Rapid identification of Quox-1 homeodomain DNA-binding sequence using SAAB.
Quox-1 is the only gene in the hox family whose expression occurs throughout the development of the central nervous system. Using the Quox-1 homeodomain produced in a bacterial expression system, we were able to identify DNA-binding targets of the Quox-1 protein from a library of randomly generated oligonucleotides by the selection and amplification binding (SAAB) technique. The results indicated that the Quox-1 protein recognizes a new consensus sequence, 5'-CAATC-3', which has not been reported for any other Hox family homeoprotein. In addition, electromobility shift assay further confirmed that the Quox-1 homeoprotein preferentially binds to the 5'-CAATC-3' sequence, but not to the binding sites for other Hox class homeoprotein (TAAT) or NKX class homeoprotein (CAAG). Based on mutation analyses of the DNA sequences, we found that the 5'-CAATC-3' core sequences are required for high affinity binding by the Quox-1 protein. Furthermore, mutation analyses of the Quox-1 homeodomain showed that one of the major determinants participating in recognition of a minor groove is the Gln6 and Thr7 in the N-terminal arm of the homeodomain.
Zhu
Fan
F
College of Life Sciences, Wuhan University, Wuhan, 430072, People's Republic of China. fanzhu@my3ia.sina.com
Liu
Zhong-Chun
ZC
Li
Wen-Xin
WX
Xu
Xiu-Ling
XL
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D009841:Oligonucleotides; C484061:Quox-1 protein, human