Structure of (5'S)-8,5'-cyclo-2'-deoxyguanosine in DNA

Abstract

Diastereomeric 8,5'-cyclopurine 2'-deoxynucleosides, containing a covalent bond between the deoxyribose and the purine base, represent an important class of DNA damage induced by ionizing radiation. The 8,5'-cyclo-2'-deoxyguanosine lesion (cdG) has been recently reported to be a strong block of replication and highly mutagenic in Escherichia coli. The 8,5'-cyclopurine-2'-deoxyriboses are suspected to play a role in the etiology of neurodegeneration in xeroderma pigmentosum patients. These lesions cannot be repaired by base excision repair, but they are substrates for nucleotide excision repair. The structure of an oligodeoxynucleotide duplex containing a site-specific S-cdG lesion placed opposite dC in the complementary strand was obtained by molecular dynamics calculations restrained by distance and dihedral angle restraints obtained from NMR spectroscopy. The S-cdG deoxyribose exhibited the O4'-exo (west) pseudorotation. Significant perturbations were observed for the β, γ, and χ torsion angles of the S-cdG nucleoside. Watson-Crick base pairing was conserved at the S-cdG·dC pair. However, the O4'-exo pseudorotation of the S-cdG deoxyribose perturbed the helical twist and base pair stacking at the lesion site and the 5'-neighbor dC·dG base pair. Thermodynamic destabilization of the duplex measured by UV melting experiments correlated with base stacking and structural perturbations involving the modified S-cdG·dC and 3'- neighbor dT·dA base pairs. These perturbations may be responsible for both the genotoxicity of this lesion and its ability to be recognized by nucleotide excision repair.

Figure 1

NOE connectivity of base H8/H6 protons with deoxyribose H1′ protons of the S-cdG containing duplex. A. Modified strand. B. Complementary strand.

Figure 2

Tile plot derived from a NOESY spectrum obtained at a mixing time of 60 ms showing the assignment of S-cdG non-exchangeable protons.

Figure 3

Assignment of the base imino and amino protons based on the NOE connectivity. NOE interactions of the imino protons with the opposite base arising from Watson-Crick base pairing are labeled as: (a) X⁵ N1H → C²⁰ N⁴H2, (b) X⁵ N1H → A¹⁹ H2, (c) X⁵ N1H → C²⁰ N⁴H1, (d) G⁷ N1H → C¹⁸ N⁴H2, (e) G⁷ N1H → A¹⁹ H2, (f) G⁷ N1H → A¹⁷ H2, (g) G⁷ N1H → C¹⁸ N⁴H1, (h) G¹¹ N1H → C¹⁴ N⁴H2, (i) G¹¹ N1H → C¹⁴ N⁴H1, (j) G³ N1H → C²² N⁴H2, (k) G³ N1H → C²² N⁴H1, (l) G²¹ N1H → C⁴ N⁴H2, (m) G²¹ N1H → C⁴ N⁴H1, (n) T² N3H → A²³ H2, (o) T¹⁰ N3H → A¹⁵ H2, (p) T⁹ N3H → A¹⁶ H2, and (q) T⁸ N3H → A¹⁷ H2.

Figure 4

An expansion of the ECOSY spectrum used for the measurement of ³J_H1′-H2′ and ³J_H1′-H2″ coupling constants. Except for X⁵, G¹¹, and T¹², all H2″ protons exhibited greater chemical shifts than H2′ protons. The geminal H2′ and H2″ protons of G¹¹ and T¹² were not resolved, and X⁵ H2″ was upfield from H2′.

Figure 5

³¹P NMR of the S-cdG containing duplex compared with the corresponding unmodified duplex. A. Unmodified duplex. B. S-cdG containing duplex.

Figure 6

Proton chemical shift perturbations of the S-cdG containing duplex compared with the unmodified duplex. A. Base protons of the S-cdG-modified strand. B. Deoxyribose protons of the S-cdG-modified strand. C. Base protons of the complementary strand. D. Deoxyribose protons of the complementary strand.

Figure 7

¹H NMR of the S-cdG containing duplex compared with the corresponding unmodified duplex at different temperatures. A. Unmodified duplex. B. S-cdG containing duplex.

Figure 8

Expanded views of the refined structure of the S-cdG containing duplex at the lesion site. A. View from the major groove. B. View from minor groove.

Figure 9

Ring conformations of the S-cdG in the refined structure. A. Six-member ring C8-N9-C1′-O4′-C4′-C5′. B. 2′-deoxyribose.

Figure 10

Base pairing and base stacking of the refined structure of the S-cdG containing duplex at the lesion site. The pink arrows indicate anticipated hydrogen bonding interactions. A. the C⁴•G²¹ and X⁵•C²⁰ base pairs. B. The X⁵•C²⁰ and T⁶•A¹⁹ base pairs.

Figure 11

Distances of guanine N1H → cytosine N3 and the thymine N3H → adenine N1 of some base pairs in the trajectories of the molecular dynamics simulations conducted in explicit solvent at 300 K. A. T²•A²³ base pair. B. C⁴•G²¹ base pair. C. X⁵•C²⁰ base pair. D. T⁶•A¹⁹ base pair. E. T⁸•A¹⁷ base pair. F. T⁹•A¹⁶ base pair.

Scheme 1

Numbering scheme of the oligodeoxynucleotide duplex containing the (5′S)-8,5′-cyclo-2′-deoxyguanosine (S-cdG) 5′-nucleotide.