Binding the Mammalian High Mobility Group Protein AT-hook 2 to AT-Rich Deoxyoligonucleotides: Enthalpy-Entropy Compensation

Joynt, Suzanne; Morillo, Victor; Leng, Fenfei

doi:10.1016/j.bpj.2009.02.015

Cited by 11 publications

(11 citation statements)

References 44 publications

(60 reference statements)

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“…These results suggest that SELEX1 and SELEX2 are indeed the preferred DNA-binding sequences of HMGA2 (21). Our results are summarized in Table 3, which are consistent with the previous published results (21,28). We next determined HMGA2-induced DNA-bending for these DNA-binding sites using the DNA fragments generated from pBendAT-SELEX1, pBendAT-SELEX2, and pBendAT-PRDII by PCR amplification.…”

Section: Resultssupporting

confidence: 93%

DNA Bending by the Mammalian High-Mobility Group Protein AT Hook 2

2010

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The mammalian high mobility group protein AT hook 2 (HMGA2) is a DNA binding protein that specifically recognizes the minor groove of AT-rich DNA sequences. Disruption of its expression pattern is directly linked to oncogenesis and obesity. In this paper, we constructed a new plasmid pBendAT to study HMGA2-induced DNA bending. pBendAT carries a 230 bp DNA segment containing 5 pairs of restriction enzyme sites, which can be used to produce a set of DNA fragments of identical length to study protein-induced DNA bending. The DNA fragments of identical length can also be generated using PCR amplification. Since pBendAT does not contain more than 3 consecutive AT base pairs, it is suitable for the determination of DNA bending induced by proteins recognizing AT-rich DNA sequences. Indeed, using pBendAT, we demonstrated that HMGA2 is a DNA bending protein and bends all three tested DNA binding sequences of HMGA2, SELEX1, SELEX2, and PRDII. The DNA bending angles were estimated to be 34.2, 33.5, and 35.4°, respectively. Keywords HMGA2; AT hook; DNA bendingThe mammalian high mobility group protein AT-hook 2 (HMGA2) is a transcriptional factor involved in mesenchymal cell differentiation and transformation (1-3). It plays an important role in fat cell proliferation (2) and is considered to be a potential target for treatment of obesity (2,4). HMGA2 is also an onco-protein associated with the formation of a variety of tumors including benign tumors, such as uterine leiomyomas (5), lipomas (6,7) and pulmonary chondroid hamartomas (8), and malignant tumors, such as lung cancers (9-11), breast cancers (12), and leukemia (13). Interestingly, the expression level of HMGA2 was demonstrated to be correlated with the degree of malignancy, the existence of metastasis, and a poor prognosis for certain cancers (14,15). These results suggest that HMGA2 is a potential target for treatment of these cancers (4,16,17).HMGA2 is a DNA binding protein containing three "AT hook" DNA binding domains that specifically recognize the minor groove of AT rich DNA sequences (18). Although each "AT hook" DNA binding domain binds to five AT base pairs (18,19), the high-affinity binding of HMGA2 requires two to three appropriated spaced 5-bp AT-rich DNA sequences as a single Here we decided to study HMGA2-induced DNA conformational change, specifically DNA bending. Since each "AT hook" DNA binding domain contains 5 to 6 positively charges, it should bend the DNA locus upon binding to the AT-rich DNA sequences (22). The challenge is to find a short DNA fragment that does not contain 4 to 5 consecutive AT base pairs for the gel permutation assay. In this case, we can clone one HMGA2 binding site into this short DNA fragment and precisely determine the HMGA2-induced DNA-bending angle without the interference caused by HMGA2 binding to other AT-rich sequences, i.e. sequences containing more than 3 consecutive AT base pairs. In this study, we constructed a new plasmid pBendAT to study HMGA2-induced DNA bending. pBendAT carries a 230 bp DNA sequence ...

