Human testis-determining factor SRY binds to the major DNA adduct of cisplatin and a putative target sequence with comparable affinities.
cis-Diamminedichloroplatinum(II) (cis-DDP or cisplatin) is a widely used anticancer drug that is most effective against tumors of the testis. Although cisplatin is believed to mediate its cytotoxicity through the formation of DNA adducts, the precise biochemical mechanisms underlying its antitumor activity and selectivity for testicular tumors remain elusive. Of significance are the high-mobility group (HMG) domain and other proteins that bind specifically to cisplatin-DNA adducts. The present study focuses on the testis-specific HMG domain protein human SRY (hSRY). The full-length hSRY protein and its HMG domain region alone were expressed in Escherichia coli and purified to homogeneity. The affinities and specificities of full-length hSRY and the hSRY-HMG domain for 20 bp DNAs containing a single cis-[Pt(NH3)2{d(GpG)-N7(1), -N7(2)}] intrastrand cross-link or a putative hSRY target site in the CD3epsilon gene enhancer (AACAAAG) were determined in electrophoretic mobility shift assays. Full-length hSRY bound to the major 1,2-d(GpG) cisplatin adduct with a Kd(app) of 120 +/- 10 nM and exhibited a 20-fold specificity over unmodified DNA. The HMG domain of hSRY was sufficient for this interaction. The hSRY-HMG domain recognized the 1,2-d(GpG) intrastrand cross-link with higher affinity [Kd(app) = 4 +/- 0.7 nM] but with lower specificity (5-fold) than the full-length protein. The affinities of full-length hSRY and the hSRY-HMG domain for a single cisplatin-DNA adduct were comparable to those for the putative target sequence AACAAAG. These data suggest that cisplatin-DNA adducts may compete with specific DNA sequences in vivo for the binding of human SRY. A possible role for this testis-specific protein in the cytotoxicity and organotropic specificity of cisplatin for testicular tumors is proposed.
Trimmer
E E
EE
Department of Chemistry and Division of Toxicology, Whitaker College of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Zamble
D B
DB
Lippard
S J
SJ
Essigmann
J M
JM
D001667:Binding, Competitive / Q000235:genetics; D002945:Cisplatin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D018736:DNA Adducts / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015342:DNA Probes / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008297:Male; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D051876:Sex-Determining Region Y Protein; D013737:Testis / Q000378:metabolism*; D014157:Transcription Factors
D018736:DNA Adducts; D015342:DNA Probes; D004268:DNA-Binding Proteins; D003848:Deoxyguanine Nucleotides; D006609:High Mobility Group Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C497745:SRY protein, human; D051876:Sex-Determining Region Y Protein; D014157:Transcription Factors; D004247:DNA; D002945:Cisplatin