dna template of prep1 hd  (GenScript corporation)

Journal: Scientific Reports

Article Title: New Insights into Cooperative Binding of Homeodomain Transcription Factors PREP1 and PBX1 to DNA

doi: 10.1038/srep40665

Figure Lengend Snippet: Panel A: Homeodomain (HD) protein constructs used for characterizations described in this study, in comparison with full-length proteins. PBX1 HD and HOXB1 HD constructs are the same used in the X-ray crystallographic study . PREP1 HD is present in four lengths, PREP1 257-325 (PREP1 hd ), PREP1 240-344 (PREP1 HD ) and two intermediate lengths (PREP1 257-344 - PREP1 hd-C - and PREP1 240-325 -PREP1 hd-N -), where respectively the N- or C-terminal extensions of the HD are omitted. Production of the proteins is described in the Materials and Methods section. PBC-A and PBC-B domains of PBX1 are those required for its dimerization with PREP1. TALE lies between helix 1 and helix 2 of the HD. MEIS-A and MEIS-B domains are PREP1 motifs required for heterodomerization with PBX1. Panel B: Consensus sequences for DNA binding by PREP1-PBX1: the very frequent decameric (PMH) and the less frequent octameric (PH) oligonucleotides , are both highlighted in red in the sequence. Control probes correspond to two sequences that do not contain any PBX1 orPREP1 binding site. Oligonucleotides used for FP were 5′ labelled with 6-carboxyfluorescein (6-FAM) (see the Materials and Methods).

Article Snippet: The DNA template of PREP1 HD in which the four residues K 231 , K 233 , K 234 , and K 235 were mutated to alanine was purchased from Genscript Corporation (Piscataway, NJ) and showed the following sequence: 5′CAGCTTCAGTTACAGTTAAACCAAGATCTCAGCATCTTGCATCAAGATGATGGTTCATCTAAGAACAAGAGGGGCGTCCTGCCAAAGCATGCCACGAACGTGATGCGGTCCTGGCTCTTCCAGCACATCGGGCATCCCTACCCAACAGAGGATGAGAAAAAACAGATTGCTGCTCAGACAAATTTGACACTACTCCAAGTCAACAACTGGTTCATCAATGCCAGAAGACGAATTCTTCAGCCAATGTTGGATTCAAGTTGTTCAGAGACCCCCGCAACAGCGGCAGCAACTGCTCAGAACCGGCCAGTTCAGAGG 3′.

Techniques: Construct, Comparison, Binding Assay, Sequencing, Control

Journal: Scientific Reports

Article Title: New Insights into Cooperative Binding of Homeodomain Transcription Factors PREP1 and PBX1 to DNA

doi: 10.1038/srep40665

Figure Lengend Snippet: K D values for individual HDs with different DNA sequences, measured by FP.

Article Snippet: The DNA template of PREP1 HD in which the four residues K 231 , K 233 , K 234 , and K 235 were mutated to alanine was purchased from Genscript Corporation (Piscataway, NJ) and showed the following sequence: 5′CAGCTTCAGTTACAGTTAAACCAAGATCTCAGCATCTTGCATCAAGATGATGGTTCATCTAAGAACAAGAGGGGCGTCCTGCCAAAGCATGCCACGAACGTGATGCGGTCCTGGCTCTTCCAGCACATCGGGCATCCCTACCCAACAGAGGATGAGAAAAAACAGATTGCTGCTCAGACAAATTTGACACTACTCCAAGTCAACAACTGGTTCATCAATGCCAGAAGACGAATTCTTCAGCCAATGTTGGATTCAAGTTGTTCAGAGACCCCCGCAACAGCGGCAGCAACTGCTCAGAACCGGCCAGTTCAGAGG 3′.

