Limits...
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.

Show MeSH

Related in: MedlinePlus

ITC shows a complex behaviorconsistent with formation of higher-ordercomplexes, even at higher salt concentrations. (a) Raw heats of bindingfor a representative titration of DBD601 into TeloA inbuffer HN50. Also included (offset) is the contributionfor the heat of dilution from a reference titration of DBD601. (b) Change in normalized heat as a function of molar ratio for20 μM TeloA titrated into 2 μM DBD601 (black)and 20 μM DBD601 titrated into 2 μM TeloA (gray)in buffer HN50 at 20 °C. (c) Change in normalizedheat as a function of molar ratio for the same experiments as in panelb but performed in buffer HN150 at 20 °C.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4263426&req=5

fig5: ITC shows a complex behaviorconsistent with formation of higher-ordercomplexes, even at higher salt concentrations. (a) Raw heats of bindingfor a representative titration of DBD601 into TeloA inbuffer HN50. Also included (offset) is the contributionfor the heat of dilution from a reference titration of DBD601. (b) Change in normalized heat as a function of molar ratio for20 μM TeloA titrated into 2 μM DBD601 (black)and 20 μM DBD601 titrated into 2 μM TeloA (gray)in buffer HN50 at 20 °C. (c) Change in normalizedheat as a function of molar ratio for the same experiments as in panelb but performed in buffer HN150 at 20 °C.

Mentions: The data in the previous sections clearly showthat depending on the solution conditions, more than one moleculeof DBD601 can bind to the model TeloA substrate. We alsonote that all the experiments presented thus far were performed ina way that enriches the population of the higher-stoichiometry complexes(e.g., increasing protein concentration). We have not yet found spectroscopicsignal changes that would allow us to reliably monitor the interactionin the other direction (e.g., increasing DNA concentration to favorformation of the singly ligated species). However, for full-lengthRap1, it has been shown that interaction with DNA can be monitoredby isothermal titration calorimetry (ITC), at least in the directionthat favors formation of a 1:1 complex (e.g., dsDNA as the titrant).23,46 Therefore, we used ITC to study the binding of DBD601 to the TeloA substrate and performed titrations to access differentend states in the reaction. Figure 5a showsthe raw heats of injection for a selected direction (the referencetitration is shown offset). Figure 5b showsthe change in normalized heat as a function of molar ratio for titrationsperformed in either direction, DBD601 titrated into DNA(gray) or DNA titrated into DBD601 (black). It is clearthat the titrations are not symmetric, as would be expected for asimple 1:1 interaction. Consistent with all the data presented inthe previous sections, these data are indicative of a complex systemin which different bound states can be achieved depending on the directionin which the experiment is performed. At a low molar ratio (i.e.,excess protein to DNA) when TeloA is titrated into DBD601, the reaction is accompanied by an initial phase with a large negativeheat (approximately −40 kcal/mol). On the basis of the experimentspresented so far, formation of a 3:1 complex of protein to DNA isexpected to be favored in this concentration regime. Further increasesin the total DNA concentration will then favor dissociation of thethird DBD molecule and allow transition to lower-stoichiometry complexes.Indeed, the normalized heat decreases linearly to a ratio of ∼0.5and then is followed by a steep drop in signal with a midpoint of ∼0.75DNA/protein. In this direction of the titration, it would be expectedthat the reaction should reach a final stoichiometry of 1:1. It is,however, possible that the observed lower than expected midpoint ofthis second phase is affected by the large differences in affinity,and possibly the ΔH between the doubly andsingly ligated species. The situation is very different when DBD601 is used as the titrant. In this direction, at a lower molarratio (i.e., excess DNA to protein), formation of the singly ligatedspecies is favored and likewise accompanied by an initial relativelyconstant ΔH value that is approximately halfof that observed in the titrations performed in the opposite direction.The observed value in this direction (approximately −20 kcal/mol)provides an estimate of the ΔH for the singlyligated species. As the protein concentration increases, the molarheat decreases with a midpoint of ∼1.3 stoichiometry. Onceagain, this is a bit surprising on the basis of the data presentedin the previous sections, because in this direction of the titrationthe 3:1 protein–DNA complex should become enriched. The lowerthan expected stoichiometry suggests that formation of the high-affinity,singly ligated complex dominates the signal of the reaction, the netresult being that any subsequent lower-affinity binding event remainsundetectable under the conditions tested.


