HMG domain proteins induce sharp bends in cisplatin-modified DNA.
Circularly permuted linear DNAs of approximately 100 bp were constructed containing the major adduct of the anticancer drug cisplatin, a cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] intrastrand cross-link, at a specific site. Gel electrophoresis mobility shift assays with these probes were used to investigate the effects of binding of HMG domain proteins to the platinated DNAs. The site-specifically platinated duplexes were recognized by six different HMG domain proteins--HMG1, mtTFA, Ixr1, and HMG domains from HMG1 (domain B), mSRY, and LEF-1--with comparable binding affinities (Kd approximately 10(-6) to 10(-7) M). In the presence of the HMG domain proteins, the platinated DNAs were bent significantly more than in their absence, the values being 86 +/- 2 degrees, 87-90 +/- 5 degrees, and 68 +/- 6 degrees, respectively, for the proteins and 65-74 +/- 4 degrees, approximately 50 degrees, and 72 +/- 6 degrees, respectively, for the domains. The variability in bend angles suggests that, although the HMG domain proteins share a common ability to bend platinated DNA, specific contacts between the proteins and the platinated duplex are different. The assay further revealed the bend loci to be centered quite near the platinum adduct. The methodology employed in the present study should be generally applicable for synthesizing other small, circularly permuted, covalently modified DNAs which cannot otherwise be readily obtained.
Chow
C S
CS
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Whitehead
J P
JP
Lippard
S J
SJ
D001483:Base Sequence; D001665:Binding Sites; D002945:Cisplatin / Q000494:pharmacology*; D016384:Consensus Sequence; D003500:Cyclization; D004247:DNA / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D015342:DNA Probes; D004262:DNA Restriction Enzymes; D004270:DNA, Circular / Q000737:chemistry / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D006609:High Mobility Group Proteins / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects*
D015342:DNA Probes; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D004247:DNA; D004262:DNA Restriction Enzymes; D002945:Cisplatin