Base-Displaced Intercalated Structure of the 3-(2-Deoxy-β-D-erythropentofuranosyl)-pyrimido[1,2- f]purine-6,10(3 H,5 H)-dione (6-oxo-M1dG) Lesion in DNA

Abstract

The genotoxic 3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M₁dG) DNA lesion arises from endogenous exposures to base propenals generated by oxidative damage and from exposures to malondialdehyde (MDA), produced by lipid peroxidation. Once formed, M₁dG may oxidize, in vivo, to 3-(2-deoxy-β-D-erythropentofuranosyl)-pyrimido[1,2-f]purine-6,10(3H,5H)-dione (6-oxo-M₁dG). The latter blocks DNA replication and is a substrate for error-prone mutagenic bypass by the Y-family DNA polymerase hpol η. To examine structural consequences of 6-oxo-M₁dG damage in DNA, we conducted NMR studies of 6-oxo-M₁dG incorporated site-specifically into 5' -d(C¹A²T³X⁴A⁵T⁶G⁷A⁸C⁹G¹⁰C¹¹T¹²)-3':5'-d(A¹³G¹⁴C¹⁵G¹⁶T¹⁷C¹⁸A¹⁹T²⁰C²¹A²²T²³G²⁴)-3' (X = 6-oxo-M₁dG). NMR spectra afforded detailed resonance assignments. Chemical shift analyses revealed that nucleobase C²¹, complementary to 6-oxo-M₁dG, was deshielded compared with the unmodified duplex. Sequential NOEs between 6-oxo-M₁dG and A⁵ were disrupted, as well as NOEs between T²⁰ and C²¹ in the complementary strand. The structure of the 6-oxo-M₁dG modified DNA duplex was refined by using molecular dynamics (rMD) calculations restrained by NOE data. It revealed that 6-oxo-M₁dG intercalated into the duplex and remained in the anti-conformation about the glycosyl bond. The complementary cytosine C²¹ extruded into the major groove, accommodating the intercalated 6-oxo-M₁dG. The 6-oxo-M₁dG H7 and H8 protons faced toward the major groove, while the 6-oxo-M₁dG imidazole proton H2 faced into the major groove. Structural perturbations to dsDNA were limited to the 6-oxo-M₁dG damaged base pair and the flanking T³:A²² and A⁵:T²⁰ base pairs. Both neighboring base pairs remained within the Watson-Crick hydrogen bonding contact. The 6-oxo-M₁dG did not stack well with the 5'-neighboring base pair T³:A²² but showed improved stacking with the 3'-neighboring base pair A⁵:T²⁰. Overall, the base-displaced intercalated structure was consistent with thermal destabilization of the 6-oxo-M₁dG damaged DNA duplex; thermal melting temperature data showed a 15 °C decrease in T_m compared to the unmodified duplex. The structural consequences of 6-oxo-M₁dG formation in DNA are evaluated in the context of the chemical biology of this lesion.