A single HMG domain in high-mobility group 1 protein binds to DNAs as small as 20 base pairs containing the major cisplatin adduct.
Proteins containing a relatively new DNA-binding motif known as the high-mobility group (HMG) domain bind specifically to DNA modified by the anticancer drug cisplatin, but not to unmodified DNA (McA'Nulty & Lippard, 1995). Southwestern-blot analyses of the binding of proteolytic fragments of HMG1 to a 123-bp globally platinated DNA demonstrate that the HMG domains A and B of HMG1 are responsible for its specific interactions with cisplatin-modified DNA. An 81 amino acid recombinant protein representing a single HMG motif, HMG1 domain B, binds with an affinity (Kd = 10(-7) M) equal to that of HMG1 itself to 92- and 100-bp DNAs containing the major adduct of cisplatin, a cis-[Pt(NH3)2-[d(GpG)-N7(1), -N7(2)]] intrastrand cross-link, at a specific site. The isolated HMG domain B binds with comparable affinity to cisplatin-modified DNAs having as few as 20 bp. The related human mitochondrial HMG domain protein mtTFA also recognizes the 123-bp globally platinated DNA, providing further evidence that HMG domains are responsible for modulating binding of this class of proteins to cisplatin-modified DNA. This work provides direct biochemical evidence in support of conclusions drawn previously from analyses of sequence conservation (Bruhn et al., 1992) that HMG domains are the key elements in protein binding to cisplatin-modified DNA.
Chow
C S
CS
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Barnes
C M
CM
Lippard
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002945:Cisplatin / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism*; D015342:DNA Probes / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006609:High Mobility Group Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018736:DNA Adducts; D015342:DNA Probes; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C499631:TFAM protein, human; C489030:Tfam protein, rat; D014157:Transcription Factors; C057270:cisplatin-DNA adduct; C422562:mitochondrial transcription factor A; D004247:DNA; D002945:Cisplatin