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Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination.

Fornander LH, Frykholm K, Reymer A, Renodon-Cornière A, Takahashi M, Nordén B - Nucleic Acids Res. (2012)

Bottom Line: In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not.It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction.A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.

ABSTRACT
Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not. A high strand exchange activity is observed for the filament formed with ATP/Ca(2+), whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.

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LD spectra of HsRad51/poly(dT)/ADP complexes. Complexes were formed by mixing HsRad51, poly(dT) and ADP in the presence of Mg2+ (red) or Ca2+ (black). LD spectra were measured after 2 h incubation at room temperature.
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gks140-F3: LD spectra of HsRad51/poly(dT)/ADP complexes. Complexes were formed by mixing HsRad51, poly(dT) and ADP in the presence of Mg2+ (red) or Ca2+ (black). LD spectra were measured after 2 h incubation at room temperature.

Mentions: The LD spectra of HsRad51/poly(dT) filaments formed in the presence of ADP/Mg2+ and ADP/Ca2+ after 2 h incubation (Figure 3) display clear differences compared with the spectra of the corresponding filaments formed with ATP. As already concluded, these observed variations cannot be explained by the accumulation of ADP, or a simple conversion of the ATP-bound complex into the corresponding ADP-bound complex. The LD spectra of complexes with ADP vary between Mg2+ and Ca2+ conditions as well. Interestingly, the LD spectrum of the HsRad51/ssDNA complex with ADP/Ca2+ is very similar to that of the complex formed with ATP/Mg2+ (during the first 6 h), suggesting that these filaments are structurally similar.Figure 3.


Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination.

Fornander LH, Frykholm K, Reymer A, Renodon-Cornière A, Takahashi M, Nordén B - Nucleic Acids Res. (2012)

LD spectra of HsRad51/poly(dT)/ADP complexes. Complexes were formed by mixing HsRad51, poly(dT) and ADP in the presence of Mg2+ (red) or Ca2+ (black). LD spectra were measured after 2 h incubation at room temperature.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367181&req=5

gks140-F3: LD spectra of HsRad51/poly(dT)/ADP complexes. Complexes were formed by mixing HsRad51, poly(dT) and ADP in the presence of Mg2+ (red) or Ca2+ (black). LD spectra were measured after 2 h incubation at room temperature.
Mentions: The LD spectra of HsRad51/poly(dT) filaments formed in the presence of ADP/Mg2+ and ADP/Ca2+ after 2 h incubation (Figure 3) display clear differences compared with the spectra of the corresponding filaments formed with ATP. As already concluded, these observed variations cannot be explained by the accumulation of ADP, or a simple conversion of the ATP-bound complex into the corresponding ADP-bound complex. The LD spectra of complexes with ADP vary between Mg2+ and Ca2+ conditions as well. Interestingly, the LD spectrum of the HsRad51/ssDNA complex with ADP/Ca2+ is very similar to that of the complex formed with ATP/Mg2+ (during the first 6 h), suggesting that these filaments are structurally similar.Figure 3.

Bottom Line: In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not.It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction.A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.

ABSTRACT
Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not. A high strand exchange activity is observed for the filament formed with ATP/Ca(2+), whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.

Show MeSH
Related in: MedlinePlus