Citation Context

...ge mutagenesis with the primer 5 0 -AAGATCTGGTTCCAGTAATAACGGGCCAAGATCAAG-3 0 and its complement. To construct EnHD-P3 and H6-EnHD-P3, the respective P8 constructs were subjected to Kunkel mutagenesis =-=(28)-=- with primer 5 0 -GCGGGCCAAGATCAAGAAGTCGACGGATTTTGATTATGAAAAGATGGC-3 0 . Phage production To obtain purified phage stocks, phages were propagated and precipitated essentially as described previously (...

Citation Context

...e (Figure 1). EnHD, the prototypical and widely studied HD, binds to the consensus sequence TAATTA (15,19). In the major groove, base-specific contacts are made primarily by residues I47, Q50 and N51 =-=(10,11,20)-=-. Residue N51 is highly conserved though all HDs and the N51A mutation in EnHD abrogate binding to DNA (15). The contributions of Q50 and I47 to binding are less pronounced; I47A and Q50A mutations le...

Citation Context

... factor (1). Despite this limitation, transcription factors with altered DNA-binding behavior have been generated by selecting the desired mutants from large libraries of mutant transcription factors =-=(2)-=-. For example, phage display systems have been developed to isolate mutant zinc finger transcription factors with altered DNA-binding specificities (2–4). From these selections, mutant zinc fingers ha...

Citation Context

...The complexity of protein–DNA interfaces presents a challenge to protein engineering; no simple set of rules is available to predictably alter the DNA-binding activity of a given transcription factor =-=(1)-=-. Despite this limitation, transcription factors with altered DNA-binding behavior have been generated by selecting the desired mutants from large libraries of mutant transcription factors (2). For ex...

Citation Context

...NA-binding domains (e.g. homeodomains have K d values on the order of 1 nM). Homeodomains, the DNA-binding domains of the homeotic proteins, play an important role in the development of all metazoans =-=(8)-=-. Over 1000 protein sequences of HDs are currently available, of which over 100 are from humans, and some mutations in these human HDs are known to cause disease (9). In order to facilitate the study ...

Citation Context

...udies of HDs reveal that these domains fold into a highly conserved structure composed of three alpha-helices in which the C-terminal helix makes sequence-specific contacts in the major groove of DNA =-=(18)-=-. The other two helices pack against the third, and the N-terminus of the HD makes additional important contacts in the DNA minor groove (Figure 1). EnHD, the prototypical and widely studied HD, binds...

Citation Context

...ate mutant zinc finger transcription factors with altered DNA-binding specificities (2–4). From these selections, mutant zinc fingers have been isolated that bind selectively to desired DNA sequences =-=(5)-=-. These engineered transcription factors hold promise for numerous biomedical applications [see Jamieson et al. (6) and references therein] and provide valuable insight into the subtle and complex rea...

Citation Context

...) with primer 5 0 -GCGGGCCAAGATCAAGAAGTCGACGGATTTTGATTATGAAAAGATGGC-3 0 . Phage production To obtain purified phage stocks, phages were propagated and precipitated essentially as described previously =-=(29)-=-. Briefly, the phagemid constructs described above were transformed into XL1-Blue Subcloning Efficiency Cells and plated on LB Agar supplemented with 100 mg/ml ampicillin. Individual colonies were pic...

Citation Context

...utant zinc fingers have been isolated that bind selectively to desired DNA sequences (5). These engineered transcription factors hold promise for numerous biomedical applications [see Jamieson et al. =-=(6)-=- and references therein] and provide valuable insight into the subtle and complex rearrangements possible at the protein–DNA interface (7). Despite the successful engineering of zinc fingers, in vitro...

Citation Context

...Table 1, library B). In light of the results from this more complex library, this work serves as the foundation to study HDs with rapid functional epitope mapping techniques, such as shotgun scanning =-=(30,35,36)-=-. Furthermore, this phage display system has been instrumental toward isolating HDs that bind specifically to unnatural, synthetic nucleotides (37). We conclude that the selection system reported here...