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Section: Resultssupporting

confidence: 93%

DNA Bending by the Mammalian High-Mobility Group Protein AT Hook 2

2010

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“…ITC experiments were performed using the MicroCal PEAK system (Malvern). Protein or DNA samples were prepared in sodium phosphate buffer with 200 mM NaCl (pH 6) and experiments were performed at 277 K to stabilize the double helix structure of DNA ( 36 ). 50 μM protein was placed in the cell and titrated with 750 μM Seq1 DNA.…”

Section: Methodsmentioning

confidence: 99%

How bacterial xenogeneic silencer rok distinguishes foreign from self DNA in its resident genome

Duan

Ding

Hughes

et al. 2018

Nucleic Acids Research

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Bacterial xenogeneic silencers play important roles in bacterial evolution by recognizing and inhibiting expression from foreign genes acquired through horizontal gene transfer, thereby buffering against potential fitness consequences of their misregulated expression. Here, the detailed DNA binding properties of Rok, a xenogeneic silencer in Bacillus subtilis, was studied using protein binding microarray, and the solution structure of its C-terminal DNA binding domain was determined in complex with DNA. The C-terminal domain of Rok adopts a typical winged helix fold, with a novel DNA recognition mechanism different from other winged helix proteins or xenogeneic silencers. Rok binds the DNA minor groove by forming hydrogen bonds to bases through N154, T156 at the N-terminal of α3 helix and R174 of wing W1, assisted by four lysine residues interacting electrostatically with DNA backbone phosphate groups. These structural features endow Rok with preference towards DNA sequences harboring AACTA, TACTA, and flexible multiple TpA steps, while rigid A-tracts are disfavored. Correspondingly, the Bacillus genomes containing Rok are rich in A-tracts and show a dramatic underrepresentation of AACTA and TACTA, which are significantly enriched in Rok binding regions. These observations suggest that the xenogeneic silencing protein and its resident genome may have evolved cooperatively.

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“…In 2009 we identified more than 400 articles after searching Web of Science, PubMed, SciDir and OVID databases using ‘isothermal AND titration AND calorimetry’ or ITC or ‘Isothermal Titration Calorimetry’ search terms. These have been classified into the following categories: Pre‐2009 references cited in the text and review articles 1–43 Protein‐protein and protein‐peptide interactions 44–124 Protein/peptide‐small molecule interactions 125–218 Protein/peptide‐metal ion interactions 219–253 Protein/peptide‐nucleic acid interactions 254–273 Protein/peptide‐lipid interactions 274–292 Protein/peptide‐polysaccharide interactions 293–316 Nucleic acid‐small molecule interactions 317–348 Small molecule interactions …”

Section: Introductionmentioning

confidence: 99%

Survey of the year 2009: applications of isothermal titration calorimetry

Falconer

Collins

2010

J. Mol. Recognit.

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Isothermal titration calorimetry (ITC) is now an established and invaluable method for determining the thermodynamic constants, association constant and stoichiometry of molecular interactions in aqueous solutions. The technique has become widely used by biochemists to study protein interaction with other proteins, small molecules, metal ions, lipids, nucleic acids and carbohydrates; and nucleic acid interaction with small molecules. The drug discovery industry has utilized this approach to measure protein (or nucleic acid) interaction with drug candidates. ITC has been used to screen candidates, guide the design of potential drugs and validate the modelling used in structure-based drug design. Emerging disciplines including nanotechnology and drug delivery could benefit greatly from ITC in enhancing their understanding and control of nano-particle assembly, and drug binding and controlled release.

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Binding the Mammalian High Mobility Group Protein AT-hook 2 to AT-Rich Deoxyoligonucleotides: Enthalpy-Entropy Compensation

Cited by 11 publications

References 44 publications

DNA Bending by the Mammalian High-Mobility Group Protein AT Hook 2

DNA Bending by the Mammalian High-Mobility Group Protein AT Hook 2

How bacterial xenogeneic silencer rok distinguishes foreign from self DNA in its resident genome

Survey of the year 2009: applications of isothermal titration calorimetry

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