| 1. |
- Ellouze, C., et al.
(författare)
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Difference between active and inactive nucleotide cofactors in the effect of DNA binding and the helical structure of RecA filament
- 1999
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 262:1, s. 88-94
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Tidskriftsartikel (refereegranskat)abstract
- The RecA protein requires ATP or dATP for its coprotease and strand exchange activities. Other natural nucleotides, such as ADP, CTP, GTP, UTP and TTP, have little or no activation effect on RecA for these activities. We have investigated the activation mechanism, and the selectivity for ATP, by studying the effect of various nucleotides on the DNA binding and the helical structure of the RecA filament. The interaction with DNA was investigated via fluorescence measurements with a fluorescent DNA analog and fluorescein-labeled oligonucleotides, assisted by linear dichroism. Filament structure was investigated via small-angle neutron scattering. There is no simple correlation between filament elongation, DNA binding affinity of RecA, and DNA structure in the RecA complex. There may be multiple conformations of RecA, Both coprotease and strand exchange activities require formation of a rigid and well organized complex. The triphosphate nucleotides which do not activate RecA, destabilize the RecA-DNA complex, indicating that the chemical nature of the nucleotide nucleobase is very important for the stability of RecA-DNA complex. Higher stability of the RecA-DNA complex in the presence of adenosine 5'-O-3-thiotriphosphate or guanosine 5'-O-3-thiotriphosphate than ATP or GTP indicates that contact between the protein and the chemical group at the gamma position of the nucleotide also affects the stability of the RecA-DNA complex. This contact appears also important for the rigid organization of DNA because ADP strongly decreases the rigidity of the complex.
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| 2. |
- Ellouze, C., et al.
(författare)
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Evidence for Elongation of the Helical Pitch of the RecA Filament Upon ATP and ADP Binding Using Small-Angle Neutron Scattering
- 1995
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 233:2, s. 579-583
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Tidskriftsartikel (refereegranskat)abstract
- Structural changes of the RecA filament upon binding of cofactors have been investigated by small-angle neutron scattering. Both ATP and ADP increased the helical pitch of the RecA homopolymer, which is observed to be 7 nm in the absence of any cofactor. The binding of ATP altered the pitch to 9 nm, whereas the binding of ADP only produced a pitch of 8.2 nm. The pitch determined for the RecA complex with the ATP analog adenosine 5'-[gamma-thio]triphosphate was similar to that found with ATP. Thus, at least three, somewhat different, RecA helical filamentous structures may form in solution. The binding of DNA to RecA did not alter the pitch significantly, indicating that the cofactor binding is the determining factor for the size of the helical pitch of the RecA filament. We also found that elongation of the helical pitch is a necessary, but not a sufficient condition, for the coprotease activity of RecA. The presence of acetate or glutamate ions is also required. The pitch of the ADP . RecA filament is in agreement with that found in the crystal structure. This correlation indicates that this structure corresponds to that of the ADP . RecA filament in solution, although this is not the species active in recombination.
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| 3. |
- Morimatsu, K., et al.
(författare)
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Roles of Tyr103 and Tyr264 in the regulation of RecA-DNA interactions by nucleotide cofactors
- 1996
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 240:1, s. 91-97
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Tidskriftsartikel (refereegranskat)abstract
- The DNA-binding mode of the RecA protein, in particular its dependence on nucleotide cofactor, has been investigated by monitoring the fluorescence and linear-dichroism signals of a tryptophan residue inserted in the RecA to replace tyrosine at position 103 or 264. These residues are important for cofactor and DNA binding, as evidenced from their fluorescence changes upon binding of cofactor and DNA [Morimatsu, K., Horii, T, & Takahashi, M. (1995) Eur. J. Biochem. 228, 779-785]. The substitution of these residues with tryptophan does not affect the structure or biological function of the complex and can therefore be exploited to gain structural information in terms of the orientation and environment of the inserted reporter chromophore. The fluorescence change upon formation of the ternary cofactor . RecA . DNA complex was much smaller than the sum of the changes induced by cofactor or DNA alone, This difference indicates that the cofactor and DNA interact with RecA via common components. The fluorescence change caused by DNA in the presence of cofactor was almost independent of the base composition of DNA, in contrast to the interaction in the absence of cofactor. Hence, the contact mode between the selected residues and DNA in the complex may depend significantly on the cofactor, Linear-dichroism measurements indicate that the cofactor does not markedly alter the organization of RecA filament. Linear dichroism shows that neither the aromatic moiety of residue 103 nor that of residue 264 is intercalated between the DNA bases. The textural changes reported for the helical pitch and contour length of RecA fiber upon interaction with cofactor and DNA may derive from a subtle change in orientation of the RecA subunits in the filament.