Citation Context

...ise for numerous biomedical applications [see Jamieson et al. (6) and references therein] and provide valuable insight into the subtle and complex rearrangements possible at the protein–DNA interface =-=(7)-=-. Despite the successful engineering of zinc fingers, in vitro selection systems for most other important families of DNAbinding domains, including the homeodomains (HDs), have not been described. Thi...

Citation Context

...st the third, and the N-terminus of the HD makes additional important contacts in the DNA minor groove (Figure 1). EnHD, the prototypical and widely studied HD, binds to the consensus sequence TAATTA =-=(15,19)-=-. In the major groove, base-specific contacts are made primarily by residues I47, Q50 and N51 (10,11,20). Residue N51 is highly conserved though all HDs and the N51A mutation in EnHD abrogate binding ...

Citation Context

...e (Figure 1). EnHD, the prototypical and widely studied HD, binds to the consensus sequence TAATTA (15,19). In the major groove, base-specific contacts are made primarily by residues I47, Q50 and N51 =-=(10,11,20)-=-. Residue N51 is highly conserved though all HDs and the N51A mutation in EnHD abrogate binding to DNA (15). The contributions of Q50 and I47 to binding are less pronounced; I47A and Q50A mutations le...

Citation Context

... To avoid biases associated with disproportionately represented orthologs in the HD Resource, we chose to compare the selected mutants to the 129 human HDs previously examined by Banerjee-Basu et al. =-=(33)-=-. The majority of sequences recovered were either similar to, or identical to the natural HDs (Figure 4); the two most frequently encountered residues at position 47 are valine and isoleucine, both fr...

Citation Context

...ACCATCACCATGACGAGAAGCGTCCAC-3 0 (for H6-EnHD-P8), and 5 0 -TTTTTTCTTAAGCGGATTTTTGGAGCCCGTCGACTTCTTGATCTT-3 0 , the product was digested with NsiI and AflII and then ligated into the phagemid pSO1148a =-=(27)-=-. The stop template was constructed using QuikChange mutagenesis with the primer 5 0 -AAGATCTGGTTCCAGTAATAACGGGCCAAGATCAAG-3 0 and its complement. To construct EnHD-P3 and H6-EnHD-P3, the respective P...

Citation Context

...n the development of all metazoans (8). Over 1000 protein sequences of HDs are currently available, of which over 100 are from humans, and some mutations in these human HDs are known to cause disease =-=(9)-=-. In order to facilitate the study and engineering of this important family of DNA-binding domains, we have developed a phage display system to select functional mutants of engrailed HD (EnHD). Struct...

Citation Context

...ld. b The high proportion of mutations at position 47 is due to the codon (RST) employed in the mutagenesis. from library A were compared with the sequences of natural HDs in The Homeodomain Resource =-=(31,32)-=-. To avoid biases associated with disproportionately represented orthologs in the HD Resource, we chose to compare the selected mutants to the 129 human HDs previously examined by Banerjee-Basu et al....

Citation Context

...l contributions to binding affinity, residues I47 and Q50 remain important to HD–DNA recognition. For example, the subtle I47L missense mutation in the HD of HOXD13 causes limb malformation in humans =-=(21)-=-. The elusive role of homeodomain residue Q50 has received considerable attention (14,15,17,22–26). Early investigations focused on a Q50K mutation that alters homeodomain DNAbinding specificity from ...

Citation Context

...ation in EnHD abrogate binding to DNA (15). The contributions of Q50 and I47 to binding are less pronounced; I47A and Q50A mutations lead to 20- and 2fold reductions in binding affinity, respectively =-=(15,16)-=-. Despite their relatively small contributions to binding affinity, residues I47 and Q50 remain important to HD–DNA recognition. For example, the subtle I47L missense mutation in the HD of HOXD13 caus...

