MersV1, Main, Exploration, bibRecord, 003694

Structural and functional comparative study of the complexes formed by viral ø29, nf and GA-1 SSB proteins with DNA1,2

Identifieur interne : 003694 ( Main/Exploration ); précédent : 003693; suivant : 003695

Structural and functional comparative study of the complexes formed by viral ø29, nf and GA-1 SSB proteins with DNA1,2

Auteurs : Irene Gasc N [Espagne] ; Crisanto Gutiérrez [Espagne] ; Margarita Salas [Espagne]

Source :

Journal of Molecular Biology [ 0022-2836 ] ; 2000.

RBID : ISTEX:F00333FB02209B6227F0FCBDB18627B422637ACF

English descriptors

Abstract

Abstract: Single-stranded DNA-binding proteins have in common their crucial roles in DNA metabolism, although they exhibit significant differences in their single-stranded DNA binding properties. To evaluate the correlation between the structure of different nucleoprotein complexes and their function, we have carried out a comparative study of the complexes that the single-stranded DNA-binding proteins of three related bacteriophages, ø29, Nf and GA-1, form with single-stranded DNA. Under the experimental conditions used, ø29 and Nf single-stranded DNA-binding proteins are stable monomers in solution, while GA-1 single-stranded DNA-binding protein presents a hexameric state, as determined in glycerol gradients. The thermodynamic parameters derived from quenching measurements of the intrinsic protein fluorescence upon single-stranded DNA binding revealed (i) that GA-1 single-stranded DNA-binding protein occludes a larger binding site (n=51 nt/oligomer) than ø29 and Nf SSBs (n=3.4 and 4.7 nt/monomer, respectively); and (ii) that it shows a higher global affinity for single-stranded DNA (GA-1 SSB, Keff=18.6 × 105 M−1; ø29 SSB, Keff=2.2 × 105 M−1; Nf SSB, Keff=2.9 × 105 M−1). Altogether, these parameters justify the differences displayed by the GA-1 single-stranded DNA-binding protein and single-stranded DNA complex under the electron microscope, and the requirement of higher amounts of ø29 and Nf single-stranded DNA-binding proteins than of GA-1 SSB in gel mobility shift assays to produce a similar effect. The structural differences of the nucleoprotein complexes formed by the three single-stranded DNA-binding proteins with single-stranded DNA correlate with their different functional stimulatory effects in ø29 DNA amplification.

Url:

https://api.istex.fr/ark:/67375/6H6-WWG5DVZC-Z/fulltext.pdf

DOI: 10.1006/jmbi.2000.3521

Affiliations:

Espagne

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Le document en format XML

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<term>TP-DNA</term>
<term>bacteriophages ø29, Nf, GA-1</term>
<term>dsDNA</term>
<term>fluorescence quenching</term>
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<term>ssDNA</term>
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<term>λem</term>
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<term>Binding protein</term>
<term>Biochem</term>
<term>Biol</term>
<term>Coli</term>
<term>Direct titration</term>
<term>Effective binding</term>
<term>Electron microscope</term>
<term>Electron microscopy</term>
<term>Error values</term>
<term>Escherichia</term>
<term>Escherichia coli</term>
<term>Experimental conditions</term>
<term>Experimental data</term>
<term>Expression qmax</term>
<term>Extensive dilution</term>
<term>Ferrari</term>
<term>Free protein</term>
<term>Free state</term>
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<term>Kornberg baker</term>
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<term>Lohman ferrari</term>
<term>Lter effect</term>
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<term>Meyer laine</term>
<term>Mobility shift assays</term>
<term>Molecular mass</term>
<term>Natl acad</term>
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<term>Nucleoprotein complexes</term>
<term>Oligomeric state</term>
<term>Oligomerization</term>
<term>Oligomerization state</term>
<term>Ordinate axis</term>
<term>Phage</term>
<term>Polymerase</term>
<term>Protein concentration</term>
<term>Qmax</term>
<term>Quenching</term>
<term>Replication</term>
<term>Salas</term>
<term>Saturation fraction</term>
<term>Scatchard plot</term>
<term>Schwartz watanabe</term>
<term>Smaller amount</term>
<term>Soengas</term>
<term>Ssbs</term>
<term>Ssbs nucleoprotein complexes</term>
<term>Ssdna</term>
<term>Ssdna length</term>
<term>Straight line</term>
<term>Terminal protein</term>
<term>Theoretical curve</term>
<term>Thermodynamic</term>
<term>Thermodynamic parameters</term>
<term>Titration</term>
<term>Tting</term>
<term>Tting procedure</term>
<term>Uorescence</term>
<term>Uorescence data</term>
<term>Uorescence emission</term>
<term>Uorescence emission constants</term>
<term>Uorescence measurements</term>
<term>Uorescence quenching</term>
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<front><div type="abstract" xml:lang="en">Abstract: Single-stranded DNA-binding proteins have in common their crucial roles in DNA metabolism, although they exhibit significant differences in their single-stranded DNA binding properties. To evaluate the correlation between the structure of different nucleoprotein complexes and their function, we have carried out a comparative study of the complexes that the single-stranded DNA-binding proteins of three related bacteriophages, ø29, Nf and GA-1, form with single-stranded DNA. Under the experimental conditions used, ø29 and Nf single-stranded DNA-binding proteins are stable monomers in solution, while GA-1 single-stranded DNA-binding protein presents a hexameric state, as determined in glycerol gradients. The thermodynamic parameters derived from quenching measurements of the intrinsic protein fluorescence upon single-stranded DNA binding revealed (i) that GA-1 single-stranded DNA-binding protein occludes a larger binding site (n=51 nt/oligomer) than ø29 and Nf SSBs (n=3.4 and 4.7 nt/monomer, respectively); and (ii) that it shows a higher global affinity for single-stranded DNA (GA-1 SSB, Keff=18.6 × 105 M−1; ø29 SSB, Keff=2.2 × 105 M−1; Nf SSB, Keff=2.9 × 105 M−1). Altogether, these parameters justify the differences displayed by the GA-1 single-stranded DNA-binding protein and single-stranded DNA complex under the electron microscope, and the requirement of higher amounts of ø29 and Nf single-stranded DNA-binding proteins than of GA-1 SSB in gel mobility shift assays to produce a similar effect. The structural differences of the nucleoprotein complexes formed by the three single-stranded DNA-binding proteins with single-stranded DNA correlate with their different functional stimulatory effects in ø29 DNA amplification.</div>
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Structural and functional comparative study of the complexes formed by viral ø29, nf and GA-1 SSB proteins with DNA1,2

Structural and functional comparative study of the complexes formed by viral ø29, nf and GA-1 SSB proteins with DNA1,2

Source :

English descriptors

Abstract

Links toward previous steps (curation, corpus...)

Le document en format XML

Pour manipuler ce document sous Unix (Dilib)

Pour mettre un lien sur cette page dans le réseau Wicri