Bulge migration of the malondialdehyde OPdG DNA adduct when placed opposite a two-base deletion in the (CpG)3 frameshift hotspot of the Salmonella typhimurium hisD3052 gene

Abstract

The OPdG adduct N (2)-(3-oxo-1-propenyl)dG, formed in DNA exposed to malondialdehyde, was introduced into 5'-d(ATCGC XCGGCATG)-3'.5'-d(CATGCCGCGAT)-3' at pH 7 (X = OPdG). The OPdG adduct is the base-catalyzed rearrangement product of the M 1dG adduct, 3-(beta- d-ribofuranosyl)pyrimido[1,2- a]purin-10(3 H)-one. This duplex, named the OPdG-2BD oligodeoxynucleotide, was derived from a frameshift hotspot of the Salmonella typhimuium hisD3052 gene and contained a two-base deletion in the complementary strand. NMR spectroscopy revealed that the OPdG-2BD oligodeoxynucleotide underwent rapid bulge migration. This hindered its conversion to the M 1dG-2BD duplex, in which the bulge was localized and consisted of the M 1dG adduct and the 3'-neighbor dC [ Schnetz-Boutaud, N. C. , Saleh, S. , Marnett, L. J. , and Stone, M. P. ( 2001) Biochemistry 40, 15638- 15649 ]. The spectroscopic data suggested that bulge migration transiently positioned OPdG opposite dC in the complementary strand, hindering formation of the M 1dG-2BD duplex, or alternatively, reverting rapidly formed intermediates in the OPdG to M 1dG reaction pathway when dC was placed opposite from OPdG. The approach of initially formed M 1dG-2BD or OPdG-2BD duplexes to an equilibrium mixture of the M 1dG-2BD and OPdG-2BD duplexes was monitored as a function of time, using NMR spectroscopy. Both samples attained equilibrium in approximately 140 days at pH 7 and 25 degrees C.

Scheme 1

(A) Formation of M₁dG from MDA or from Base Propenals and (B) Depiction of M₁dG Being Stable in Single-Stranded DNA ^a

Scheme 2

2BD Oligodeoxynucleotide Duplex Containing a Two-Nucleotide 5′-GpC-3′ Deletion in the Complementary Strand ^a

Figure 1

(A) COSY spectrum of the unmodified 2BD duplex. (B) COSY spectrum of the freshly prepared OPdG-2BD duplex. (C) COSY spectrum of the freshly prepared M₁dG-2BD duplex. Cross-peaks: a, C¹² H5 → C¹² H6; b, C¹ H5 → C¹ H6; c, C³ H5 → C³ H6; d, C¹⁷ H5 → C¹⁷ H6; e, C¹⁶ H5 → C¹⁶ H6; f, C⁸ H5 → C⁸ H6; g, C⁵ H5 → C⁵ H6; h, C¹⁹ H5 → C¹⁹ H6; i, M₁dG H7 → H8; j, M₁dG H6 → H7. The experiments were performed at 800 MHz and 25 °C.

Figure 2

(A) Expanded plot showing sequential NOE connectivity between aromatic and anomeric protons for nucleotides A⁻² → G¹¹ of the unmodified 2BD duplex. (B) Expanded plot showing sequential NOE connectivity between aromatic and anomeric protons for nucleotides C¹² → T²² of the unmodified 2BD duplex. (C) Expanded plot showing sequential NOE connectivity between aromatic and anomeric protons for nucleotides A⁻² → G¹¹ of the OPdG-2BD duplex. (D) Expanded plot showing sequential NOE connectivity between aromatic and anomeric protons for nucleotides C¹² → T²² of the OPdG-2BD duplex. The 800 MHz 250 ms mixing time NOESY experiments were conducted at 25 °C.

