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Quantitative characterization of protein-protein complexes involved in base excision DNA repair.

Moor NA, Vasil'eva IA, Anarbaev RO, Antson AA, Lavrik OI - Nucleic Acids Res. (2015)

Bottom Line: The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ.Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein-protein binding affinities.Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process.

View Article: PubMed Central - PubMed

Affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.

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Fluorescence titration of FAM-PARP1 with PARP1, Polβ, XRCC1 and TDP1 (A), and of the FAM-labeled APE1, PARP1 and Polβ with p24 protein (B). The FAM-labeled protein (40 nM) was excited at 485 nm in the absence or presence of increasing concentrations of the binding partner and the relative fluorescence intensities were monitored at 520 nm. Curves show the best fits of the four-parameter equation (with R2 values exceeding 0.98).
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Figure 2: Fluorescence titration of FAM-PARP1 with PARP1, Polβ, XRCC1 and TDP1 (A), and of the FAM-labeled APE1, PARP1 and Polβ with p24 protein (B). The FAM-labeled protein (40 nM) was excited at 485 nm in the absence or presence of increasing concentrations of the binding partner and the relative fluorescence intensities were monitored at 520 nm. Curves show the best fits of the four-parameter equation (with R2 values exceeding 0.98).

Mentions: The fluorescence intensity of FAM-PARP1 increased in the presence of four different proteins, namely PARP1, Polβ, XRCC1 or TDP1 (Figure 2A), while addition of APE1 had no effect (data not shown). Interestingly, the highest change of fluorescence intensity in titration experiments with the FAM-labeled PARP1 and the FAM-labeled Polβ was induced by self-association of the protein (Supplementary Table S3). The EC50 values determined for the four binding partners of FAM-PARP1 are comparable (Table 1), indicating that PARP1 can interact with homologous and many heterologous proteins with closely similar affinities. The apparent dissociation constants measured by titration of FAM-Polβ with PARP1 or of FAM-PARP1 with Polβ are practically identical, suggesting that the fluorescent labeling of either partner did not disturb their interaction.


Quantitative characterization of protein-protein complexes involved in base excision DNA repair.

Moor NA, Vasil'eva IA, Anarbaev RO, Antson AA, Lavrik OI - Nucleic Acids Res. (2015)

Fluorescence titration of FAM-PARP1 with PARP1, Polβ, XRCC1 and TDP1 (A), and of the FAM-labeled APE1, PARP1 and Polβ with p24 protein (B). The FAM-labeled protein (40 nM) was excited at 485 nm in the absence or presence of increasing concentrations of the binding partner and the relative fluorescence intensities were monitored at 520 nm. Curves show the best fits of the four-parameter equation (with R2 values exceeding 0.98).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499159&req=5

Figure 2: Fluorescence titration of FAM-PARP1 with PARP1, Polβ, XRCC1 and TDP1 (A), and of the FAM-labeled APE1, PARP1 and Polβ with p24 protein (B). The FAM-labeled protein (40 nM) was excited at 485 nm in the absence or presence of increasing concentrations of the binding partner and the relative fluorescence intensities were monitored at 520 nm. Curves show the best fits of the four-parameter equation (with R2 values exceeding 0.98).
Mentions: The fluorescence intensity of FAM-PARP1 increased in the presence of four different proteins, namely PARP1, Polβ, XRCC1 or TDP1 (Figure 2A), while addition of APE1 had no effect (data not shown). Interestingly, the highest change of fluorescence intensity in titration experiments with the FAM-labeled PARP1 and the FAM-labeled Polβ was induced by self-association of the protein (Supplementary Table S3). The EC50 values determined for the four binding partners of FAM-PARP1 are comparable (Table 1), indicating that PARP1 can interact with homologous and many heterologous proteins with closely similar affinities. The apparent dissociation constants measured by titration of FAM-Polβ with PARP1 or of FAM-PARP1 with Polβ are practically identical, suggesting that the fluorescent labeling of either partner did not disturb their interaction.

Bottom Line: The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ.Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein-protein binding affinities.Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process.

View Article: PubMed Central - PubMed

Affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.

Show MeSH
Related in: MedlinePlus