Techniques: Control

Journal: Scientific Reports

Article Title: New Insights into Cooperative Binding of Homeodomain Transcription Factors PREP1 and PBX1 to DNA

doi: 10.1038/srep40665

Figure Lengend Snippet: DNA probes were incubated with different amounts of HD. Protein:DNA ratios were 0.5 (DNA excess), 1 (equimolar ratio), or 2 (protein excess). Gels were stained both with ethidium bromide and coomassie blue to visualize DNA or proteins, respectively. Panel A: PREP1 HD forms two different complexes with PMH: the first is visible, at protein:DNA ratio of 0.5 (lanes 1 and 2, left panel), and the second as a smeared band, at protein:DNA ratio of 2 (lane 3, left panel). This slower migrating band might represent binding of two PREP1 HD proteins to DNA. In the case of PH (central panel) and control probes (right panel), a specific DNA complex is not formed. Panel B: PREP1 hd forms a single complex with PMH; whereas binding to control and PH oligos is weak and no specific bands are formed. Panel C: PBX1 HD forms a single complex with PMH and PH oligos at protein:DNA ratio of 0.5 (lane 1); by increasing the protein concentration, a slower migrating band becomes visible, possibly due to binding of a second monomer to DNA. PBX1 binds also the control oligo.

Article Snippet: The DNA template of PREP1 HD in which the four residues K 231 , K 233 , K 234 , and K 235 were mutated to alanine was purchased from Genscript Corporation (Piscataway, NJ) and showed the following sequence: 5′CAGCTTCAGTTACAGTTAAACCAAGATCTCAGCATCTTGCATCAAGATGATGGTTCATCTAAGAACAAGAGGGGCGTCCTGCCAAAGCATGCCACGAACGTGATGCGGTCCTGGCTCTTCCAGCACATCGGGCATCCCTACCCAACAGAGGATGAGAAAAAACAGATTGCTGCTCAGACAAATTTGACACTACTCCAAGTCAACAACTGGTTCATCAATGCCAGAAGACGAATTCTTCAGCCAATGTTGGATTCAAGTTGTTCAGAGACCCCCGCAACAGCGGCAGCAACTGCTCAGAACCGGCCAGTTCAGAGG 3′.

Techniques: Incubation, Staining, Binding Assay, Control, Protein Concentration

Journal: Scientific Reports

Article Title: New Insights into Cooperative Binding of Homeodomain Transcription Factors PREP1 and PBX1 to DNA

doi: 10.1038/srep40665

Figure Lengend Snippet: FP K D measurement of PREP1 HDs in the presence of a preformed PBX1 HD : DNA complex.

Article Snippet: The DNA template of PREP1 HD in which the four residues K 231 , K 233 , K 234 , and K 235 were mutated to alanine was purchased from Genscript Corporation (Piscataway, NJ) and showed the following sequence: 5′CAGCTTCAGTTACAGTTAAACCAAGATCTCAGCATCTTGCATCAAGATGATGGTTCATCTAAGAACAAGAGGGGCGTCCTGCCAAAGCATGCCACGAACGTGATGCGGTCCTGGCTCTTCCAGCACATCGGGCATCCCTACCCAACAGAGGATGAGAAAAAACAGATTGCTGCTCAGACAAATTTGACACTACTCCAAGTCAACAACTGGTTCATCAATGCCAGAAGACGAATTCTTCAGCCAATGTTGGATTCAAGTTGTTCAGAGACCCCCGCAACAGCGGCAGCAACTGCTCAGAACCGGCCAGTTCAGAGG 3′.

