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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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Higher salt concentration abolishes bindingof the third DBD601 molecule. (a) Change in fluorescenceanisotropy (top) andrelative total intensity (bottom) as a function of DBD601:DNA ratio for 255 nM TeloA FAM-labeled at the 3′-end of thetop strand in buffer H at 50, 100, and 150 mM NaCl. (b) Change inrelative total intensity as a function of DBD601:DNA ratioin buffer HN150 of 255 nM (black square) and 760 nM (graysquare) HMRE FAM-labeled at the 3′-end of the top strand. Thetitration for 255 nM TeloA is shown as a reference (triangles).
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fig4: Higher salt concentration abolishes bindingof the third DBD601 molecule. (a) Change in fluorescenceanisotropy (top) andrelative total intensity (bottom) as a function of DBD601:DNA ratio for 255 nM TeloA FAM-labeled at the 3′-end of thetop strand in buffer H at 50, 100, and 150 mM NaCl. (b) Change inrelative total intensity as a function of DBD601:DNA ratioin buffer HN150 of 255 nM (black square) and 760 nM (graysquare) HMRE FAM-labeled at the 3′-end of the top strand. Thetitration for 255 nM TeloA is shown as a reference (triangles).

Mentions: The data presented in the previous sections were determined inthe presence of a relatively low concentration of NaCl (50 mM) toamplify the presence of all possible bound states of DBD601. Next, we explored the effect of increasing NaCl concentrationson the ability of DBD601 to access higher stoichiometries.The top panel of Figure 4a shows the changein fluorescence anisotropy of the TeloA substrate (labeled at the3′-end of the top strand with FAM) as a function of the ratioof the total protein to DNA concentrations in buffer H with 50, 100,and 150 mM NaCl. It is evident that as the concentration of NaCl increases,there is a strong effect on the anisotropy corresponding to bindingof the second and third DBD601 molecules. At the same time,binding of the first DBD601 molecule, as monitored by theinitial phase of the anisotropy, is little affected by a 3-fold increasein NaCl concentration. Similar behavior is also observed when therelative total fluorescence intensity is monitored (Figure 4a, bottom panel). Increasing the NaCl concentrationaffects the binding of only the second and third DBD601 molecules (loss of the large fluorescence increase). Taken at facevalue, these data would suggest that higher salt concentrations inhibitformation of the higher-stoichiometry complexes. However, Figure 4b shows the change in the relative total fluorescenceintensity for both TeloA and the lower-affinity HMRE, determined at150 mM NaCl. At the same concentration of DNA for both substrates,DBD601 induces an initial quenching of the fluorophorefollowed by a small yet detectable fluorescence increase. This secondphase of fluorescence enhancement becomes more evident when the concentrationof HMRE is increased 3-fold, suggesting that this signal originatesfrom a low-affinity binding phase. The simple observation of a changefrom quenching to an enhancement of the relative fluorescence intensityindicates that at least one additional molecule of DBD601 must bind even at this higher NaCl concentration. This conclusionis further reinforced by analytical equilibrium ultracentrifugationexperiments with DBD601–DNA complexes formed inbuffer H at different NaCl concentrations (Table 2). While at 100 mM NaCl DBD601 is still able toaccess a stoichiometry of 3:1 on any of the substrates used, at 150mM NaCl it is clear that only two molecules of DBD601 canbind at saturation. Interestingly, for the TeloA substrate, a 2:1stoichiometry becomes more evident with a larger protein excess, suggestingthat at this NaCl concentration it is more difficult to populate thedoubly ligated species with this high-affinity Rap1 recognition sequence.Indeed, the doubly ligated species for the lower-affinity HMRE sequenceis more easily detectable at a smaller excess of DBD601. These data clearly show that higher NaCl concentrations preventbinding of a third DBD601 molecule but still allow forformation of at least a 2:1 complex even when a Rap1 recognition sequenceis present in the substrate.


