| 1. |
- Kim, Hye-Kyung, 1970, et al.
(författare)
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ADP Stabilizes the Human Rad51-single stranded DNA Complex and Promotes Its DNA Annealing Activity
- 2002
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Ingår i: Genes to Cells. - : Wiley. - 1356-9597 .- 1365-2443. ; 7:11, s. 1125-1134
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Tidskriftsartikel (refereegranskat)abstract
- Background: Human Rad51 protein (HsRad51) is a homologue of Escherichia coli RecA protein, and involved in homologous recombination. These eukaryotic and bacterial proteins catalyse strand exchange between two homologous DNA molecules, each forming a complex with single-stranded DNA (ssDNA) and ATP as the initial step. Both proteins hydrolyse ATP; however, the role of ATP hydrolysis appears to vary between the two proteins.Results: Measurements using the fluorescence ssDNA analogue, poly(1,N (6) -etheno-deoxyadenosine), indicate that ATP affects the HsRad51-ssDNA complex, promoting two conformational states: one transient, rather rigid transition state and a final more flexible state. While ADP lowers the affinity of RecA protein to ssDNA, it is found to rather stabilize the HsRad51-ssDNA complex. ADP does not activate the strand exchange by HsRad51 but instead stimulates annealing between complementary ssDNAs.Conclusions: The hydrolysis of ATP promotes a transition of the HsRad51-ssDNA complex from a stiff state to less stiff state. The first state may be important for the strand separation of dsDNA in the initial step of strand exchange, while the second state may be important for annealing in the next step. However, hydrolysis does not dissociate HsRad51 from DNA as a component step of its recycling.
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| 2. |
- 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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| 3. |
- Ellouze, C., et al.
(författare)
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Nucleotide Cofactor-Dependent Structural Change of Xenopus laevis Rad51 Protein Filament Detected by Small-Angle Neutron Scattering Measurements in Solution
- 1997
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 36:44, s. 13524-13529
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Tidskriftsartikel (refereegranskat)abstract
- Rad51 protein, a eukaryotic homologue of RecA protein, forms a filamentous complex with DNA and catalyzes homologous recombination. We have analyzed the structure of Xenopus Rad51 protein (XRad51.1) in solution by small-angle neutron scattering (SANS). The measurements showed that XRad51.1 forms a helical filament independently of DNA. The sizes of the cross-sectional and helical pitch of the filament could be determined, respectively, from a Guinier plot and the position of the subsidiary maximum of SANS data. We observed that the helical structure is modified by nucleotide binding as in the case of RecA. Upon ATP binding under high-salt conditions (600 mM NaCl), the helical pitch of XRad51.1 filament was increased from 8 to 10 nm and the cross-sectional diameter decreased from 7 to 6 nm. The pitch sizes of XRad51.1 are similar to, though slightly larger than, those of RecA filament under corresponding conditions. A similar helical pitch size was observed by electron microscopy for budding yeast Rad51 [Ogawa, T., et al. (1993) Science 259, 1896-1899]. In contrast to the RecA filament, the structure of XRad51.1 filament with ADP is not significantly different from that with ATP. Thus, the hydrolysis of ATP to ADP does not modify the helical filament of XRad51.1. Together with our recent observation that ADP does not weaken the XRad51.1/DNA interaction, the effect of ATP hydrolysis on XRad51.1 nucleofilament should be very different from that on RecA.
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| 4. |
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| 5. |
- Kim, Hye-Kyung, 1970, et al.
(författare)
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Co-Ion Dependence of DNA Nuclease Activity Suggests Hydrophobic Cavitation as a Potential Source of Activation Energy
- 2001
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Ingår i: European Physical Journal E. - : Springer Science and Business Media LLC. - 1292-8941 .- 1292-895X. ; 4:4, s. 411-417
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Tidskriftsartikel (refereegranskat)abstract
- The source of the activation energy that allows cutting of DNA by restriction enzymes is unclear. A systematic study of the cutting efficiency of the type-II restriction endonuclease EcoRI, with varying background electrolyte ion pair and buffer reported here, shows only a modest dependence of efficiency on cation type. Surprisingly, efficiency does depend strongly on the presumed indifferent anion of the background salt. What emerges is that competition between the background salt anion and the buffer anion for the enzyme and DNA surfaces is crucial. The results are unexpected and counterintuitive from the point of view of conventional electrolyte theory. However, taken together with recent developments in surface chemistry, the results do fall into place and could also suggest a novel mechanism for enzyme activity as an alternative to metal-activated hydrolysis: microscopic cavitation in a hydrophobic pocket might be the source of activation energy.