Techniques:

Journal: Scientific Reports

Article Title: New Insights into Cooperative Binding of Homeodomain Transcription Factors PREP1 and PBX1 to DNA

doi: 10.1038/srep40665

Figure Lengend Snippet: Panel A: A sample containing a preformed PBX1 HD :PMH complex (at 4 and 8 μM, respectively) was titrated with increasing amounts of PREP1 HD (2, 4 and 8 μM). PREP1 HD binds preferentially to the PBX1 HD :PMH complex rather than to the free DNA; the DNA pool decrease appreciably upon an increase in PREP1 HD concentration. At the lower PREP1 HD concentrations (lane 3) a slower migrating band is observed above the monomeric PREP1 HD :DNA and PBX1 HD :DNA complexes (identified in lanes 1 and 2, respectively). The band marked with a star in lane 5 was analyzed by mass spectrometry. Panel B: Titration of PREP1 HD : PMH complex with PBX1 HD . PREP1 HD (4 μM) and PMH (4 μM) were titrated with increasing concentrations of PBX1 hHD (2, 4 and 8 μM). Upon increase of PBX1 HD a slower migrating band appears, corresponding to PREP1 HD :DNA:PBX1 HD complex. Panel C: Titration of PBX1 HD : PMH complex with PREP1 hd . PBX1 HD (4 μM) and PMH (4 μM) were titrated with increasing concentrations of PREP1 hd (2, 4 and 8 μM). Upon increase of PREP1 hd only a band corresponding to PREP1 hd :DNA is visible, therefore, PREP1 hd in EMSA does not show clear binding to the preformed PBX1 HD :DNA complex. Panel D: Titration of PMH:PBX1 HD with PREP1 hd-N . PMH and PBX1 HD at a fixed concentration (8 μM and 4 μM, respectively) were titrated with increasing amounts of PREP1 hd-N (2, 4, 8, and 16 μM). Upon an increase in PREP1 hd-N no retarded band above the individual PMH:PREP1 hd-N and PMH:PBX11 HD complexes (lanes 3-5) are observed Panel E: Titration of PBX1 HD :PMH with PREP1 hd-C . PMH oligo and PBX1 HD at a fixed concentration (8 μM and 4 μM, respectively) were titrated with increasing concentrations of PREP1 hd-C (2, 4 and 8 μM). Upon an increase in PREP1 hd - C , a slower migrating band corresponding to PREP1 hd-C :DNA: PBX1 HD complex is visible.

Article Snippet: The DNA template of PREP1 HD in which the four residues K 231 , K 233 , K 234 , and K 235 were mutated to alanine was purchased from Genscript Corporation (Piscataway, NJ) and showed the following sequence: 5′CAGCTTCAGTTACAGTTAAACCAAGATCTCAGCATCTTGCATCAAGATGATGGTTCATCTAAGAACAAGAGGGGCGTCCTGCCAAAGCATGCCACGAACGTGATGCGGTCCTGGCTCTTCCAGCACATCGGGCATCCCTACCCAACAGAGGATGAGAAAAAACAGATTGCTGCTCAGACAAATTTGACACTACTCCAAGTCAACAACTGGTTCATCAATGCCAGAAGACGAATTCTTCAGCCAATGTTGGATTCAAGTTGTTCAGAGACCCCCGCAACAGCGGCAGCAACTGCTCAGAACCGGCCAGTTCAGAGG 3′.

Techniques: Concentration Assay, Mass Spectrometry, Titration, Binding Assay

Journal: Scientific Reports

Article Title: New Insights into Cooperative Binding of Homeodomain Transcription Factors PREP1 and PBX1 to DNA

doi: 10.1038/srep40665

Figure Lengend Snippet: Mass spectrometry analysis of EMSA titrations bands from Fig. 3 .

Article Snippet: The DNA template of PREP1 HD in which the four residues K 231 , K 233 , K 234 , and K 235 were mutated to alanine was purchased from Genscript Corporation (Piscataway, NJ) and showed the following sequence: 5′CAGCTTCAGTTACAGTTAAACCAAGATCTCAGCATCTTGCATCAAGATGATGGTTCATCTAAGAACAAGAGGGGCGTCCTGCCAAAGCATGCCACGAACGTGATGCGGTCCTGGCTCTTCCAGCACATCGGGCATCCCTACCCAACAGAGGATGAGAAAAAACAGATTGCTGCTCAGACAAATTTGACACTACTCCAAGTCAACAACTGGTTCATCAATGCCAGAAGACGAATTCTTCAGCCAATGTTGGATTCAAGTTGTTCAGAGACCCCCGCAACAGCGGCAGCAACTGCTCAGAACCGGCCAGTTCAGAGG 3′.