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| 4. |
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| 5. |
- Takahashi, Masayuki, 1949, et al.
(författare)
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ACCESSIBILITY TO MODIFICATION OF HISTIDINE-RESIDUES OF RECA PROTEIN UPON DNA AND COFACTOR BINDING
- 1993
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 217:2, s. 665-670
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Tidskriftsartikel (refereegranskat)abstract
- The potential role of histidine residues of RecA protein in binding DNA has been investigated by monitoring their accessibility to diethylpyrocarbonate. In the absence of both DNA and cofactor, only one of two histidine residues is modified by the reagent, indicating that the other residue is buried. However, both histidine residues become accessible after addition of cofactor analog adenosine 5'-O-(3-thiotriphosphate) (ATP[S]) indicating a change in the organization of the RecA filament and/or a change in the conformation of protein. The diethylpyrocarbonate-modified RecA is found to be able to polymerize just as the unmodified protein. The binding of double-stranded DNA, in the presence of ATP[S], reduces the reactivity of both histidine residues to diethylpyrocarbonate. The binding of single-stranded DNA (with ATP[S]) has a similar, though smaller, protective effect. However, no significant dissociation of either of the complexes as a result of the modification was observed and a RecA molecule which had been modified in the absence of DNA could still bind DNA. A protection of the histidine residues is also effected by high salt concentration which promotes, just as DNA binding, ATPase and coprotease activity in RecA. The protection of histidine residues to diethylpyrocarbonate upon DNA binding probably relates to a conformational change of RecA and may not be any direct effect of shielding by the DNA. Nonetheless, the domains including the histidine residues could be centers of allosteric effects and are concluded to be close to the DNA binding site.
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| 6. |
- Takahashi, Masayuki, 1949, et al.
(författare)
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COORDINATION AND INTERNAL EXCHANGE OF 2 DNA-MOLECULES IN A RECA FILAMENT IN THE PRESENCE OF HYDROLYZING ATP - INFORMATION ON ATP-RECA-DNA STRUCTURE FROM LINEAR DICHROISM SPECTROSCOPY
- 1992
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 210:1, s. 87-92
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Tidskriftsartikel (refereegranskat)abstract
- Solution structure of complexes between DNA and recombinase RecA from Escherchia coli, in the presence of the physiological cofactor ATP, is probed by flow linear dichroism (LD) spectroscopy. A problem of ADP accumulation which promotes dissociation of DNA-RecA is circumvented by using an ATP-regenerating system. The LD features indicate that the local structure of the complex is very similar to that found in the presence of the non-hydrolysable analog of ATP, adenosine-5'-O-[gamma-thio]triphosphate (ATP[gammaS]); the DNA bases are oriented with their planes preferentially perpendicular to the long axis of the filament, while the indole chromophores of the two tryptophan residues of RecA are rather parallel to this reference direction. A much smaller overall amplitude of the LD spectrum, compared to ATP[gammaS], is interpreted as a result of fast dissociation of RecA due to hydrolysis of ATP, producing transiently naked DNA regions which act like flexible joints, diminishing the macroscopic orientation of the RecA filaments. However, the ATP hydrolysis is not found to prevent simultaneous accommodation of two non-complementary DNA molecules in the RecA complex, as judged from the LD behaviour upon successive addition of two different polynucleotides or modified DNA strands. A notable difference from corresponding complexes formed with ATP[gammaS] is that, in the presence of ATP hydrolysis, the order in which the two DNA molecules have been added is insignificant as judged from virtually identical resulting structures; this observation indicates that exchange of DNA occurs between the two DNA accommodation sites within the RecA filament.
