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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/DNA/ATP complexes: comparison between Ca2+ and Mg2+ conditions. (A) Complexes were formed by mixing HsRad51, poly(dT) and ATP in buffer containing Ca2+ (black) or Mg2+ (red) as described in text. LD spectra were measured after 2 h incubation at room temperature. (B) Complexes were formed by mixing HsRad51 and ATP together with calf thymus DNA in the presence of Ca2+ (black) or Mg2+ (red) and incubated for 2 h before LD measurement. (C) Transition moments responsible for LD signals at indicated wavelengths: thymine (1), adenine (2), tyrosine (3) and phenylalanine (4).
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gks140-F1: LD spectra of HsRad51/DNA/ATP complexes: comparison between Ca2+ and Mg2+ conditions. (A) Complexes were formed by mixing HsRad51, poly(dT) and ATP in buffer containing Ca2+ (black) or Mg2+ (red) as described in text. LD spectra were measured after 2 h incubation at room temperature. (B) Complexes were formed by mixing HsRad51 and ATP together with calf thymus DNA in the presence of Ca2+ (black) or Mg2+ (red) and incubated for 2 h before LD measurement. (C) Transition moments responsible for LD signals at indicated wavelengths: thymine (1), adenine (2), tyrosine (3) and phenylalanine (4).

Mentions: Neither ssDNA nor HsRad51 alone gives rise to any significant LD signal, regardless of the presence or absence of nucleotide cofactors (results not shown). ssDNA, in contrast to dsDNA, has a flexible structure so that its bases on the whole appear unoriented even at high shear-flow gradients, while HsRad51 alone, in the concentration range used here, does not form stable, sufficiently long filaments to be significantly oriented (47). However, in combination, the HsRad51/poly(dT)/ATP complexes with Ca2+ as well as with Mg2+ exhibit relatively strong LD signals (Figure 1A), although weaker than that observed for the RecA/ poly(dT)/ATPγS complex (35). This observation indicates that HsRad51 forms quite a long and regular filament around poly(dT). If discontinuous short filaments were formed, with naked DNA regions in between, the filament stretches would act as flexible joints and the sample would not align in the shear flow, resulting in a much weaker, if any, LD signal (48). Interestingly, the formation of stiff nucleoprotein filaments, which are aligned by shear flow, requires both ATP and divalent ions. In the absence of either, we observed only weak LD signals (results not shown).Figure 1.


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/DNA/ATP complexes: comparison between Ca2+ and Mg2+ conditions. (A) Complexes were formed by mixing HsRad51, poly(dT) and ATP in buffer containing Ca2+ (black) or Mg2+ (red) as described in text. LD spectra were measured after 2 h incubation at room temperature. (B) Complexes were formed by mixing HsRad51 and ATP together with calf thymus DNA in the presence of Ca2+ (black) or Mg2+ (red) and incubated for 2 h before LD measurement. (C) Transition moments responsible for LD signals at indicated wavelengths: thymine (1), adenine (2), tyrosine (3) and phenylalanine (4).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks140-F1: LD spectra of HsRad51/DNA/ATP complexes: comparison between Ca2+ and Mg2+ conditions. (A) Complexes were formed by mixing HsRad51, poly(dT) and ATP in buffer containing Ca2+ (black) or Mg2+ (red) as described in text. LD spectra were measured after 2 h incubation at room temperature. (B) Complexes were formed by mixing HsRad51 and ATP together with calf thymus DNA in the presence of Ca2+ (black) or Mg2+ (red) and incubated for 2 h before LD measurement. (C) Transition moments responsible for LD signals at indicated wavelengths: thymine (1), adenine (2), tyrosine (3) and phenylalanine (4).
Mentions: Neither ssDNA nor HsRad51 alone gives rise to any significant LD signal, regardless of the presence or absence of nucleotide cofactors (results not shown). ssDNA, in contrast to dsDNA, has a flexible structure so that its bases on the whole appear unoriented even at high shear-flow gradients, while HsRad51 alone, in the concentration range used here, does not form stable, sufficiently long filaments to be significantly oriented (47). However, in combination, the HsRad51/poly(dT)/ATP complexes with Ca2+ as well as with Mg2+ exhibit relatively strong LD signals (Figure 1A), although weaker than that observed for the RecA/ poly(dT)/ATPγS complex (35). This observation indicates that HsRad51 forms quite a long and regular filament around poly(dT). If discontinuous short filaments were formed, with naked DNA regions in between, the filament stretches would act as flexible joints and the sample would not align in the shear flow, resulting in a much weaker, if any, LD signal (48). Interestingly, the formation of stiff nucleoprotein filaments, which are aligned by shear flow, requires both ATP and divalent ions. In the absence of either, we observed only weak LD signals (results not shown).Figure 1.

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