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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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Monomers of DBD601 bind DNA with a higher than expectedstoichiometry. (a) Modular organization of the full-length Rap1 proteinsequence with selected domains highlighted: BRCT, BRCA1 C-terminaldomain; DBD, DNA-binding domain; Tox, toxicity region; Act, activationregion; RCT, Rap1 C-terminal domain. (b) Sodium dodecyl sulfate–polyacrylamidegel electrophoresis of purified DBD601 stained with CoomassieBlue. (c) Distribution of sedimentation coefficients for 20 μMDBD601 in buffer HN50 showing a single speciesof 2.4 S. (d) Sedimentation equilibrium profiles of 20 μM DBD601 in buffer HN50 at rotor speeds of 16000, 20000,and 24000 rpm. The solid gray lines are the global analyses of thedata fit with a single-species model with an observed Mw of 29.8 kDa, consistent with the Mw of a monomer. (e) Gel electrophoretic mobility shift assaysperformed at the indicated excesses of DBD601 with either30 nM (left) or 300 nM (middle) TeloA and RND labeled at the 5′-endof the top strand with FAM. The right panel shows an EMSA performedat 2 μM unlabeled TeloA and RND, stained postelectrophoresis.
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fig1: Monomers of DBD601 bind DNA with a higher than expectedstoichiometry. (a) Modular organization of the full-length Rap1 proteinsequence with selected domains highlighted: BRCT, BRCA1 C-terminaldomain; DBD, DNA-binding domain; Tox, toxicity region; Act, activationregion; RCT, Rap1 C-terminal domain. (b) Sodium dodecyl sulfate–polyacrylamidegel electrophoresis of purified DBD601 stained with CoomassieBlue. (c) Distribution of sedimentation coefficients for 20 μMDBD601 in buffer HN50 showing a single speciesof 2.4 S. (d) Sedimentation equilibrium profiles of 20 μM DBD601 in buffer HN50 at rotor speeds of 16000, 20000,and 24000 rpm. The solid gray lines are the global analyses of thedata fit with a single-species model with an observed Mw of 29.8 kDa, consistent with the Mw of a monomer. (e) Gel electrophoretic mobility shift assaysperformed at the indicated excesses of DBD601 with either30 nM (left) or 300 nM (middle) TeloA and RND labeled at the 5′-endof the top strand with FAM. The right panel shows an EMSA performedat 2 μM unlabeled TeloA and RND, stained postelectrophoresis.

Mentions: Genetic and biochemical studies show that Rap1has a modular domainorganization21 (Figure 1a). Deletion of the N-terminal region containing a singleBRCT domain does not have evident phenotypes.22 Also, it has been shown that this region does not display any detectableinteraction with the rest of the protein23 but is required for interaction with Gcr1;24 however, the precise role of the BRCT is still not fully understood.Intriguingly, overexpression of Rap1 is toxic, and part of this putative“Tox” domain comprises residues 598–616, overlappingwith the C-terminus of the DNA-binding domain.25 Deletion of the Tox domain in vivo rescuesthe toxic phenotype upon overexpression.25 The DNA-binding domain (DBD) of yeast Rap1 is centrally positionedwithin the full-length protein sequence, spanning residues 358–601based on electron density provided from the most recent crystal structureof the DBD–DNA complex.23 The regioncomprising residues 591–597, the end of the C-terminal tailof the DNA-binding domain, appears to be important for viability,although it does not dramatically affect DNA binding.23 Finally, the C-terminal region of the protein (RCT) iswhere most of the functional interactions are believed to occur,15,18,26,27 yet little is known of the linkage between Rap1 DNA binding andinteraction of the RCT with interacting factors.


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

Feldmann EA, Galletto R - Biochemistry (2014)

Monomers of DBD601 bind DNA with a higher than expectedstoichiometry. (a) Modular organization of the full-length Rap1 proteinsequence with selected domains highlighted: BRCT, BRCA1 C-terminaldomain; DBD, DNA-binding domain; Tox, toxicity region; Act, activationregion; RCT, Rap1 C-terminal domain. (b) Sodium dodecyl sulfate–polyacrylamidegel electrophoresis of purified DBD601 stained with CoomassieBlue. (c) Distribution of sedimentation coefficients for 20 μMDBD601 in buffer HN50 showing a single speciesof 2.4 S. (d) Sedimentation equilibrium profiles of 20 μM DBD601 in buffer HN50 at rotor speeds of 16000, 20000,and 24000 rpm. The solid gray lines are the global analyses of thedata fit with a single-species model with an observed Mw of 29.8 kDa, consistent with the Mw of a monomer. (e) Gel electrophoretic mobility shift assaysperformed at the indicated excesses of DBD601 with either30 nM (left) or 300 nM (middle) TeloA and RND labeled at the 5′-endof the top strand with FAM. The right panel shows an EMSA performedat 2 μM unlabeled TeloA and RND, stained postelectrophoresis.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Monomers of DBD601 bind DNA with a higher than expectedstoichiometry. (a) Modular organization of the full-length Rap1 proteinsequence with selected domains highlighted: BRCT, BRCA1 C-terminaldomain; DBD, DNA-binding domain; Tox, toxicity region; Act, activationregion; RCT, Rap1 C-terminal domain. (b) Sodium dodecyl sulfate–polyacrylamidegel electrophoresis of purified DBD601 stained with CoomassieBlue. (c) Distribution of sedimentation coefficients for 20 μMDBD601 in buffer HN50 showing a single speciesof 2.4 S. (d) Sedimentation equilibrium profiles of 20 μM DBD601 in buffer HN50 at rotor speeds of 16000, 20000,and 24000 rpm. The solid gray lines are the global analyses of thedata fit with a single-species model with an observed Mw of 29.8 kDa, consistent with the Mw of a monomer. (e) Gel electrophoretic mobility shift assaysperformed at the indicated excesses of DBD601 with either30 nM (left) or 300 nM (middle) TeloA and RND labeled at the 5′-endof the top strand with FAM. The right panel shows an EMSA performedat 2 μM unlabeled TeloA and RND, stained postelectrophoresis.
Mentions: Genetic and biochemical studies show that Rap1has a modular domainorganization21 (Figure 1a). Deletion of the N-terminal region containing a singleBRCT domain does not have evident phenotypes.22 Also, it has been shown that this region does not display any detectableinteraction with the rest of the protein23 but is required for interaction with Gcr1;24 however, the precise role of the BRCT is still not fully understood.Intriguingly, overexpression of Rap1 is toxic, and part of this putative“Tox” domain comprises residues 598–616, overlappingwith the C-terminus of the DNA-binding domain.25 Deletion of the Tox domain in vivo rescuesthe toxic phenotype upon overexpression.25 The DNA-binding domain (DBD) of yeast Rap1 is centrally positionedwithin the full-length protein sequence, spanning residues 358–601based on electron density provided from the most recent crystal structureof the DBD–DNA complex.23 The regioncomprising residues 591–597, the end of the C-terminal tailof the DNA-binding domain, appears to be important for viability,although it does not dramatically affect DNA binding.23 Finally, the C-terminal region of the protein (RCT) iswhere most of the functional interactions are believed to occur,15,18,26,27 yet little is known of the linkage between Rap1 DNA binding andinteraction of the RCT with interacting factors.

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