Citation Context

...city compared with the wild-type protein (15). Furthermore, recent structural comparisons between the Q50 (WT) and Q50A crystal structures demonstrate only subtle changes at the protein–DNA interface =-=(22)-=-. Together, these data suggest that Q50 plays only a modest role in *To whom correspondence should be addressed. Tel: +1 415 514 0472; Fax: +1 415 514 0822; Email: shokat@cmp.ucsf.edu Nucleic Acids Re...

Citation Context

...ATA20. Clones that bound to TAATTA20, but not TATATA20, were sequenced. The EnHD-encoding portion of the selected phage clones was sequenced using two sequential PCR reactions as described previously =-=(30)-=-. Expression of mutant homeodomains Mutant homeodomains were expressed off-phage as maltosebinding protein fusions, and purified to near homogeneity using amylose resin. Mutations were introduced usin...

Citation Context

...e (Figure 1). EnHD, the prototypical and widely studied HD, binds to the consensus sequence TAATTA (15,19). In the major groove, base-specific contacts are made primarily by residues I47, Q50 and N51 =-=(10,11,20)-=-. Residue N51 is highly conserved though all HDs and the N51A mutation in EnHD abrogate binding to DNA (15). The contributions of Q50 and I47 to binding are less pronounced; I47A and Q50A mutations le...

Citation Context

...properties of the EnHD makes it a valuable groundwork toward the engineering of the EnHD–DNA interface. MATERIALS AND METHODS Plasmid construction Residues 1 to 60 of EnHD were amplified from pMal-En =-=(17)-=- using primers with the sequence 5 0 -GGCTATCGGATGCATCGGACGAGAAGCGTCCAC-3 0 (EnHD-P8 without hexa-histidine) or 5 0 -GGCTATCGGATGCATCGCACCATCACCATCACCATGACGAGAAGCGTCCAC-3 0 (for H6-EnHD-P8), and 5 0 -...

Citation Context

...st the third, and the N-terminus of the HD makes additional important contacts in the DNA minor groove (Figure 1). EnHD, the prototypical and widely studied HD, binds to the consensus sequence TAATTA =-=(15,19)-=-. In the major groove, base-specific contacts are made primarily by residues I47, Q50 and N51 (10,11,20). Residue N51 is highly conserved though all HDs and the N51A mutation in EnHD abrogate binding ...

Citation Context

...ld. b The high proportion of mutations at position 47 is due to the codon (RST) employed in the mutagenesis. from library A were compared with the sequences of natural HDs in The Homeodomain Resource =-=(31,32)-=-. To avoid biases associated with disproportionately represented orthologs in the HD Resource, we chose to compare the selected mutants to the 129 human HDs previously examined by Banerjee-Basu et al....

Citation Context

...Table 1, library B). In light of the results from this more complex library, this work serves as the foundation to study HDs with rapid functional epitope mapping techniques, such as shotgun scanning =-=(30,35,36)-=-. Furthermore, this phage display system has been instrumental toward isolating HDs that bind specifically to unnatural, synthetic nucleotides (37). We conclude that the selection system reported here...

Citation Context

...homolog in yeast. It was found that Pho2 Q50A binds to an upstream promoter site at 70% of wild-type levels in vitro but only activated expression from this promoter at 5% of wild-type levels in vivo =-=(34)-=-. As found for EnHD in the current study, it appears the importance of Q50 in Pho2 is greater than expected from biochemical experiments alone. These findings challenges3630 Nucleic Acids Research, 20...

Citation Context

...e mapping techniques, such as shotgun scanning (30,35,36). Furthermore, this phage display system has been instrumental toward isolating HDs that bind specifically to unnatural, synthetic nucleotides =-=(37)-=-. We conclude that the selection system reported herein provides powerful new opportunities for the study and engineering of this important class of transcription factors, the homeodomains. ACKNOWLEDG...