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The DNA-binding domain of yeast Rap1 interacts with double-stranded DNA in multiple binding modes.

Feldmann EA, Galletto R - Biochemistry (2014)

Bottom Line: Unexpectedly, we found that while Rap1(DBD) forms a high-affinity 1:1 complex with its DNA recognition site, it can also form lower-affinity complexes with higher stoichiometries on DNA.In the other alternative lower-affinity binding mode, we propose that a single Myb-like domain of the Rap1(DBD) makes interactions with DNA, allowing for more than one protein molecule to bind to the DNA substrates.Our findings suggest that the Rap1(DBD) does not simply target the protein to its recognition sequence but rather it might be a possible point of regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine , St. Louis, Missouri 63110, United States.

ABSTRACT
Saccharomyces cerevisiae repressor-activator protein 1 (Rap1) is an essential protein involved in multiple steps of DNA regulation, as an activator in transcription, as a repressor at silencer elements, and as a major component of the shelterin-like complex at telomeres. All the known functions of Rap1 require the known high-affinity and specific interaction of the DNA-binding domain with its recognition sequences. In this work, we focus on the interaction of the DNA-binding domain of Rap1 (Rap1(DBD)) with double-stranded DNA substrates. Unexpectedly, we found that while Rap1(DBD) forms a high-affinity 1:1 complex with its DNA recognition site, it can also form lower-affinity complexes with higher stoichiometries on DNA. These lower-affinity interactions are independent of the presence of the recognition sequence, and we propose they originate from the ability of Rap1(DBD) to bind to DNA in two different binding modes. In one high-affinity binding mode, Rap1(DBD) likely binds in the conformation observed in the available crystal structures. In the other alternative lower-affinity binding mode, we propose that a single Myb-like domain of the Rap1(DBD) makes interactions with DNA, allowing for more than one protein molecule to bind to the DNA substrates. Our findings suggest that the Rap1(DBD) does not simply target the protein to its recognition sequence but rather it might be a possible point of regulation.

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DBD601 binding to FAM-labeled dsDNA substrates monitoredby fluorescence anisotropy confirms the formation of high-stoichiometrycomplexes. (a) Change in the fluorescence anisotropy and relativetotal intensity (inset) of 255 nM dsDNAs labeled at the 5′-endof the top strand in buffer HN50 as a function of proteinto DNA total concentration ratio for TeloA (black), RND (gray), HMRE(blue), and RPG (red). (b) Change in the fluorescence anisotropy ofFAM-labeled TeloA where the fluorophore is positioned at various endsof the dsDNA duplex: circles for the 5′-end (black) or 3′-end(gray) of the top strand and triangles for the 5′-end (black)or 3′-end (gray) of the bottom strand. (c) Change in the relativetotal intensity in buffer HN50 as a function of DBD601:DNA ratio for 255 nM dsDNA labeled with FAM at the 3′-endof the top strand: TeloA (black), RND (gray), and HMRE (blue). (d)Change in the fluorescence anisotropy in buffer HN50 asa function of DBD601 concentration for 10 nM dsDNA labeledwith FAM at the 5′-end of the top strand: TeloA (black), RPG(red), and HMRE (blue).
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fig3: DBD601 binding to FAM-labeled dsDNA substrates monitoredby fluorescence anisotropy confirms the formation of high-stoichiometrycomplexes. (a) Change in the fluorescence anisotropy and relativetotal intensity (inset) of 255 nM dsDNAs labeled at the 5′-endof the top strand in buffer HN50 as a function of proteinto DNA total concentration ratio for TeloA (black), RND (gray), HMRE(blue), and RPG (red). (b) Change in the fluorescence anisotropy ofFAM-labeled TeloA where the fluorophore is positioned at various endsof the dsDNA duplex: circles for the 5′-end (black) or 3′-end(gray) of the top strand and triangles for the 5′-end (black)or 3′-end (gray) of the bottom strand. (c) Change in the relativetotal intensity in buffer HN50 as a function of DBD601:DNA ratio for 255 nM dsDNA labeled with FAM at the 3′-endof the top strand: TeloA (black), RND (gray), and HMRE (blue). (d)Change in the fluorescence anisotropy in buffer HN50 asa function of DBD601 concentration for 10 nM dsDNA labeledwith FAM at the 5′-end of the top strand: TeloA (black), RPG(red), and HMRE (blue).

Mentions: Next we examined the binding ofDBD601 in solution using fluorescence spectroscopy whilemonitoring the signals from fluorescently labeled DNA substrates.Figure 3a shows the change in fluorescenceanisotropy of 255 nM dsDNA substrates [TeloA, HMRE, RPG, and RND labeledat the 5′-end of the top strand with FAM (see Table 1)] as a function of the ratio of the total proteinto DNA concentration in buffer HN50 [20 mM HEPES (pH 7.4),50 mM NaCl, 2 mM MgCl2, and 10% (v/v) glycerol]. Bindingof DBD601 to these 5′-labeled dsDNAs is accompaniedby a large increase in fluorescence anisotropy, providing a largesignal change to monitor the reaction. Although tight binding conditionsfor the higher-stoichiometry complexes cannot be fully achieved, thesedata strongly suggest that at saturation approximately three DBD601 molecules bind to the DNA, regardless of the presence orabsence of a Rap1 recognition sequence. This provides further supportto the conclusions from analytical ultracentrifugation experimentsperformed at higher DNA concentrations (Table 2).


