Rapid Identification of Quox-1 Homeodomain DNA-Binding Sequence Using SAAB

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Fan Zhu^1#, Zhong-Chun Liu^2#, Wen-Xin Li^1*, and Xiu-Ling Xu¹

¹College of Life Sciences, Wuhan University, Wuhan 430072, People's Republic of China; fax: 86-027-87669560; E-mail: fanzhu@my3ia.sina.com

²Ren-Ming Hospital, Wuhan University, Wuhan 430060, People's Republic of China

^* To whom correspondence should be addressed.

^# These authors contributed equally to the work.

Received August 30, 2004; Revision received September 30, 2004

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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.

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KEY WORDS: Quox-1, DNA-protein interaction, DNA-binding site, homeodomain protein

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