Techniques: Mass Spectrometry

Journal: Scientific Reports

Article Title: New Insights into Cooperative Binding of Homeodomain Transcription Factors PREP1 and PBX1 to DNA

doi: 10.1038/srep40665

Figure Lengend Snippet: Panel A: Superposition of 1 H- 15 N HSQC spectra of PREP1 HD (magenta) and PREP1 hd (blue). Panel B: Combined amide chemical shift difference (Δ δ ) between PREP1 HD and PREP1 hd corresponding residues. Panel C: Cartoon representation of the PREP1 HD model: the residues (in blue) shared with PREP1 hd , the N- and C- terminal extensions (in cyan) of PREP1 HD . Spheres indicate PREP1 HD residues showing significant (>average) amide Δ δ with respect to PREP1 hd . Panel D: Normalized CD melting curves for PREP1 HD (magenta) and PREP1 hd (blue) at 222 nm.

Article Snippet: The DNA template of PREP1 HD in which the four residues K 231 , K 233 , K 234 , and K 235 were mutated to alanine was purchased from Genscript Corporation (Piscataway, NJ) and showed the following sequence: 5′CAGCTTCAGTTACAGTTAAACCAAGATCTCAGCATCTTGCATCAAGATGATGGTTCATCTAAGAACAAGAGGGGCGTCCTGCCAAAGCATGCCACGAACGTGATGCGGTCCTGGCTCTTCCAGCACATCGGGCATCCCTACCCAACAGAGGATGAGAAAAAACAGATTGCTGCTCAGACAAATTTGACACTACTCCAAGTCAACAACTGGTTCATCAATGCCAGAAGACGAATTCTTCAGCCAATGTTGGATTCAAGTTGTTCAGAGACCCCCGCAACAGCGGCAGCAACTGCTCAGAACCGGCCAGTTCAGAGG 3′.

Techniques:

Journal: Scientific Reports

Article Title: New Insights into Cooperative Binding of Homeodomain Transcription Factors PREP1 and PBX1 to DNA

doi: 10.1038/srep40665

Figure Lengend Snippet: Data reported correspond to a protein:PMH molar ratio of 1:0.5. Panel A: Superposition of 1 H- 15 N HSQC spectra of free (black) and PMH-bound (red) PREP1 HD . (left) and PREP1 hd (right) Panel B: Plot showing the peaks intensity reduction observed upon PMH binding to PREP1 hd and PREP1 HD . Panel C: Residues disappearing or showing significant amide chemical shift displacement (as reported in the CSD plot in ) are highlighted on PREP1 HD structural model (top, generated using CS23D2.0 web server) and PREP1 hd NMR structure (bottom, 1 × 2 N.pdb,).

Article Snippet: The DNA template of PREP1 HD in which the four residues K 231 , K 233 , K 234 , and K 235 were mutated to alanine was purchased from Genscript Corporation (Piscataway, NJ) and showed the following sequence: 5′CAGCTTCAGTTACAGTTAAACCAAGATCTCAGCATCTTGCATCAAGATGATGGTTCATCTAAGAACAAGAGGGGCGTCCTGCCAAAGCATGCCACGAACGTGATGCGGTCCTGGCTCTTCCAGCACATCGGGCATCCCTACCCAACAGAGGATGAGAAAAAACAGATTGCTGCTCAGACAAATTTGACACTACTCCAAGTCAACAACTGGTTCATCAATGCCAGAAGACGAATTCTTCAGCCAATGTTGGATTCAAGTTGTTCAGAGACCCCCGCAACAGCGGCAGCAACTGCTCAGAACCGGCCAGTTCAGAGG 3′.

Techniques: Binding Assay, Generated