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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–DNA complexesof higher strochiometrymonitored by analytical ultracentrifugation. (a) Sedimentation coefficientdistributions from velocity experiments with 2 μM Cy3-labeledTeloA in buffer HN50 at different DBD601:DNAratios: 0.5 (···), 1 (---), 2 (thin solid line), and10 (thick solid line). The data for Cy3-labeled TeloA alone are shown,as well. The distribution of sedimentation coefficients with unlabeledTeloA and an 8-fold excess of DBD601 is included, as well(diamonds). (b) Same experiments as in panel a but with Cy3-labeledRND. (c) Sedimentation equilibrium profile of 1.5 μM Cy3-labeledTeloA in the presence of an 8-fold excess of DBD601 inbuffer HN50 at rotor speeds of 14000, 16000, and 18000rpm. The solid gray lines are the global analyses of the data fitwith a single-species model (see Table 2).(d) EMSA of protein–DNA complexes formed at a 1:1 ratio withdifferent 21 bp substrates at 300 nM.
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fig2: DBD601–DNA complexesof higher strochiometrymonitored by analytical ultracentrifugation. (a) Sedimentation coefficientdistributions from velocity experiments with 2 μM Cy3-labeledTeloA in buffer HN50 at different DBD601:DNAratios: 0.5 (···), 1 (---), 2 (thin solid line), and10 (thick solid line). The data for Cy3-labeled TeloA alone are shown,as well. The distribution of sedimentation coefficients with unlabeledTeloA and an 8-fold excess of DBD601 is included, as well(diamonds). (b) Same experiments as in panel a but with Cy3-labeledRND. (c) Sedimentation equilibrium profile of 1.5 μM Cy3-labeledTeloA in the presence of an 8-fold excess of DBD601 inbuffer HN50 at rotor speeds of 14000, 16000, and 18000rpm. The solid gray lines are the global analyses of the data fitwith a single-species model (see Table 2).(d) EMSA of protein–DNA complexes formed at a 1:1 ratio withdifferent 21 bp substrates at 300 nM.

Mentions: Whilethe EMSAs show evidence of the formation of higher-order DBD601–DNA complexes, the data also show that at the higher DBD601 concentrations there is a distribution of multiply ligatedspecies (Figure 1e, right panel). To test whetherthis distribution is also present in solution, we performed analyticalsedimentation velocity experiments using TeloA labeled at the 5′-endof the top strand with Cy3, monitoring Cy3 absorbance at 545 nm wherethere is no contribution from protein to the signal. Figure 2a shows the distribution of sedimentation coefficients[c(s)] obtained at different DBD601 loading concentrations in buffer HN50 [20 mMHEPES (pH 7.4), 50 mM NaCl, 2 mM MgCl2, and 10% (v/v) glycerol].At substoichiometric concentrations of DBD601, the peakcorresponding to free dsDNA is clearly observable and, within error,migrates at the same position as free DNA. At stoichiometric concentrations,only a slight population of free TeloA can be observed, while themajority of the DNA is bound in a singly ligated complex with an s20,w of 3.6 S. At a 2:1 DBD601:DNAloading ratio, only subtle changes in the s20,w value are detectable, whereas at a 10-fold excess of DBD601, the DNA-bound species shows a single peak that sediments with ahigh s20,w value (5.6 S). The molecularweight of this species estimated from the s20,w (∼99.7 kDa; P/Dcalc ∼ 2.9) suggests that approximately three DBD601 molecules bind at saturation. In Figure 2a, we also show the distribution of c(s) obtained for unlabeled TeloA in the presence of an 8-fold excessof DBD601 while monitoring the absorbance at 260 nm, wherethe protein signal is minimal at this DNA concentration. Consistentwith the data for Cy3-labeled TeloA, the protein–DNA complexsediments with a high s20,w value (5.55S). Together with the EMSA in Figure 1e, thesedata indicate that even if the label were to affect the detailed energeticsof the interaction, formation of the larger DBD601–TeloAcomplexes is label-independent. Finally, the appearance of a singlepeak in the c(s) distribution observedat this high DBD601 loading ratio strongly suggests a relativelyhomogeneous distribution of bound species. Therefore, the apparentdistribution of multiply ligated species that are observed by EMSAmost likely results from the dissociation of the higher-stoichiometrycomplexes during electrophoresis.