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| 7. |
- Takahashi, M., et al.
(författare)
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Locations of functional domains in the RecA protein - Overlap of domains and regulation of activities
- 1996
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 242:1, s. 20-28
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Tidskriftsartikel (refereegranskat)abstract
- `We review the locations of various functional domains of the RecA protein of Escherichia coli, including how they have been assigned, and discuss the potential regulatory roles of spatial overlap between different domains. RecA is a multifunctional and ubiquitous protein involved both in general genetic recombination and in DNA repair: it regulates the synthesis and activity of DNA repair enzymes (SOS induction) and catalyses homologous recombination and mutagenesis. For these activities RecA interacts with a nucleotide cofactor. single-stranded and double-stranded DNAs, the LexA repressor, UmuD protein, the UmuD(2)'C complex as well as with RecA itself in forming the catalytically active nucleofilament. Attempts to locate the respective interaction sites have been advanced in order to understand the various functions of RecA. An intriguing question is how these numerous functional sites an contained within this rather small protein (38 kDa). To assess more clearly the roles of the respective sites and to what extent the sites may be interacting with each other, we review and compare the results obtained from various biological, biochemical and physico-chemical approaches. From a three-dimensional model it is concluded that all sites are concentrated to one part of the protein. As a consequence there are significant overlaps between the sites and it is speculated that corresponding interactions may play important roles in regulating RecA activities.
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| 8. |
- Tuite, Eimer, 1966, et al.
(författare)
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Effects of minor and major groove-binding drugs and intercalators on the DNA association of minor groove-binding proteins RecA and deoxyribonuclease I detected by flow linear dichroism
- 1997
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 243:1-2, s. 482-492
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Tidskriftsartikel (refereegranskat)abstract
- Linear and circular dichroic spectroscopies have been employed to investigate the effects of small DNA ligands on the interactions of two proteins which bind to the minor groove of DNA, viz. RecA protein from Escherichia coli and deoxyribonuclease I (bovine pancreas). Ligands representing three specific non-covalent binding modes were investigated: 4',6-diamidino-2-phenylindole and distamycin A (minor groove binders), methyl green (major groove binder), and methylene blue, ethidium bromide and ethidium dimer (intercalators). Linear dichroism was demonstrated to be an excellent detector, in real time, of DNA double-strand cleavage by deoxyribonuclease I. Ligands bound in all three modes interfered with the deoxyribonuclease I digestion of dsDNA, although the level of interference varied in a manner which could be related to the ligand binding site, the ligand charge appearing to be less important. In particular, the retardation of deoxyribonuclease I cleavage by the major groove binder methyl green demonstrates that accessibility to the minor groove can be affected by occupancy of the opposite groove. Binding of all three types of ligand also had marked effects on the interaction of RecA with dsDNA in the presence of non-hydrolyzable cofactor adenosine 5'-O-3-thiotriphosphate, decreasing the association rate to varying extents but with the strongest effects from ligands having some minor groove occupancy. Finally, each ligand was displaced from its DNA binding site upon completion of RecA association, again demonstrating that modification of either groove can affect the properties and behaviour of the other. The conclusions are discussed against the background of previous work on the use of small DNA ligands to probe DNA-protein interactions.
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| 9. |
- Wittung, Pernilla, 1968, et al.
