The central three-zinc finger connection of the native nine-zinc finger protein transcription factor IIIA (TFIIIA) is composed of unique linker sequences, -NIKICV-. -TQQLP- -AG-. and -QDL-. New artificial nine-zinc finger proteins, Sp1ZF9TC and Sp1ZF9TN, which use the TFIIIA-type linker for their C- and N-terminal three-zinc finger connections have been created. To investigate the influence of TFIIIA-type linker sequences by their different locations in the proteins, gel mobility shift assays (GMSA), DNase I footprinting assays. methylation interference analyses, and hydroxyl radical footprinting assays were performed. The GMSA revealed similar DNA-binding affinities of these two proteins. The footprinting analyses indicated that the two zinc finger proteins recognize the same part of GCII or GCIII DNA. Moreover. the specific base contacts were observed in the same sites of the substrate DNA. In the present proteins. Sp1ZF9TC and Sp1ZF9TN. the four zinc fingers (fingers 1-4 or 5-9) situated in the site opposite to the TFIIIA-type linker position participate in their DNA bindings, The position of the TFIIIA-type linker is important in DNA recognition by multi-zinc finger proteins. (C) 2002 Elsevier Science (USA). All rights reserved.