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

Feldmann EA, Galletto R - Biochemistry (2014)

ITC shows a complex behaviorconsistent with formation of higher-ordercomplexes, even at higher salt concentrations. (a) Raw heats of bindingfor a representative titration of DBD601 into TeloA inbuffer HN50. Also included (offset) is the contributionfor the heat of dilution from a reference titration of DBD601. (b) Change in normalized heat as a function of molar ratio for20 μM TeloA titrated into 2 μM DBD601 (black)and 20 μM DBD601 titrated into 2 μM TeloA (gray)in buffer HN50 at 20 °C. (c) Change in normalizedheat as a function of molar ratio for the same experiments as in panelb but performed in buffer HN150 at 20 °C.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: ITC shows a complex behaviorconsistent with formation of higher-ordercomplexes, even at higher salt concentrations. (a) Raw heats of bindingfor a representative titration of DBD601 into TeloA inbuffer HN50. Also included (offset) is the contributionfor the heat of dilution from a reference titration of DBD601. (b) Change in normalized heat as a function of molar ratio for20 μM TeloA titrated into 2 μM DBD601 (black)and 20 μM DBD601 titrated into 2 μM TeloA (gray)in buffer HN50 at 20 °C. (c) Change in normalizedheat as a function of molar ratio for the same experiments as in panelb but performed in buffer HN150 at 20 °C.
Mentions: The data in the previous sections clearly showthat depending on the solution conditions, more than one moleculeof DBD601 can bind to the model TeloA substrate. We alsonote that all the experiments presented thus far were performed ina way that enriches the population of the higher-stoichiometry complexes(e.g., increasing protein concentration). We have not yet found spectroscopicsignal changes that would allow us to reliably monitor the interactionin the other direction (e.g., increasing DNA concentration to favorformation of the singly ligated species). However, for full-lengthRap1, it has been shown that interaction with DNA can be monitoredby isothermal titration calorimetry (ITC), at least in the directionthat favors formation of a 1:1 complex (e.g., dsDNA as the titrant).23,46 Therefore, we used ITC to study the binding of DBD601 to the TeloA substrate and performed titrations to access differentend states in the reaction. Figure 5a showsthe raw heats of injection for a selected direction (the referencetitration is shown offset). Figure 5b showsthe change in normalized heat as a function of molar ratio for titrationsperformed in either direction, DBD601 titrated into DNA(gray) or DNA titrated into DBD601 (black). It is clearthat the titrations are not symmetric, as would be expected for asimple 1:1 interaction. Consistent with all the data presented inthe previous sections, these data are indicative of a complex systemin which different bound states can be achieved depending on the directionin which the experiment is performed. At a low molar ratio (i.e.,excess protein to DNA) when TeloA is titrated into DBD601, the reaction is accompanied by an initial phase with a large negativeheat (approximately −40 kcal/mol). On the basis of the experimentspresented so far, formation of a 3:1 complex of protein to DNA isexpected to be favored in this concentration regime. Further increasesin the total DNA concentration will then favor dissociation of thethird DBD molecule and allow transition to lower-stoichiometry complexes.Indeed, the normalized heat decreases linearly to a ratio of ∼0.5and then is followed by a steep drop in signal with a midpoint of ∼0.75DNA/protein. In this direction of the titration, it would be expectedthat the reaction should reach a final stoichiometry of 1:1. It is,however, possible that the observed lower than expected midpoint ofthis second phase is affected by the large differences in affinity,and possibly the ΔH between the doubly andsingly ligated species. The situation is very different when DBD601 is used as the titrant. In this direction, at a lower molarratio (i.e., excess DNA to protein), formation of the singly ligatedspecies is favored and likewise accompanied by an initial relativelyconstant ΔH value that is approximately halfof that observed in the titrations performed in the opposite direction.The observed value in this direction (approximately −20 kcal/mol)provides an estimate of the ΔH for the singlyligated species. As the protein concentration increases, the molarheat decreases with a midpoint of ∼1.3 stoichiometry. Onceagain, this is a bit surprising on the basis of the data presentedin the previous sections, because in this direction of the titrationthe 3:1 protein–DNA complex should become enriched. The lowerthan expected stoichiometry suggests that formation of the high-affinity,singly ligated complex dominates the signal of the reaction, the netresult being that any subsequent lower-affinity binding event remainsundetectable under the conditions tested.

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