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Molecular determinants of the DprA-RecA interaction for nucleation on ssDNA.

Lisboa J, Andreani J, Sanchez D, Boudes M, Collinet B, Liger D, van Tilbeurgh H, Guérois R, Quevillon-Cheruel S - Nucleic Acids Res. (2014)

Bottom Line: At the core of these patches, (DprA)M238 and (RecA)F230 are involved in the interaction.Our data favor a model of DprA acting as a cap of the RecA filament, involving a DprA-RecA interplay at two levels: their own oligomeric states and their respective interaction with DNA.Our model forms the basis for a mechanistic explanation of how DprA can act as a mediator for the loading of RecA on ssDNA.

View Article: PubMed Central - PubMed

Affiliation: Université Paris-Sud, Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, UMR 8619, F-91405 Orsay, France.

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DprA−RecA interaction tests using Y2H assays. Yeasts expressing wild-type or mutant SpDprA as Gal4 binding domain fusion (BD-SpDprA) and variants of RecA as Gal4 activation domain fusions (AD-SpRecA) were spotted as a series of 1/5th dilutions on selective medium lacking histidine. Plates were incubated for 5 days at 28°C. The left column indicates the nature of DprA, the upper line defines the length of RecA and the right column shows its content in terms of mutations.
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Figure 4: DprA−RecA interaction tests using Y2H assays. Yeasts expressing wild-type or mutant SpDprA as Gal4 binding domain fusion (BD-SpDprA) and variants of RecA as Gal4 activation domain fusions (AD-SpRecA) were spotted as a series of 1/5th dilutions on selective medium lacking histidine. Plates were incubated for 5 days at 28°C. The left column indicates the nature of DprA, the upper line defines the length of RecA and the right column shows its content in terms of mutations.

Mentions: A contrario the full-length RecA does not display any detectable interaction with DprA in Y2H experiments (Supplementary Figure S6). The origin for this lack of interaction might be that full-length RecA preferentially self-associates rendering RecA−DprA interaction undetectable in Y2H. However, DprA−RecA interaction becomes clearly observable in the Δ28RecA context and to a lesser extent in the Δ50RecA context, suggesting additionally that the [28–50] segment of RecA might be involved in the interaction with DprA. As previously noted, this region has been omitted in the docking simulation due to its high mobility. The M238ADprA mutant (described below), actually leading to the strongest interaction between DprA and RecA, follows the same tendency with a higher interaction with Δ28RecA than with Δ50RecA (Figure 4).


Molecular determinants of the DprA-RecA interaction for nucleation on ssDNA.

Lisboa J, Andreani J, Sanchez D, Boudes M, Collinet B, Liger D, van Tilbeurgh H, Guérois R, Quevillon-Cheruel S - Nucleic Acids Res. (2014)

DprA−RecA interaction tests using Y2H assays. Yeasts expressing wild-type or mutant SpDprA as Gal4 binding domain fusion (BD-SpDprA) and variants of RecA as Gal4 activation domain fusions (AD-SpRecA) were spotted as a series of 1/5th dilutions on selective medium lacking histidine. Plates were incubated for 5 days at 28°C. The left column indicates the nature of DprA, the upper line defines the length of RecA and the right column shows its content in terms of mutations.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4066776&req=5

Figure 4: DprA−RecA interaction tests using Y2H assays. Yeasts expressing wild-type or mutant SpDprA as Gal4 binding domain fusion (BD-SpDprA) and variants of RecA as Gal4 activation domain fusions (AD-SpRecA) were spotted as a series of 1/5th dilutions on selective medium lacking histidine. Plates were incubated for 5 days at 28°C. The left column indicates the nature of DprA, the upper line defines the length of RecA and the right column shows its content in terms of mutations.
Mentions: A contrario the full-length RecA does not display any detectable interaction with DprA in Y2H experiments (Supplementary Figure S6). The origin for this lack of interaction might be that full-length RecA preferentially self-associates rendering RecA−DprA interaction undetectable in Y2H. However, DprA−RecA interaction becomes clearly observable in the Δ28RecA context and to a lesser extent in the Δ50RecA context, suggesting additionally that the [28–50] segment of RecA might be involved in the interaction with DprA. As previously noted, this region has been omitted in the docking simulation due to its high mobility. The M238ADprA mutant (described below), actually leading to the strongest interaction between DprA and RecA, follows the same tendency with a higher interaction with Δ28RecA than with Δ50RecA (Figure 4).

Bottom Line: At the core of these patches, (DprA)M238 and (RecA)F230 are involved in the interaction.Our data favor a model of DprA acting as a cap of the RecA filament, involving a DprA-RecA interplay at two levels: their own oligomeric states and their respective interaction with DNA.Our model forms the basis for a mechanistic explanation of how DprA can act as a mediator for the loading of RecA on ssDNA.

View Article: PubMed Central - PubMed

Affiliation: Université Paris-Sud, Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, UMR 8619, F-91405 Orsay, France.

Show MeSH
Related in: MedlinePlus