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

Feldmann EA, Galletto R - Biochemistry (2014)

DBD601–DNA complexesof higher strochiometrymonitored by analytical ultracentrifugation. (a) Sedimentation coefficientdistributions from velocity experiments with 2 μM Cy3-labeledTeloA in buffer HN50 at different DBD601:DNAratios: 0.5 (···), 1 (---), 2 (thin solid line), and10 (thick solid line). The data for Cy3-labeled TeloA alone are shown,as well. The distribution of sedimentation coefficients with unlabeledTeloA and an 8-fold excess of DBD601 is included, as well(diamonds). (b) Same experiments as in panel a but with Cy3-labeledRND. (c) Sedimentation equilibrium profile of 1.5 μM Cy3-labeledTeloA in the presence of an 8-fold excess of DBD601 inbuffer HN50 at rotor speeds of 14000, 16000, and 18000rpm. The solid gray lines are the global analyses of the data fitwith a single-species model (see Table 2).(d) EMSA of protein–DNA complexes formed at a 1:1 ratio withdifferent 21 bp substrates at 300 nM.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4263426&req=5

fig2: DBD601–DNA complexesof higher strochiometrymonitored by analytical ultracentrifugation. (a) Sedimentation coefficientdistributions from velocity experiments with 2 μM Cy3-labeledTeloA in buffer HN50 at different DBD601:DNAratios: 0.5 (···), 1 (---), 2 (thin solid line), and10 (thick solid line). The data for Cy3-labeled TeloA alone are shown,as well. The distribution of sedimentation coefficients with unlabeledTeloA and an 8-fold excess of DBD601 is included, as well(diamonds). (b) Same experiments as in panel a but with Cy3-labeledRND. (c) Sedimentation equilibrium profile of 1.5 μM Cy3-labeledTeloA in the presence of an 8-fold excess of DBD601 inbuffer HN50 at rotor speeds of 14000, 16000, and 18000rpm. The solid gray lines are the global analyses of the data fitwith a single-species model (see Table 2).(d) EMSA of protein–DNA complexes formed at a 1:1 ratio withdifferent 21 bp substrates at 300 nM.
Mentions: Whilethe EMSAs show evidence of the formation of higher-order DBD601–DNA complexes, the data also show that at the higher DBD601 concentrations there is a distribution of multiply ligatedspecies (Figure 1e, right panel). To test whetherthis distribution is also present in solution, we performed analyticalsedimentation velocity experiments using TeloA labeled at the 5′-endof the top strand with Cy3, monitoring Cy3 absorbance at 545 nm wherethere is no contribution from protein to the signal. Figure 2a shows the distribution of sedimentation coefficients[c(s)] obtained at different DBD601 loading concentrations in buffer HN50 [20 mMHEPES (pH 7.4), 50 mM NaCl, 2 mM MgCl2, and 10% (v/v) glycerol].At substoichiometric concentrations of DBD601, the peakcorresponding to free dsDNA is clearly observable and, within error,migrates at the same position as free DNA. At stoichiometric concentrations,only a slight population of free TeloA can be observed, while themajority of the DNA is bound in a singly ligated complex with an s20,w of 3.6 S. At a 2:1 DBD601:DNAloading ratio, only subtle changes in the s20,w value are detectable, whereas at a 10-fold excess of DBD601, the DNA-bound species shows a single peak that sediments with ahigh s20,w value (5.6 S). The molecularweight of this species estimated from the s20,w (∼99.7 kDa; P/Dcalc ∼ 2.9) suggests that approximately three DBD601 molecules bind at saturation. In Figure 2a, we also show the distribution of c(s) obtained for unlabeled TeloA in the presence of an 8-fold excessof DBD601 while monitoring the absorbance at 260 nm, wherethe protein signal is minimal at this DNA concentration. Consistentwith the data for Cy3-labeled TeloA, the protein–DNA complexsediments with a high s20,w value (5.55S). Together with the EMSA in Figure 1e, thesedata indicate that even if the label were to affect the detailed energeticsof the interaction, formation of the larger DBD601–TeloAcomplexes is label-independent. Finally, the appearance of a singlepeak in the c(s) distribution observedat this high DBD601 loading ratio strongly suggests a relativelyhomogeneous distribution of bound species. Therefore, the apparentdistribution of multiply ligated species that are observed by EMSAmost likely results from the dissociation of the higher-stoichiometrycomplexes during electrophoresis.

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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