title: The combination of sequence-specific and nonspecific DNA-binding modes of transcription factor SATB1.
abstract: Transcription factor SATB1 (special AT-rich sequence binding protein 1) contains multiple DNA-binding domains (DBDs), i.e. two CUT-domain repeats (CUTr1 and CUTr2 from the N-terminus) and a homeodomain, and binds to the matrix attachment region (MAR) of DNA. Although CUTr1 and the homeodomain, but not CUTr2, are known to contribute to DNA binding, different research groups have not reached a consensus on which DBD is responsible for recognition of the target sequence in MAR, 5'-TAATA-3'. Here, we used isothermal titration calorimetry to demonstrate that CUTr1 has binding specificity to this motif, whereas the homeodomain shows affinity for a variety of DNAs without specificity. In line with nonspecific DNA-binding properties of the homeodomain, a mutation of the invariant Asn at position 51 of the homeodomain (typically in contact with the A base in a sequence-specific binding mode) did not affect the binding affinity significantly. The NMR analyses and computational modeling of the homeodomain, however, revealed the tertiary structure and DNA-binding mode that are typical of homeodomains capable of sequence-specific binding. We believe that the lack of highly conserved basic residues in the helix relevant to the base recognition loosens its fitting into the DNA groove and impairs the specific binding. The two DBDs, when fused in tandem, showed strong binding to DNA containing the 5'-TAATA-3' motif with an affinity constant >10(8) M(-1) and retained nonspecific binding activity. The combination of the sequence-specific and nonspecific DNA-binding modes of SATB1 should be advantageous in a search for target loci during transcriptional regulation.
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Yamasaki
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Yamasaki
mesh_terms: D000595:Amino Acid Sequence; D002151:Calorimetry; D004247:DNA / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D036961:Matrix Attachment Region Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid
chemical_list: D018398:Homeodomain Proteins; D036961:Matrix Attachment Region Binding Proteins; C076189:SATB1 protein, human; D004247:DNA
keywords: DNA-binding domain; NMR; homeodomain; isothermal titration calorimetry; nonspecific binding; sequence-specific binding