(författare)
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Secondary Structure of RecA in Solution - The Effects of Cofactor, DNA and Ionic Conditions
- 1995
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 228:1, s. 149-154
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Tidskriftsartikel (refereegranskat)abstract
- The interactions of RecA with double-stranded DNA and with cofactor adenosine 5'-[3-thio]triphosphate (ATP[S] an analog of ATP) have been characterized by circular dichroism (CD) spectroscopy in a search for conformational changes associated with the formation of helical RecA ATP . DNA . fibers. Upon interaction with the RecA protein the cofactor is found to be structurally perturbed, possibly towards the syn ribose form of ATP[S], while the secondary structure of RecA remains unaffected. By contrast, when the ATP[S] . RecA . DNA complex is formed, a distinct change of the protein CD spectrum near 200 nm is observed as a result of interaction of RecA with DNA. The main change occurs upon the binding of the first DNA molecule [RecA can bind up to three DNA molecules simultaneously; Takahashi, M., Kubista, M. and Norden, B. (1991) Biochimie (Paris) 73, 219-226] and the effect appears to be independent of DNA sequence, suggesting a general change of protein conformation upon DNA binding. The CD of DNA is changed, indicating an alteration of the DNA structure, possibly related to stretching and unwinding. A small, reversible, decrease in the CD amplitude of RecA was observed when raising the temperature from 4 degrees C to 30 degrees C. The CD of RecA increases slightly with pH (up to 7.8) but is constant between pH 6.0 and 6.8. At pH below 6.0 or higher temperature (40 degrees C) slow irreversible denaturation of RecA occurs. The CD signal is effectively independent of salt, even in 2.2 M NaCl or 1 M sodium acetate, which is relevant regarding reported ATPase and coprotease activities promoted by salt. For high concentrations of magnesium (10 mM) at 30 degrees C the CD of RecA changes markedly and the appearance of light scattering indicates aggregation.
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| 10. |
- Wittung, Pernilla, 1968, et al.
(författare)
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SPECTROSCOPIC OBSERVATION OF RENATURATION BETWEEN POLYNUCLEOTIDES INTERACTING WITH RECA IN THE PRESENCE OF ATP HYDROLYSIS
- 1994
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 224:1, s. 39-45
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Tidskriftsartikel (refereegranskat)abstract
- To obtain mechanistic insights about RecA-promoted base pairing between complementary polynucleotides, the complex formation of RecA with poly(dA) and poly(dT) in the presence of ATP (and ATP-regenerating system) has been studied. The reaction was followed using a fluorescent probe, benzopyrenediolepoxide (BPDE), covalently attached to less than 1% of the adenine bases of poly(dA). BPDE is sensitive to its environment and has been found useful for detection of interactions between DNA strands, in the three binding positions of the RecA filament, in the presence of adenosine 5'-O-3-thiotriphosphate (ATP[S]) [Wittung, P., Norden, B. and Takahashi, M. (1994) J. Biol. Chern. 269, 5799-5803]. The emission spectrum of RecA:BPDE-poly(dA) formed in the presence of ATP is similar to that observed with ATP[S] supporting similar structures of the complexes. However, the fluorescence anisotropy is considerably reduced, suggesting a higher degree of freedom of DNA in the presence of ATP hydrolysis. Upon addition of a complementary strand, poly(dT), to a preformed filament of RecA:BPDE-poly(dA) in the presence of ATP, the fluorescence intensity slowly decreases and a change of emission profile consistent with Watson-Crick base pairing is observed. This contrasts with the case of ATP[S] in which normal base pairing is never observed. Hence, ATP hydrolysis appears necessary for the RecA filament to be able to promote true renaturation. The renaturation reaction is found more effective when one of the complementary DNA strands is bound in the primary RecA DNA-binding position and the other is added as the third strand, but the reaction can also occur between DNA strands in any combination of binding positions in the RecA filament. This observation suggests the importance of the third DNA-binding position of the RecA filament. Renaturation between DNA strands in the other two combinations of binding positions is speculated to have a role in aborting the strand-exchange reaction when the strands are insufficiently complementary.
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