Mutagenic Replication of the Major Oxidative Adenine Lesion 7,8-Dihydro-8-oxoadenine by Human DNA Polymerases

Abstract

Reactive oxygen species attack DNA to produce 7,8-dihyro-8-oxoguanine (oxoG) and 7,8-dihydro-8-oxoadenine (oxoA) as major lesions. The structural basis for the mutagenicity of oxoG, which induces G to T mutations, is well understood. However, the structural basis for the mutagenic potential of oxoA, which induces A to C mutations, remains poorly understood. To gain insight into oxoA-induced mutagenesis, we conducted kinetic studies of human DNA polymerases β and η replicating across oxoA and structural studies of polβ incorporating dTTP/dGTP opposite oxoA. While polη readily bypassed oxoA, it incorporated dGTP opposite oxoA with a catalytic specificity comparable to that of correct insertion, underscoring the promutagenic nature of the major oxidative adenine lesion. Polη and polβ incorporated dGTP opposite oxoA ~170-fold and ~100-fold more efficiently than that opposite dA, respectively, indicating that the 8-oxo moiety greatly facilitated error-prone replication. Crystal structures of polβ showed that, when paired with an incoming dTTP, the templating oxoA adopted an anti conformation and formed Watson-Crick base pair. When paired with dGTP, oxoA adopted a syn conformation and formed a Hoogsteen base pair with Watson-Crick-like geometry, highlighting the dual-coding potential of oxoA. The templating oxoA was stabilized by Lys280-mediated stacking and hydrogen bonds. Overall, these results provide insight into the mutagenic potential and dual-coding nature of the major oxidative adenine lesion.

Structural insights into the promutagenic bypass of the major cisplatin-induced DNA lesion

“…Overall, the empty space within the nascent base pair binding pocket is best filled with adenine due to its steric bulkiness as a purine and abilities to form pi stacking interactions. With a role for polβ in mutagenic replication past other DNA lesions [49,53,54], once again we are poised with evidence to appreciate the sensitivity of polβ in binding inaccurate nucleotides across bulky DNA lesions with minor alterations in protein conformation.…”

DNA interstrand cross-links induced by the major oxidative adenine lesion 7,8-dihydro-8-oxoadenine

“…Later studies showed that oxoA is significantly mutagenic in mammalian cells, underscoring the differential mutagenicity of oxoA in prokaryotes and eukaryotes. Indeed, recent kinetic and structural studies reveal that bypass of oxoA by human DNA polymerases is highly error prone 69 , 70 . Unlike oxoG, the mutagenicity of oxoA in human cells may be partially due to minor groove contacts by DNA polymerases 69 , 70 .…”

Mechanisms of telomerase inhibition by oxidized and therapeutic dNTPs

“…Although 8-oxo-dATP is a very poor telomerase substrate, it inhibits processivity. Polymerase studies with 8-oxo-dATP are lacking, but mammalian DNA polymerases insert dTTP or dGTP opposite a template 8-oxodA and accommodate the base pairs through base tautomerization and interactions with active site residues 49 . Telomerase may lack contacts required to stabilize an 8-oxodA base pair for catalysis, or 8-oxo-dATP may occupy the active site in a non-productive manner blocking natural dNTP access, similar to telomerase inhibitor 5-MeCITP 50 .…”