Cisplatin (cis-Pt(NH(3))(2)Cl(2)) and cis-[Pt(NH(3))(2)(H(2)O)(2)](2+) Intrastrand Cross-Linking Reactions at the Telomere GGGT DNA Sequence Embedded in a Duplex, a Hairpin, and a Bulged Duplex: Use of Mg(2+) and Zn(2+) to Convert a Hairpin to a Bulged Duplex.
In the past, we showed that metal species have a high affinity for the central G in the GGG sequence of the duplex d(A(1)T(2)G(3)G(4)G(5)T(6)A(7)C(8)C(9)C(10)A(11)T(12))(2) (G3-D) and that cisplatin (cis-Pt(NH(3))(2)Cl(2)) and G3-D formed an N7-Pt-N7 G(4),G(5) intrastrand cross-link preferentially over the G(3),G(4) adduct ( approximately 25:1). Thus, a putative G(4) monoadduct was postulated to cross-link in the 3'- rather than the normally more favorable 5'-direction. To evaluate this hypothesis and also to explore why the G3-D G(4),G(5) adduct had an unusual hairpin structure, we have now introduced the use of N,N'-dimethylthiourea (DMTU) as a monoadduct trap and have extended the study to a G3-D analogue with a hairpin form, d(A(1)T(2)G(3)G(4)G(5)T(6)T(7)C(8)C(9)C(10)A(11)T(12)) (G3-H). Chemical shift and 2D (1)H and (13)C NMR data indicated that the G3-H hairpin has a stem region with B-form structure and a nonhelical loop region. Zn(2+) or Mg(2+) ions transformed G3-H into a bulged duplex. Downfield shifts of G(4)H8 and G(4)C8 NMR signals indicated that Zn(2+) binds preferentially to G(4)N7. Reaction of cisplatin or cis-[Pt(NH(3))(2)(H(2)O)(2)](2+) with the bulged duplex and hairpin forms of G3-H gave a similar intrastrand cross-link ratio, G(4),G(5):G(3),G(4) = 7:3. This ratio is insensitive to DNA form or Pt leaving group. For G3-D this ratio is lower in the cis-[Pt(NH(3))(2)(H(2)O)(2)](2+) reaction ( approximately 1:1) than in the cisplatin reaction (25:1), indicating that the leaving group influences the cross-linking step for G3-D. The G(4) monoadducts of the cis-Pt(NH(3))(2)Cl(2)-G3-H and -G3-D and the cis-[Pt(NH(3))(2)(H(2)O)(2)](2+)-G3-D reactions were trapped with DMTU, but no monoadduct was trapped in the cis-[Pt(NH(3))(2)(H(2)O)(2)](2+)-G3-H reaction. The results suggest that the respective monoadducts are more long-lived for G3-D. We postulate that the G(5) in the G3-D Cl-G(4) monoadduct is placed in a favorable position to form the cross-link because of a prior conformational change induced by G(4)-A(7) stacking. This accounts for the very high selectivity for 3'-cross-linking. Nevertheless, in all other cases, regardless of the form or conformation, 3'-direction cross-linking is unusually favored at GGGT sequences, suggesting that the sequence itself contributes greatly to the 3'-cross-linking preference; since telomeres have multiple repeats of this GGGT sequence, this finding may have biological relevance.
Villanueva
Julie M.
JM
Departments of Chemistry and Biochemistry, Emory University, Atlanta, Georgia 30322, and Division of Cancer Biology and Department of Radiation Oncology, Emory University School of Medicine, Atlanta, Georgia 30322.
Jia
Xin
X
Yohannes
Paulos G.
PG
Doetsch
Paul W.
PW
Marzilli
Luigi G.
LG