DNA binding by single HMG box model proteins.
The HMG1/2 family is a large group of proteins that share a conserved sequence of approximately 80 amino acids rich in basic, aromatic and proline side chains, referred to as an HMG box. Previous studies show that HMG boxes can bind to DNA in a structure-specific manner. To define the basis for DNA recognition by HMG boxes, we characterize the interaction of two model HMG boxes, one a structure-specific box, rHMGb from the rat HMG1 protein, the other a sequence-specific box, Rox1 from yeast, with oligodeoxynucleotide substrates. Both proteins interact with single-stranded oligonucleotides in this study to form 1:1 complexes. The stoichiometry of binding of rHMGb to duplex or branched DNAs differs: for a 16mer duplex we find a weak 2:1 complex, while a 4:1 protein:DNA complex is detected with a four-way DNA junction of 16mers in the presence of Mg(2+). In the case of the sequence-specific Rox1 protein we find tight 1:1 and 2:1 complexes with its cognate duplex sequence and again a 4:1 complex with four-way branched DNA. If the DNA branching is reduced to three arms, both proteins form 3:1 complexes. We believe that these multimeric complexes are relevant for HMG1/2 proteins in vivo, since Mg(2+) is present in the nucleus and these proteins are expressed at a very high level.
Xin
H
H
Department of Chemistry, New York University, 31 Washington Place, New York, NY 10003, USA.
Taudte
S
S
Kallenbach
N R
NR
Limbach
M P
MP
Zitomer
R S
RS
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D005453:Fluorescence; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000737:chemistry* / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D013816:Thermodynamics; D017186:Titrimetry; D014461:Ultracentrifugation
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D006609:High Mobility Group Proteins; D009838:Oligodeoxyribonucleotides; C083348:ROX1 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D008274:Magnesium