Abstract
N1-methyl-deoxyadenosine (1-MeA) is formed by methylation of deoxyadenosine at the N1 atom. 1-MeA presents a block to replicative DNA polymerases due to its inability to participate in Watson-Crick (W-C) base pairing. Here we determine how human DNA polymerase-ι (Polι) promotes error-free replication across 1-MeA. Steady state kinetic analyses indicate that Polι is ~100 fold more efficient in incorporating the correct nucleotide T versus the incorrect nucleotide C opposite 1-MeA. To understand the basis of this selectivity, we determined ternary structures of Polι bound to template 1-MeA and incoming dTTP or dCTP. In both structures, template 1-MeA rotates to the syn conformation but pairs differently with dTTP versus dCTP. Thus, whereas dTTP partakes in stable Hoogsteen base pairing with 1-MeA, dCTP fails to gain a “foothold” and is largely disordered. Together, our kinetic and structural studies show how Polι maintains discrimination between correct and incorrect incoming nucleotide opposite 1-MeA in preserving genome integrity.
Highlights
Alkylating agents are common reactive chemicals in the environment[1,2,3] and in cells (e.g. S-adenosylmethionine) that can modify the structures of biological macromolecules by transferring alkyl carbon groups[4]
We show that template 1-MeA adopts the syn conformation in both structures, though with significant differences. dTTP and dCTP insert differently opposite template 1-MeA with dTTP participating in Hoogsteen base pairing, while dCTP is largely disordered, consistent with multiple conformations
Alkylating agents modify DNA by adding alkyl groups to both the ring nitrogens and the exocyclic oxygen atoms, generating adducts that have cytotoxic effects5,6,8. 1-MeA is a common adduct generated by the transfer of methyl group to the N1 nitrogen atom of deoxyadenosine
Summary
Alkylating agents are common reactive chemicals in the environment (e.g. tobacco smoke)[1,2,3] and in cells (e.g. S-adenosylmethionine) that can modify the structures of biological macromolecules by transferring alkyl carbon groups[4]. Cells have evolved a variety of mechanisms to repair alkylated DNA bases[6,7,8] This includes the classical multi-step pathways invoking base excision repair (BER), mismatch repair (MMR), and nucleotide excision repair (NER), as well as specific enzymes that can directly dealkylate the bases. Amongst the latter, AlkB in E. coli[9,10] and ABH2 in mammals[11,12,13] use a mononuclear iron (II) center and cofactors such as 2-ketoglutarate and dioxygen to demethylate the 1-MeA adduct directly[8,14]. Our kinetic and structural studies show that Polιcan accommodate lesions such as 1-MeA with impaired W-C edges, but that it can maintain discrimination between correct and incorrect incoming nucleotides opposite the lesion
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