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| 6. |
- Kim, Hye-Kyung, 1970
(författare)
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Structural Adaptation of DNA to Small Ligands and Recombination Proteins
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In order to contribute to the understanding of mechanisms of recognition in nucleic acid systems, the interaction of DNA with a variety of ligands, representing different cases of adaptation of the double-helix structure, was studied using flow linear dichroism (LD), circular dichroism (CD), fluorescence and absorption spectroscopic techniques. A chiral hydrophobic metal complex, [Ru(dpphen)3]2+ was examined, since this had been reported to be the most efficient molecule to discriminate DNA handedness. However, opposite enantiomers of the complex were found to have practically identical binding affinities and binding modes for B-DNA in a random non-intercalative binding. Various other small ligands, such as intercalators (9-aminoacridine (9AA), bis-9AA) and groove binders (DAPI, Hoechst 33258), were also examined with respect to their binding mode and ability to stabilise double and triple helical DNA. Intercalators and groove binders were found to interact with triplex DNA in the same binding modes as those observed with duplex DNA. However, while intercalators stabilise both triplex and duplex DNA, the minor groove binders studied seem to destabilise triplex but stabilise duplex DNA. Structural adaptation is believed to be important for the recognition between homologous DNAs and DNA strand exchange reactions mediated by recombination enzymes. Both eukaryotic and prokaryotic recombination proteins (RecA, XRad51, HsRad51) were studied in the presence of various nucleotide cofactors and double or single stranded DNAs with respect to structural changes and stabilities. A secondly bound, complementary ssDNA entering into the RecA fiber is found to have its bases slightly more tilted than those of the first bound template ssDNA according to LD and smallangle neutron scattering (SANS) data. Active cofactors were confirmed to elongate the filament of RecA and XRad51 more effectively than inactive cofactors and DNA adapts its structure by elongating its length by approximately 50% compared to B-DNA according to the SANS results. Fluorescence measurements indicate that active cofactors generally promote more stable protein-ssDNA complexes than inactive cofactors, with the exception of the inactive cofactor ADP which stabilises the HsRad51-ssDNA complex as much as the active cofactor ATP. This suggests that the roles of nucleotide cofactors in the strand exchange reaction may vary among the RecA, XRad51 and HsRad51systems.
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| 7. |
- Nordén, Bengt, 1945, et al.
(författare)
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Base Orientation of Second DNA in RecA-DNA Filaments. Analysis by combination of linear dichroism and small angle neutron scattering in flow-oriented solution
- 1998
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 273:25, s. 15682-15686
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Tidskriftsartikel (refereegranskat)abstract
- 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 RecA DNA filament by combined measurements of linear dichroism and small angle neutron scattering on flow-oriented samples. An etheno-modified DNA, poly(d epsilon 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 RecA DNA 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.
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| 8. |
- Orru, Anna Maria, 1976, et al.
(författare)
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AHA! festival 2016
- 2016
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- For the third year, the AHA festival investigates the meeting between art and science in a three-day event at the Chalmers University of Technology hosted by the Department of Architecture and the Department of Physics. An international festival intended to provide enlightening experiences, staging surprises, new thoughts and displaced perspectives that lead to alternative modes of thinking about exploring the world through art and science. We invite scientists (physicists, historians, astronomers, engineers), artists (dancers, musicians, painters, poets, acrobats) who reside in these borderlands and wish to share their vision and work. The key intention is to celebrate both art and science as key knowledge building devices.The first year’s theme ’Embodiment’ (2014) explored the body as our anchor in the world, followed by the 2015 theme on ’Numbers’, a delightful net we cast over the world. This year's theme is ’Uni-verse,’ again a natural consequence of our interest in the relation between art and science. The elemental force that drives science as well as art is curiosity. Come be curious with us! During the festival we have chosen to divide the word universe into three: uni and "-" and verse. Uni means that something is combined into a whole. Verse means that we are turned in a direction, the origin of the word tells us that it is the plow that turns at the end of the field. And the dash "-" is all the spaces and cracks where new discoveries can grow. Art and science unfolds in the gap between what we know and what we want to know.
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