Nucleic Acids Research, 2001, Vol. 29, No. 24 4920-4929
© 2001 Oxford University Press
Selected base sequence outside the target binding site of zinc finger protein Sp1
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
Human transcription factor Sp1 contains three contiguous repeats of the C2H2-type zinc finger motif and binds to the decanucleotide sequence 5'-(G/T)GGGCGG(G/A)(G/A)(C/T)-3' (GC box). In order to determine whether the three-zinc finger peptide Sp1(530–623) has selectivity for sequence outside the GC box, we used a selection and amplification of binding experiment. The high affinity sequence generated from this selection is 5'-GGGTGGGCGTGGC-3' (s-GC box), which is flanked by a novel conserved guanine triplet on the 5'-side of the core decanucleotide. Gel mobility shift assays reveal that Sp1(530–623) binds to the s-GC box with 2.3-fold higher affinity than to the wild-type GC box, 5'-GGGGCGGGGC-3' (c-GC box). DNase I and hydroxyl radical footprinting analyses show that the area of the s-GC box protected by binding of Sp1(530–623) is wider by 1 nt than that of the c-GC box. On the other hand, alkylation interference analyses demonstrate that Sp1(530–623) forms only one special base contact at the guanine triplet. With respect to cleavage of the c-GC and s-GC boxes by the 1,10-phenanthroline–copper complex (OP-Cu), binding of Sp1(530–623) has no effect on the cleavage pattern of the s-GC box, whereas OP-Cu actually enhances cleavage of the c-GC box. Additionally, the extent of cleavage of the s-GC box by DNase I and OP-Cu is clearly different from that of the c-GC box under peptide-free conditions. The results strongly indicate that: (i) the conformation of the s-GC box is evidently distinct from that of the c-GC box; (ii) Sp1(530–623) binds to the s-GC box without induction of a conformational change in DNA detectable by cleavage with OP-Cu. The present study provides useful information for the design of multi-zinc finger proteins with various sequence specificities.
* To whom correspondence should be addressed. Tel: +81 774 38 3210; Fax: +81 774 32 3038; Email: sugiura{at}scl.kyoto-u.ac.jp
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