exchange. Our hypothesis is that a homogeneously stretched DNA undergoes a 'disproportionation' into an inhomogeneous Σ-form consisting of triplets of locally B-like perpendicularly stacked bases. This structure may ensure improved fidelity of base-pair recognition and promote rejection in case of mismatch during homologous recombination reaction. Because a triplet is the length of a gene codon, we speculate that the structural physics of nucleic acids may have biased the evolution of recombinase proteins to exploit triplet base stacks and also the genetic code.

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“…Base-base interaction can occur between all three DNA strands in the RecA⅐DNA complex, supporting formation of a triplex DNA structure, as has been suggested by other studies (9,10). Several models of different base-triads, involved in triplex-structure of recombination intermediates have been proposed (11,12) In order to investigate the base-base interactions and understand more about the mechanism of recognition between complementary DNA strands in RecA, we earlier assessed the orientation (roll and tilt angles) of DNA bases in RecA⅐DNA complexes by linear dichroism (LD) 1 spectroscopy combined with small-angle neutron scattering (SANS) and chromophorereplacement studies (13)(14)(15). LD measures, on an aligned sample, the absorption difference of light polarized parallel and perpendicular to the sample orientation direction (16).…”

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confidence: 99%

Base Orientation of Second DNA in RecA·DNA Filaments

Nordén

Wittung-Stafshede

Ellouze

et al. 1998

Journal of Biological Chemistry

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To gain insight into the mechanism of pairing two complementary DNA strands by the RecA protein, we have determined the nucleobase orientation of the first and the second bound DNA strands in the RecA⅐DNA filament by combined measurements of linear dichroism and small angle neutron scattering on flow-oriented samples. An etheno-modified DNA, poly(d⑀A) was adapted as the first DNA and an oligo(dT) as the second DNA, making it possible to distinguish between the linear dichroism signals of the two DNA strands. The results indicate that binding of the second DNA does not alter the nucleobase orientation of the first bound strand and that the bases of the second DNA are almost coplanar to the bases of the first strand although somewhat more tilted (60 degrees relative to the fiber axis compared with 70 degrees for the first DNA strand). Similar results were obtained for the RecA⅐DNA complex formed with unmodified poly(dA) and oligo(dT). An almost coplanar orientation of nucleobases of two DNA strands in a RecA-DNA filament would facilitate scanning for, and recognition of, complementary base sequences. The slight deviation from co-planarity could increase the free energy of the duplex to facilitate dissociation in case of mismatching base sequences.The RecA protein plays a crucial role in homologous recombination in Escherichia coli: it regulates the synthesis of proteins involved in recombination reaction, including RecA itself, and catalyzes strand-exchange reaction (1). RecA can, in the presence of cofactor ATP, promote pairing of two complementary DNA strands (2) and also perform strand exchange between two homologous DNA molecules in vitro (3, 4). In these reactions, RecA first binds with high cooperativity to a singlestranded DNA (5) forming a nucleoprotein filament (6). In the complex, the protein monomers are arranged in a helical manner around the DNA, and the DNA is elongated about 50% in length. This RecA single-stranded DNA nucleofilament can then bind a second DNA (either single-or double-stranded DNA) and pair complementary parts.From fluorescence measurements of various probes attached to the DNA bases, we have concluded that direct base-base interaction of the Watson-Crick type contributes to the sequence recognition by RecA (7, 8). Base-base interaction can occur between all three DNA strands in the RecA⅐DNA complex, supporting formation of a triplex DNA structure, as has been suggested by other studies (9, 10). Several models of different base-triads, involved in triplex-structure of recombination intermediates have been proposed (11,12) In order to investigate the base-base interactions and understand more about the mechanism of recognition between complementary DNA strands in RecA, we earlier assessed the orientation (roll and tilt angles) of DNA bases in RecA⅐DNA complexes by linear dichroism (LD) 1 spectroscopy combined with small-angle neutron scattering (SANS) and chromophorereplacement studies (13-15). LD measures, on an aligned sample, the absorption difference of light polarized parallel a...

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Structure of DNA-RecA complexes studied by residue differential linear dichroism and fluorescence spectroscopy for a genetically engineered RecA protein

Cited by 30 publications

References 21 publications

Arrangement of RecA protein in its active filament determined by polarized-light spectroscopy

Arrangement of RecA protein in its active filament determined by polarized-light spectroscopy

A stretched conformation of DNA with a biological role?

Base Orientation of Second DNA in RecA·DNA Filaments

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