Altered DNA Recognition and Bending by Insertions in the α2 Tail of the Yeast a1/α2 Homeodomain Heterodimer

Jin, Yisheng; Mead, Janet E.; Li, Thomas; Wolberger, Cynthia; Vershon, Andrew K.

doi:10.1126/science.270.5234.290

Cited by 41 publications

(21 citation statements)

References 27 publications

(9 reference statements)

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“…The a1 and ␣2 homeodomain proteins, which bind cooperatively as a heterodimer to hsg sites, interact via a carboxy-terminal tail in ␣2 that tethers the a1 and ␣2 homeodomains together (11,29,30). Amino acid insertions in the extended region of the ␣2 tail suppress defective a1-␣2 sites with increased spacing between the a1 and ␣2 half sites (21). The spatial constraints between ␣2 and Mcm1 are also in contrast with the distance requirements of the human ETS domain proteins, Elk-1 and SAP-1, which bind cooperatively with the MADS box protein, serum response factor (SRF) (47).…”

Section: Discussionmentioning

confidence: 99%

The Yeast α2 and Mcm1 Proteins Interact through a Region Similar to a Motif Found in Homeodomain Proteins of Higher Eukaryotes

Mead

Zhong

Acton

et al. 1996

Molecular and Cellular Biology

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Homeodomain proteins are transcriptional regulatory factors that, in general, bind DNA with relatively low sequence specificity and affinity. One mechanism homeodomain proteins use to increase their biological specificity is through interactions with other DNA-binding proteins. We have examined how the yeast (Saccharomyces cerevisiae) homeodomain protein ␣2 specifically interacts with Mcm1, a MADS box protein, to bind DNA specifically and repress transcription. A patch of predominantly hydrophobic residues within a region preceding the homeodomain of ␣2 has been identified that specifies direct interaction with Mcm1 in the absence of DNA. This hydrophobic patch is required for cooperative DNA binding with Mcm1 in vitro and for transcriptional repression in vivo. We have also found that a conserved motif, termed YPWM, frequently found in homeodomain proteins of insects and mammals, partially functions in place of the patch in ␣2 to interact with Mcm1. These findings suggest that homeodomain proteins from diverse organisms may use analogous interaction motifs to associate with other proteins to achieve high levels of DNA binding affinity and specificity.

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Section: Discussionmentioning

confidence: 99%

The Yeast α2 and Mcm1 Proteins Interact through a Region Similar to a Motif Found in Homeodomain Proteins of Higher Eukaryotes

Mead

Zhong

Acton

et al. 1996

Molecular and Cellular Biology

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“…They have shown that, as expected, circularization of the duplex template so as to compress the major groove by 17-20°over 7 bp results in an increase in TBP binding affinity. The (36) investigated the binding of the al/a2 homeodomain protein heterodimer, which is known to bend duplex DNA so as to compress the minor groove near the center of its bound complex (37). Protein mutants were also studied which possessed one, two or three glycine residues inserted into the linker region which connects the two monomer subunits.…”

Section: Discussionmentioning

confidence: 99%

The design of an agent to bend DNA.

Akiyama¹,

Hogan²

1996

Proc. Natl. Acad. Sci. U.S.A.

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An artificial DNA bending agent has been designed to assess helix flexibility over regions as small as a protein binding site. Bending was obtained by linking a pair of 15-base-long triple helix forming oligonucleotides (TFOs) by an adjustable polymeric linker. By design, DNA bending was introduced into the double helix within a 10-bp spacer region positioned between the two sites of 15-base triple helix formation. The existence of this bend has been confirmed by circular permutation and phase-sensitive electrophoresis, and the directionality of the bend has been determined as a compression of the minor helix groove. The magnitude of the resulting duplex bend was found to be dependent on the length of the polymeric linker in a fashion consistent with a simple geometric model. Data suggested that a 50-70°bend was achieved by binding of the TFO chimera with the shortest linker span (18 rotatable bonds). Equilibrium analysis showed that, relative to a chimera which did not bend the duplex, the stability of the triple helix possessing a 50-70°b end was reduced by less than 1 kcal/mol of that of the unbent complex. Based upon this similarity, it is proposed that duplex DNA may be much more flexible with respect to minor groove compression than previously assumed. It is shown that this unusual flexibility is consistent with recent quantitation of protein-induced minor groove bending.DNA bending is essential for packaging into the nucleosome (1), for transcription (2, 3), and recombination (4). Many examples of protein-induced bending (5-8) and structural deformation of DNA by chemical modification (9, 10) have been reported. Ring closure, hydrodynamics, and polarization methods have provided a good estimate of the average work required to bend long duplex segments (11,12). Although elegant calculational methods have been described, experimental methods are not yet generally available to probe the mechanical properties of DNA over segments the size of a protein binding site. As a first step toward the development of tools for microscopic DNA flexibility analysis, we describe here the use of a nucleic acid chimera, comprising a pair of triple helix forming oligonucleotide (TFOs) connected by an inert, adjustable polymeric linker.Triple helix formation has been widely studied as a method to achieve the rational design of ligands which bind to duplex DNA in a site-selective fashion (13-15). As a variation on that general theme, it was reported that two TFOs connected by a flexible polymeric linker displayed cooperative binding to a pair of cognate duplex binding sites separated by one helical turn (16). At that binding site, the intervening turn of duplex was spanned by the polymeric linker at the surface of the duplex, traversing a path which passes over the top of the minor groove (Fig. la Right). In that study, molecular modeling and experiment suggested that a polymeric linker of 25 rotatable bonds would be sufficient to span the duplex without distortion. As a corollary of that observation, we have reasoned t...