Figure 3

(A) Expanded plot showing sequential NOE connectivity of the base-paired imino protons for the unmodified 2BD duplex. (B) Expanded plot showing sequential NOE connectivity for the base-paired imino protons of the freshly prepared OPdG-2BD duplex. Cross-peaks: a, C¹⁶ N⁴H2 → C¹⁶ H5; b, C¹⁶ H6 → C¹⁶ H5; c, C¹⁶ N⁴H1 → C¹⁶ H5; d, C⁸ N⁴H2 → C⁸ H5; e, C⁸ H6 → C⁸ H5; f, C⁸ N⁴H1 → C⁸ H5; g, C⁵ N⁴H2 → C⁵ H5; h, C⁵ H6 → C⁵ H5; i, C⁵ N⁴H1 → C⁵ H5; j, C¹⁹ N⁴H2 → C¹⁹ H5; k, C¹⁹ H6 → C¹⁹ H5; l, C¹⁹ N⁴H1 → C¹⁹ H5; m, C¹⁷ N⁴H2 → C¹⁷ H5; n, C¹⁷ H6 → C¹⁷ H5; o, C¹⁷ N⁴H1 → C¹⁷ H5; p, C³ N⁴H2 → C³ H5; q, C³ H6 → C³ H5; r, C³ N⁴H1 → C³ H5; s, C¹ N⁴H2 → C¹ H5; t, C¹ H6 → C¹ H5; u, C¹ N⁴H1 → C¹ H5; v, C¹² N⁴H2 → C¹² H5; w, C¹² H6 → C¹² H5; x, C¹² N⁴H1 → C¹² H5; y, G¹⁵ N1H → C⁸ N⁴H2; z, G¹⁵ N1H → C⁸ N⁴H1; aa, G⁷ N1H → C¹⁶ N⁴H2; bb, G⁷ N1H → C¹⁶ N⁴H1; cc, G¹⁸ N1H → C⁵ N⁴H2; dd, G² N1H and X⁴ N1H → C¹⁹ N⁴H2; ee, G⁶ N1H and X⁴ N1H → C¹⁷ N⁴H2; ff, G¹⁸ N1H → C⁵ N⁴H1; gg, G² N1H and X⁴ N1H → C¹⁹ N⁴H1; hh, G⁶ N1H and X⁴ N1H → C¹⁷ N⁴H1; ii, G¹⁸ N1H and G²⁰ N1H → C³ N⁴H2; jj, G¹⁸ N1H and G²⁰ N1H → C³ N⁴H1. The 800 MHz, 250 ms mixing time NOESY experiments were performed at 10 °C.

Figure 4

Tile plot showing NOE cross-peaks between OPdG protons and DNA protons in the OPdG-2BD duplex. Cross-peaks: a, OPdG H8 → C⁵ H5′; b, OPdG H8 → C⁵ H4′; c, OPdG H8 → C⁵ H1′; d, OPdG H8 → OpdG H6; e, OPdG H8 → X⁴ H1′; f, OPdG H8 → OPdG H7; g, OPdG H8 → OPdG H8; h, OPdG H7 → C⁵ H5′; i, OPdG H7 → C⁵ H1′; j, OPdG H7 → OPdG H6. The 800 MHz, 250 ms mixing time NOESY experiment was conducted at 25 °C.

Figure 5

(A) Chemical shift differences of cytosine H5 and H6 protons of the OPdG-2BD duplex relative to the unmodified 2BD duplex. (B) Chemical shift differences of cytosine H5 and H6 protons of the M₁dG-2BD duplex relative to the unmodified 2BD duplex. The data for the cytosine H5 protons are colored gray and the data for the cytosine H6 protons black. (C) Chemical shift differences of nucleotide base protons A⁻² → G¹¹ of the OPdG-2BD duplex relative to the M₁dG-2BD duplex. (D) Chemical shift differences of nucleotide base protons C¹² → T²² of the OPdG-2BD duplex relative to the M₁dG-2BD duplex. The data for the nucleotide base aromatic H8/H6 protons are colored black, the data for the cytosine aromatic H5 protons gray, and the data for the sugar H1′ protons white. In all instances, Δδ = δ_{modified oligodeoxynucleotide} – δ_{unmodified oligodeoxynucleotide} (parts per million).

Figure 6

Intensity ratios of the two C¹ H5 → C¹ H6 cross-peaks in the COSY spectrum, arising from M₁dG and OPdG, as a function of time: (A) freshly prepared OpdG-2BD duplex and (B) freshly prepared M₁dG-2BD duplex. The solid lines represent the best fits through the data in both plots. The errors in measuring the ratios are estimated to be ±2%.