| 1. |
- Takahashi, M., et al.
(author)
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Calorimetric analysis of binding of two consecutive DNA strands to RecA protein illuminates mechanism for recognition of homology
- 2007
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In: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 365:3, s. 603-611
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Journal article (peer-reviewed)abstract
- RecA protein recognises two complementary DNA strands for homologous recombination. To gain insight into the molecular mechanism, the thermodynamic parameters of the DNA binding have been characterised by isothermal calorimetry. Specifically, conformational changes of protein and DNA were searched for by measuring variations in enthalpy change, (Delta H) with temperature (heat capacity change, Delta C-p). In the presence of the ATP analogue ATP gamma S, the Delta H for the binding of the first DNA strand depends upon temperature (large Delta C-p) and the type of buffer, in a way that is consistent with the organisation of disordered parts and the protonation of RecA upon complex formation. In contrast, the binding of the second DNA strand occurs without any pronounced Delta C-p, indicating the absence of further reorganisation of the RecA-DNA filament. In agreement with these findings, a significant change in the CD spectrum of RecA was observed only upon the binding of the first DNA strand. In the absence of nucleotide cofactor, the Delta H of DNA binding is almost independent of temperature, indicating a requirement for ATP in the reorganisation of RecA. When the second DNA strand is complementary to the first, the Delta H is larger than that for non-complementary DNA strand, but less than the Delta H of the annealing of the complementary DNA without RecA. This small Delta H could reflect a weak binding that may facilitate the dissociation of only partly complementary DNA and thus speed the search for complementary DNA. The Delta H of binding DNA sequences displaying strong base-base stacking is small for both the first and second binding DNA strand, suggesting that the second is also stretched upon interaction with RecA. These results support the proposal that the RecA protein restructures DNA, preparing it for the recognition of a complementary second DNA strand, and that the recognition is due mainly to direct base-base contacts between DNA strands. (c) 2006 Elsevier Ltd. All rights reserved.
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| 2. |
- Morimatsu, K., et al.
(author)
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Roles of Tyr103 and Tyr264 in the regulation of RecA-DNA interactions by nucleotide cofactors
- 1996
-
In: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 240:1, s. 91-97
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Journal article (peer-reviewed)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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| 3. |
- Selmane, T., et al.
(author)
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The L2 Loop Peptide of recA Stiffens and restricts Base Motions of Single-stranded DNA Similar to Intact Protein
- 1999
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In: FEBS Letters. - 1873-3468 .- 0014-5793. ; 446:1, s. 30-34
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Journal article (peer-reviewed)abstract
- The L2 loop in the RecA protein is the catalytic center for DNA strand exchange, Here we investigate the DMA binding properties of the L2 loop peptide using optical spectroscopy with polarized light. Both fluorescence intensity and anisotropy of an etheno-modified poly(dA) increase upon peptide binding, indicate that the base motions of single-stranded DNA are restricted in the complex. In agreement with this conclusion, the peptide-poly(dT) complex exhibits a significant linear dichroism signal. The peptide is also found to modify the structure of double-stranded DNA, but does not denature it. It is inferred that strand separation may not be required for the formation of a joint molecule.
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| 4. |
- Takahashi, M., et al.
(author)
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Locations of functional domains in the RecA protein - Overlap of domains and regulation of activities
- 1996
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In: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 242:1, s. 20-28
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Journal article (peer-reviewed)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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| 5. |
- Wittung, Pernilla, 1968, et al.
(author)
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Thermochemical and kinetic evidence for nucleotide-sequence-dependent RecA-DNA interactions
- 1997
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In: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 245:3, s. 715-719
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Journal article (peer-reviewed)abstract
- RecA catalyses homologous recombination in Escherichia coli by promoting pairing of homologous DNA molecules after formation of a helical nucleoprotein filament with single-stranded DNA. The primary reaction of RecA with DNA is generally assumed to be unspecific. We show here, by direct measurement of the interaction enthalpy by means of isothermal titration calorimetry, that the polymerisation of RecA on single-stranded DNA depends on the DNA sequence, with a high exothermic preference for thymine bases. This enthalpic sequence preference of thymines by RecA correlates with faster binding kinetics of RecA to thymine DNA. Furthermore, the enthalpy of interaction between the RecA.DNA filament and a second DNA strand is large only when the added DNA is complementary to the bound DNA in RecA. This result suggests a possibility for a rapid search mechanism by RecA.DNA filaments for homologous DNA molecules.
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