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“…The fact that in the presence of Mcm1 sites were selected from the random pool which have both 5-and 6-bp spacing further shows that binding by the ␣2-Mcm1 complex can accommodate either spacing. In contrast, the spacing requirements between the ␣2 and a1 binding sites of haploid-specific operators, as well as the positions of the binding sites in other homeodomain complexes such as the Drosophila Paired homodimer and the Hox-Pbx heterodimer are rigidly fixed (43,53,54). These results suggest that either the proteinprotein or protein-DNA interactions in the ␣2-Mcm1-DNA complex can adjust, to some extent, to accommodate the alterations in spacing between the binding sites.…”

Section: Discussionmentioning

confidence: 41%

“…Electrophoretic Mobility Shift Assays (EMSAs)-The relative ␣2-Mcm1 DNA binding affinities for the AMSC and mutant operators were determined by EMSA as described previously (43). Labeled DNA fragments containing ␣2-Mcm1 sites were incubated with purified ␣2-(92-210) and Mcm1-(1-96) proteins at the concentrations given in the figure legends at room temperature for 3 h. The ␣2 and Mcm1 proteins used in these experiments were purified as described previously and were greater than 90% pure as judged on Coomassie-stained SDS-polyacrylamide gels (40).…”

Section: Methodsmentioning

confidence: 99%

The Yeast Homeodomain Protein MATα2 Shows Extended DNA binding Specificity in Complex with Mcm1

Zhong

Vershon

1997

Journal of Biological Chemistry

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The MAT␣2 (␣2) repressor interacts with the Mcm1 protein to turn off a-cell type-specific genes in the yeast Saccharomyces cerevisiae. We compared five natural ␣2-Mcm1 sites with an ␣2-Mcm1 symmetric consensus site (AMSC) for their relative strength of repression and found that the AMSC functions slightly better than any of the natural sites. To further investigate the DNA binding specificity of ␣2 in complex with Mcm1, symmetric substitutions at each position in the ␣2 half-sites of AMSC were constructed and assayed for their effect on repression in vivo and DNA binding affinity in vitro. As expected, substitutions at positions in which there are base-specific contacts decrease the level of repression. Interestingly, substitutions at other positions, in which there are no apparent base-specific contacts made by the protein in the ␣2-DNA co-crystal structure, also significantly decrease repression. As an alternative method to examining the DNA binding specificity of ␣2, we performed in vitro ␣2 binding site selection experiments in the presence and absence of Mcm1. In the presence of Mcm1, the consensus sequences obtained were extended and more closely related to the natural ␣2 sites than the consensus sequence obtained in the absence of Mcm1. These results demonstrate that in the presence of Mcm1 the sequence specificity of ␣2 is extended to these positions.Homeodomain proteins are a family of transcription factors involved in many developmental and cellular processes and have been found in almost every eukaryotic organism (1-4). The natural target sites for many homeodomain proteins are unknown; therefore, their DNA-binding sites have been defined through site selection experiments in vitro (5-9). Although these studies provide important information on homeodomain-DNA recognition in vitro, in some cases it appears that the homeodomain proteins do not function well at these sites in vivo (10 -14). One possible explanation for this discrepancy is that in vivo the DNA binding specificity and affinity of some homeodomain proteins may be influenced by interactions with cofactors (13,(15)(16)(17)(18)(19). Since many of the studies which examine homeodomain binding sites have been done in the absence of cofactors, this may explain why in some cases sites selected in vitro may not be functional sites for the homeodomain protein complexes in vivo. To address this issue and to understand how homeodomain proteins recognize specific sites, we have investigated the DNA binding specificity of ␣2, a yeast homeodomain protein, in which the natural target sites and cofactors are well known.The ␣2 protein is involved in the regulatory system that specifies cell mating type in Saccharomyces cerevisiae (20 -24). In diploid cells, the ␣2 and a1 proteins form a heterodimer to repress expression of haploid-specific genes (25, 26). In haploid ␣ cells and diploid cells, ␣2 interacts with a general transcription regulatory factor, Mcm1, to repress expression of a-specific genes (asg) 1 (27-29). DNase I protection and deletion experimen...

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Altered DNA Recognition and Bending by Insertions in the α2 Tail of the Yeast a1/α2 Homeodomain Heterodimer

Cited by 41 publications

References 27 publications

The Yeast α2 and Mcm1 Proteins Interact through a Region Similar to a Motif Found in Homeodomain Proteins of Higher Eukaryotes

The Yeast α2 and Mcm1 Proteins Interact through a Region Similar to a Motif Found in Homeodomain Proteins of Higher Eukaryotes

The design of an agent to bend DNA.

The Yeast Homeodomain Protein MATα2 Shows Extended DNA binding Specificity in Complex with Mcm1

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