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

Feldmann EA, Galletto R - Biochemistry (2014)

Higher salt concentration abolishes bindingof the third DBD601 molecule. (a) Change in fluorescenceanisotropy (top) andrelative total intensity (bottom) as a function of DBD601:DNA ratio for 255 nM TeloA FAM-labeled at the 3′-end of thetop strand in buffer H at 50, 100, and 150 mM NaCl. (b) Change inrelative total intensity as a function of DBD601:DNA ratioin buffer HN150 of 255 nM (black square) and 760 nM (graysquare) HMRE FAM-labeled at the 3′-end of the top strand. Thetitration for 255 nM TeloA is shown as a reference (triangles).
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Show All Figures
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fig4: Higher salt concentration abolishes bindingof the third DBD601 molecule. (a) Change in fluorescenceanisotropy (top) andrelative total intensity (bottom) as a function of DBD601:DNA ratio for 255 nM TeloA FAM-labeled at the 3′-end of thetop strand in buffer H at 50, 100, and 150 mM NaCl. (b) Change inrelative total intensity as a function of DBD601:DNA ratioin buffer HN150 of 255 nM (black square) and 760 nM (graysquare) HMRE FAM-labeled at the 3′-end of the top strand. Thetitration for 255 nM TeloA is shown as a reference (triangles).
Mentions: The data presented in the previous sections were determined inthe presence of a relatively low concentration of NaCl (50 mM) toamplify the presence of all possible bound states of DBD601. Next, we explored the effect of increasing NaCl concentrationson the ability of DBD601 to access higher stoichiometries.The top panel of Figure 4a shows the changein fluorescence anisotropy of the TeloA substrate (labeled at the3′-end of the top strand with FAM) as a function of the ratioof the total protein to DNA concentrations in buffer H with 50, 100,and 150 mM NaCl. It is evident that as the concentration of NaCl increases,there is a strong effect on the anisotropy corresponding to bindingof the second and third DBD601 molecules. At the same time,binding of the first DBD601 molecule, as monitored by theinitial phase of the anisotropy, is little affected by a 3-fold increasein NaCl concentration. Similar behavior is also observed when therelative total fluorescence intensity is monitored (Figure 4a, bottom panel). Increasing the NaCl concentrationaffects the binding of only the second and third DBD601 molecules (loss of the large fluorescence increase). Taken at facevalue, these data would suggest that higher salt concentrations inhibitformation of the higher-stoichiometry complexes. However, Figure 4b shows the change in the relative total fluorescenceintensity for both TeloA and the lower-affinity HMRE, determined at150 mM NaCl. At the same concentration of DNA for both substrates,DBD601 induces an initial quenching of the fluorophorefollowed by a small yet detectable fluorescence increase. This secondphase of fluorescence enhancement becomes more evident when the concentrationof HMRE is increased 3-fold, suggesting that this signal originatesfrom a low-affinity binding phase. The simple observation of a changefrom quenching to an enhancement of the relative fluorescence intensityindicates that at least one additional molecule of DBD601 must bind even at this higher NaCl concentration. This conclusionis further reinforced by analytical equilibrium ultracentrifugationexperiments with DBD601–DNA complexes formed inbuffer H at different NaCl concentrations (Table 2). While at 100 mM NaCl DBD601 is still able toaccess a stoichiometry of 3:1 on any of the substrates used, at 150mM NaCl it is clear that only two molecules of DBD601 canbind at saturation. Interestingly, for the TeloA substrate, a 2:1stoichiometry becomes more evident with a larger protein excess, suggestingthat at this NaCl concentration it is more difficult to populate thedoubly ligated species with this high-affinity Rap1 recognition sequence.Indeed, the doubly ligated species for the lower-affinity HMRE sequenceis more easily detectable at a smaller excess of DBD601. These data clearly show that higher NaCl concentrations preventbinding of a third DBD601 molecule but still allow forformation of at least a 2:1 complex even when a Rap1 recognition sequenceis present in the substrate.

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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