The DNA-binding domain of yeast Rap1 interacts with double-stranded DNA in multiple binding modes.

Feldmann EA, Galletto R - Biochemistry (2014)

DBD601 binding to FAM-labeled dsDNA substrates monitoredby fluorescence anisotropy confirms the formation of high-stoichiometrycomplexes. (a) Change in the fluorescence anisotropy and relativetotal intensity (inset) of 255 nM dsDNAs labeled at the 5′-endof the top strand in buffer HN50 as a function of proteinto DNA total concentration ratio for TeloA (black), RND (gray), HMRE(blue), and RPG (red). (b) Change in the fluorescence anisotropy ofFAM-labeled TeloA where the fluorophore is positioned at various endsof the dsDNA duplex: circles for the 5′-end (black) or 3′-end(gray) of the top strand and triangles for the 5′-end (black)or 3′-end (gray) of the bottom strand. (c) Change in the relativetotal intensity in buffer HN50 as a function of DBD601:DNA ratio for 255 nM dsDNA labeled with FAM at the 3′-endof the top strand: TeloA (black), RND (gray), and HMRE (blue). (d)Change in the fluorescence anisotropy in buffer HN50 asa function of DBD601 concentration for 10 nM dsDNA labeledwith FAM at the 5′-end of the top strand: TeloA (black), RPG(red), and HMRE (blue).
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Related In: Results  -  Collection

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Show All Figures
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fig3: DBD601 binding to FAM-labeled dsDNA substrates monitoredby fluorescence anisotropy confirms the formation of high-stoichiometrycomplexes. (a) Change in the fluorescence anisotropy and relativetotal intensity (inset) of 255 nM dsDNAs labeled at the 5′-endof the top strand in buffer HN50 as a function of proteinto DNA total concentration ratio for TeloA (black), RND (gray), HMRE(blue), and RPG (red). (b) Change in the fluorescence anisotropy ofFAM-labeled TeloA where the fluorophore is positioned at various endsof the dsDNA duplex: circles for the 5′-end (black) or 3′-end(gray) of the top strand and triangles for the 5′-end (black)or 3′-end (gray) of the bottom strand. (c) Change in the relativetotal intensity in buffer HN50 as a function of DBD601:DNA ratio for 255 nM dsDNA labeled with FAM at the 3′-endof the top strand: TeloA (black), RND (gray), and HMRE (blue). (d)Change in the fluorescence anisotropy in buffer HN50 asa function of DBD601 concentration for 10 nM dsDNA labeledwith FAM at the 5′-end of the top strand: TeloA (black), RPG(red), and HMRE (blue).
Mentions: Next we examined the binding ofDBD601 in solution using fluorescence spectroscopy whilemonitoring the signals from fluorescently labeled DNA substrates.Figure 3a shows the change in fluorescenceanisotropy of 255 nM dsDNA substrates [TeloA, HMRE, RPG, and RND labeledat the 5′-end of the top strand with FAM (see Table 1)] as a function of the ratio of the total proteinto DNA concentration in buffer HN50 [20 mM HEPES (pH 7.4),50 mM NaCl, 2 mM MgCl2, and 10% (v/v) glycerol]. Bindingof DBD601 to these 5′-labeled dsDNAs is accompaniedby a large increase in fluorescence anisotropy, providing a largesignal change to monitor the reaction. Although tight binding conditionsfor the higher-stoichiometry complexes cannot be fully achieved, thesedata strongly suggest that at saturation approximately three DBD601 molecules bind to the DNA, regardless of the presence orabsence of a Rap1 recognition sequence. This provides further supportto the conclusions from analytical ultracentrifugation experimentsperformed at higher DNA concentrations (Table 2).

Bottom Line: Unexpectedly, we found that while Rap1(DBD) forms a high-affinity 1:1 complex with its DNA recognition site, it can also form lower-affinity complexes with higher stoichiometries on DNA.In the other alternative lower-affinity binding mode, we propose that a single Myb-like domain of the Rap1(DBD) makes interactions with DNA, allowing for more than one protein molecule to bind to the DNA substrates.Our findings suggest that the Rap1(DBD) does not simply target the protein to its recognition sequence but rather it might be a possible point of regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine , St. Louis, Missouri 63110, United States.

ABSTRACT
Saccharomyces cerevisiae repressor-activator protein 1 (Rap1) is an essential protein involved in multiple steps of DNA regulation, as an activator in transcription, as a repressor at silencer elements, and as a major component of the shelterin-like complex at telomeres. All the known functions of Rap1 require the known high-affinity and specific interaction of the DNA-binding domain with its recognition sequences. In this work, we focus on the interaction of the DNA-binding domain of Rap1 (Rap1(DBD)) with double-stranded DNA substrates. Unexpectedly, we found that while Rap1(DBD) forms a high-affinity 1:1 complex with its DNA recognition site, it can also form lower-affinity complexes with higher stoichiometries on DNA. These lower-affinity interactions are independent of the presence of the recognition sequence, and we propose they originate from the ability of Rap1(DBD) to bind to DNA in two different binding modes. In one high-affinity binding mode, Rap1(DBD) likely binds in the conformation observed in the available crystal structures. In the other alternative lower-affinity binding mode, we propose that a single Myb-like domain of the Rap1(DBD) makes interactions with DNA, allowing for more than one protein molecule to bind to the DNA substrates. Our findings suggest that the Rap1(DBD) does not simply target the protein to its recognition sequence but rather it might be a possible point of regulation.

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