Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota

Abstract

The 8-oxo-guanine (8-oxo-G) lesion is the most abundant and mutagenic oxidative DNA damage existing in the genome. Due to its dual coding nature, 8-oxo-G causes most DNA polymerases to misincorporate adenine. Human Y-family DNA polymerase iota (polι) preferentially incorporates the correct cytosine nucleotide opposite 8-oxo-G. This unique specificity may contribute to polι's biological role in cellular protection against oxidative stress. However, the structural basis of this preferential cytosine incorporation is currently unknown. Here we present four crystal structures of polι in complex with DNA containing an 8-oxo-G lesion, paired with correct dCTP or incorrect dATP, dGTP, and dTTP nucleotides. An exceptionally narrow polι active site restricts the purine bases in a syn conformation, which prevents the dual coding properties of 8-oxo-G by inhibiting syn/anti conformational equilibrium. More importantly, the 8-oxo-G base in a syn conformation is not mutagenic in polι because its Hoogsteen edge does not form a stable base pair with dATP in the narrow active site. Instead, the syn 8-oxo-G template base forms the most stable replicating base pair with correct dCTP due to its small pyrimidine base size and enhanced hydrogen bonding with the Hoogsteen edge of 8-oxo-G. In combination with site directed mutagenesis, we show that Gln59 in the finger domain specifically interacts with the additional O 8 atom of the lesion base, which influences nucleotide selection, enzymatic efficiency, and replication stalling at the lesion site. Our work provides the structural mechanism of high-fidelity 8-oxo-G replication by a human DNA polymerase.DNA replication | Y family polymerase | translesion DNA synthesis | oxidative lesion | DNA mutagenesis

Translesion and Repair DNA Polymerases: Diverse Structure and Mechanism

“…4d). A similar Hoogsteen configuration is also adopted by Pol ι when correctly incorporating dCTP opposite 8-oxo-G, a prevalent lesion caused by oxidation (94). …”

Translesion DNA polymerases in eukaryotes: what makes them tick?

“…Since an incoming dA adopts a syn conformation and exhibits reduced base stacking, pol ι gives preference to dG remaining in an anti conformation that is stabilized via hydrogen bonding with glutamine (Q59) in the finger subdomain (Nair et al , 2006a). At the same time, the enzyme’s narrow groove width imposition on the nascent base pair makes pol ι more accurate than most polymerases while copying DNA containing the oxidative lesion, 8-oxoG (Kirouac and Ling, 2011). In this case, pol ι firmly holds the damaged base in the syn conformation that would normally cause pairing with the incorrect dA.…”

“…In this case, pol ι firmly holds the damaged base in the syn conformation that would normally cause pairing with the incorrect dA. However, the restrictive active site prevents this from occurring and instead promotes stable pairing with the smaller and correct dC (Kirouac and Ling, 2011). …”

A historical account of hoogsteen base‐pairs in duplex DNA

“…99 Aggarwal et al later put the matter to rest by (i) solving X-ray structures of Polι, unambiguously showing a protonated G-C + HG base-pair at pH 6.5, reinforcing their hypothesis that Polι has evolved to favor HG base-pairing by constraining the backbone C1′-C1′ distance between template and incoming nucleotide 100 and (ii) showing selective inhibition of DNA synthesis by Polι but not other polymerases when using 7-deazaadenine or 7-deazaguanine, which are incapable of forming HG base-pairing, as the templating residue. 101 Several other structures capturing DNA synthesis by Polι followed, ultimately demonstrating that major purine alkylation and oxidation lesions, including 1,N6-ethenoadenine, 102 N2-ethylguanine, 103 O6-methylguanine 104 and 8-oxoguanine, 105 adopted a syn conformation and, where possible, formed HG type base-pairs with incoming complementary pyrimidine and purine nucleotides (Figure 6) (reviewed in Makarova et al . 106 ).…”