Relationship of solution and protein-bound structures of DNA duplexes with the major intrastrand cross-link lesions formed on cisplatin binding to DNA.
DNA bases in the three-base-pair (3bp) region of duplexes with the two major lesions of cisplatin (cis-PtCl(2)(NH(3))(2)) with DNA, namely d(XGG) and d(XAG) ( = N7-platinated base), differ in their relative positions by as much as approximately 3.5 A in structures in the literature. Such large differences impede drug design and assessments of the effects of protein binding on DNA structure. One recent and several past structures based on NMR-restrained molecular dynamics (RMD) differ significantly from the reported X-ray structure of an HMG-bound XGG 16-mer DNA duplex (Ohndorf, U.-M.; Rould, M. A.; He, Q.; Pabo, C. O.; Lippard, S. J. Nature 1999, 399, 708). This 16-mer structure has several significant novel and unique features (e.g., a bp step with large positive shift and slide). Hypothesizing that novel structural features in the XGG or XAG region of duplexes elude discovery by NMR methods (especially because of the flexible nature of the 3bp region), we studied an oligomer with only G.C bp's in the XGGY site by NMR methods for the first time. This 9-mer gave a 5'-G N1H signal with a normal shift and intensity and showed clear NOE cross-peaks to C NHb and NHe. We assigned for the first time (13)C NMR signals of a duplex with a GG lesion. These data, by adding NMR-based criteria to those inherent in NOESY and COSY data, have more specifically defined the structural features that should be present in an acceptable model. In particular, our data indicated that the sugar of the X residue has an N pucker and that the GG cross-link should have a structure similar to the original X-ray structure of cis-Pt(NH(3))(2)(d(pGpG)) (Sherman S. E.; Gibson, D.; Wang, A. H.-J.; Lippard, S. J. J. Am. Chem. Soc. 1988, 110, 7368). With these restrictions added to NOE restraints, an acceptable model was obtained only when we started our modeling with the 16-mer structural features. The new X-ray/NMR-based model accounted for the NOESY data better than NOE-based models, was very similar in structure to the 16-mer, and differed from solely NOE-based models. We conclude that all XGG and XAG (X = C or T) duplexes undoubtedly have structures similar to those of the 16-mer and our model. Thus, protein binding does not change greatly the structure of the 3bp region. The structure of this region can now be used in understanding structure-activity relationships needed in the design of new carrier ligands for improving Pt anticancer drug activity.
Marzilli
L G
LG
Department of Chemistry, Emory University, Atlanta, Georgia 30322, USA. lmarzil@emory.edu
Saad
J S
JS
Kuklenyik
Z
Z
Keating
K A
KA
Xu
Y
Y
D002945:Cisplatin / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D011485:Protein Binding
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D004247:DNA; D002945:Cisplatin