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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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Rap1DBD binds to dsDNA in multiple DNA binding modes.(a) Cartoon model of a possible pathway from the singly ligated complex(PDB entry 3UKG), bound in the high-affinity binding mode where both Myb domainsinteract with the recognition sequence, to the higher-stoichiometrycomplexes, where now only a single Myb domain binds to the dsDNA.(b) Partition functions that describe the different models used foranalysis of the data. (c) Anisotropy binding data collected in bufferHN50 at 10 and 255 nM RND labeled at the 5′-endof the top strand. Blue lines are the fits with model 1 and red linesthose with model 2. (d) Binding data as in panel c collected for TeloA.Black, blue, and red lines are the fits with model 3, and the greenline is a fit with model 4. For details of the models, assumptions,and parameters, see the Supporting Information.
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fig6: Rap1DBD binds to dsDNA in multiple DNA binding modes.(a) Cartoon model of a possible pathway from the singly ligated complex(PDB entry 3UKG), bound in the high-affinity binding mode where both Myb domainsinteract with the recognition sequence, to the higher-stoichiometrycomplexes, where now only a single Myb domain binds to the dsDNA.(b) Partition functions that describe the different models used foranalysis of the data. (c) Anisotropy binding data collected in bufferHN50 at 10 and 255 nM RND labeled at the 5′-endof the top strand. Blue lines are the fits with model 1 and red linesthose with model 2. (d) Binding data as in panel c collected for TeloA.Black, blue, and red lines are the fits with model 3, and the greenline is a fit with model 4. For details of the models, assumptions,and parameters, see the Supporting Information.

Mentions: Wepropose that the DNA-binding domain of Rap1 can bind dsDNA inat least two different binding modes. In one high-affinity mode, bothMyb-like domains make contact with the entire recognition sequence,whereas in the other lower-affinity mode, only one of the two Myb-likedomains binds DNA. Figure 6a shows a simplemodel for a dsDNA substrate containing a Rap1 recognition sequencedepicting a possible pathway from the singly ligated complex to thehigher-stoichiometry complexes. The most recent crystal structureof the DBD bound to a TeloA sequence shows that the ∼30 aminoacids in the C-terminal region of the DBD (wrapping loop) fold backonto the N-terminal Myb-like domain to form a closed complex on DNA.23 We propose that in solution, one possible pathleading to the transition between the two binding modes is the transientopening of the C-terminal wrapping loop (Figure 6a, complex Ia-c). In the presence of excess protein, formation ofcomplex Ic would then allow a second or third molecule of Rap1DBD to bind. At this stage, we do not know which one of thetwo Myb-like domains would be bound in complex Ic. The sequence conservationof the two half-sites in the Rap1 recognition motif and the crystalstructures29,49−51 suggest thatthe N-terminal Myb domain might be a possibility. On the basis ofthe model in Figure 6a, we analyzed the anisotropybinding data for the RND and TeloA substrates with different bindingmodels (Figure 6b–d and Supporting Information). The estimated equilibriumdissociation constant of binding of DBD601 to the specificTeloA site in complex Ia (0.2–0.6 nM) is consistent with thereported value for this system29 and isat least one order of magnitude lower than that for formation of complexIc. It remains to be determined whether the transition from complexIa to Ic is accompanied by and perhaps driven by cooperative bindingof the second and third proteins in complexes II and III. At thisstage, quantitative estimates of the equilibrium constants for complexesIc–III are strongly dependent on the choice of model and assumptionsand they will require additional information about the system (seethe Supporting Information).


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

Feldmann EA, Galletto R - Biochemistry (2014)

Rap1DBD binds to dsDNA in multiple DNA binding modes.(a) Cartoon model of a possible pathway from the singly ligated complex(PDB entry 3UKG), bound in the high-affinity binding mode where both Myb domainsinteract with the recognition sequence, to the higher-stoichiometrycomplexes, where now only a single Myb domain binds to the dsDNA.(b) Partition functions that describe the different models used foranalysis of the data. (c) Anisotropy binding data collected in bufferHN50 at 10 and 255 nM RND labeled at the 5′-endof the top strand. Blue lines are the fits with model 1 and red linesthose with model 2. (d) Binding data as in panel c collected for TeloA.Black, blue, and red lines are the fits with model 3, and the greenline is a fit with model 4. For details of the models, assumptions,and parameters, see the Supporting Information.
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fig6: Rap1DBD binds to dsDNA in multiple DNA binding modes.(a) Cartoon model of a possible pathway from the singly ligated complex(PDB entry 3UKG), bound in the high-affinity binding mode where both Myb domainsinteract with the recognition sequence, to the higher-stoichiometrycomplexes, where now only a single Myb domain binds to the dsDNA.(b) Partition functions that describe the different models used foranalysis of the data. (c) Anisotropy binding data collected in bufferHN50 at 10 and 255 nM RND labeled at the 5′-endof the top strand. Blue lines are the fits with model 1 and red linesthose with model 2. (d) Binding data as in panel c collected for TeloA.Black, blue, and red lines are the fits with model 3, and the greenline is a fit with model 4. For details of the models, assumptions,and parameters, see the Supporting Information.
Mentions: Wepropose that the DNA-binding domain of Rap1 can bind dsDNA inat least two different binding modes. In one high-affinity mode, bothMyb-like domains make contact with the entire recognition sequence,whereas in the other lower-affinity mode, only one of the two Myb-likedomains binds DNA. Figure 6a shows a simplemodel for a dsDNA substrate containing a Rap1 recognition sequencedepicting a possible pathway from the singly ligated complex to thehigher-stoichiometry complexes. The most recent crystal structureof the DBD bound to a TeloA sequence shows that the ∼30 aminoacids in the C-terminal region of the DBD (wrapping loop) fold backonto the N-terminal Myb-like domain to form a closed complex on DNA.23 We propose that in solution, one possible pathleading to the transition between the two binding modes is the transientopening of the C-terminal wrapping loop (Figure 6a, complex Ia-c). In the presence of excess protein, formation ofcomplex Ic would then allow a second or third molecule of Rap1DBD to bind. At this stage, we do not know which one of thetwo Myb-like domains would be bound in complex Ic. The sequence conservationof the two half-sites in the Rap1 recognition motif and the crystalstructures29,49−51 suggest thatthe N-terminal Myb domain might be a possibility. On the basis ofthe model in Figure 6a, we analyzed the anisotropybinding data for the RND and TeloA substrates with different bindingmodels (Figure 6b–d and Supporting Information). The estimated equilibriumdissociation constant of binding of DBD601 to the specificTeloA site in complex Ia (0.2–0.6 nM) is consistent with thereported value for this system29 and isat least one order of magnitude lower than that for formation of complexIc. It remains to be determined whether the transition from complexIa to Ic is accompanied by and perhaps driven by cooperative bindingof the second and third proteins in complexes II and III. At thisstage, quantitative estimates of the equilibrium constants for complexesIc–III are strongly dependent on the choice of model and assumptionsand they will require additional information about the system (seethe Supporting Information).

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.

Show MeSH
Related in: MedlinePlus