Structure of DNA Polymerase β with the Mutagenic DNA Lesion 8-Oxodeoxyguanine Reveals Structural Insights into Its Coding Potential

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

Section: Resultsmentioning

confidence: 60%

Section: Resultsmentioning

confidence: 99%

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

Kirouac

2011

“…However, the ability of purine bases to stack with aromatic rings suggests that dATP or dGTP may simply be stacking with the displaced guanine or with the phenanthrene ring system in the productive complex for purine nucleotide misinsertion. Such a stacking arrangement of purine bases was observed in a structure of a ternary complex of Pol ␤ with a templating 8-oxodG that had been incubated with dATP (46). In another possible mechanism, the syn-conformation of the adducted deoxyguanosine could serve as a template for purine nucleotide misinsertion.…”

Section: Discussionmentioning

confidence: 92%

Structure of DNA polymerase β with a benzo[ c ]phenanthrene diol epoxide-adducted template exhibits mutagenic features

Batra

Shock

Prasad

et al. 2006

We have determined the crystal structure of the human base excision repair enzyme DNA polymerase ␤ (Pol ␤) in complex with a 1-nt gapped DNA substrate containing a template N 2 -guanine adduct of the tumorigenic (؊)-benzo[c]phenanthrene 4R,3S-diol 2S,1R-epoxide in the gap. Nucleotide insertion opposite this adduct favors incorrect purine nucleotides over the correct dCMP and hence can be mutagenic. The structure reveals that the phenanthrene ring system is stacked with the base pair immediately 3 to the modified guanine, thereby occluding the normal binding site for the correct incoming nucleoside triphosphate. The modified guanine base is displaced downstream and prevents the polymerase from achieving the catalytically competent closed conformation. The incoming nucleotide binding pocket is distorted, and the adducted deoxyguanosine is in a syn conformation, exposing its Hoogsteen edge, which can hydrogen-bond with dATP or dGTP. In a reconstituted base excision repair system, repair of a deaminated cytosine (i.e., uracil) opposite the adducted guanine was dramatically decreased at the Pol ␤ insertion step, but not blocked. The efficiency of gap-filling dCMP insertion opposite the adduct was diminished by >6 orders of magnitude compared with an unadducted templating guanine. In contrast, significant misinsertion of purine nucleotides (but not dTMP) opposite the adducted guanine was observed. Pol ␤ also misinserts a purine nucleotide opposite the adduct with ungapped DNA and exhibits limited bypass DNA synthesis. These results indicate that Pol ␤-dependent base excision repair of uracil opposite, or replication through, this bulky DNA adduct can be mutagenic.DNA adduct ͉ DNA repair ͉ fidelity ͉ mutagenesis P olycyclic aromatic hydrocarbons (PAHs) such as benzo- , and the adduct derived from trans-opening of the epoxide ring by the exocyclic 2-amino group of guanine in DNA, which is the subject of the present investigation.Bulky PAH-derived lesions can persist in human tissues when cellular repair enzymes fail to correct these lesions (13). The presence of these lesions in the genome blocks replicative DNA polymerases. If these lesions are not repaired, they must be bypassed for replication to continue. Bypass DNA synthesis occurs when a translesional DNA polymerase is recruited to the site of DNA damage and inserts a nucleotide opposite the adduct. DNA synthesis can be error-free or error-prone. The resulting base pair is then extended by either the same DNA polymerase or an auxiliary polymerase. Because replication of these adducts is known to result in mutations (14-16), mutagenic bypass provides a mechanism for adduct-induced tumor initiation and progression.Several DNA repair mechanisms protect cells from the deleterious effects of DNA lesions. For example, bulky DNA adducts such as PAH adducts and the formamidopyrimidine adduct of aflatoxin are primarily repaired by nucleotide excision repair (17, 18), although some unstable B[a]P DE adducts might promote depurination and elicit the base excision repair (BER...

“…UDG and APE are relatively small enzymes that bend the helix axis of DNA Ϸ20°and 35°, respectively, on binding (36,37). This distortion is considerably less than that of a nicked DNA molecule bound to pol ␤, which causes a bend of Ϸ90° (38,39). Moreover, the catalytic activity of the V(D)J recombinase enzymes recombination-activating gene (RAG) 1 and RAG2 was tested on a nucleosome substrate, in combination with the nonhistone proteins HMG1 and HMG2 (7).…”

Section: Discussionmentioning

confidence: 99%

Suppressed catalytic activity of base excision repair enzymes on rotationally positioned uracil in nucleosomes

Beard

Wilson

Smerdon

2003

143

141

The majority of DNA in eukaryotic cells exists in the highly condensed structural hierarchy of chromatin, which presents a challenge to DNA repair enzymes in that recognition, incision, and restoration of the original sequence at most sites must take place within these structural constraints. To test base excision repair (BER) activities on chromatin substrates, an in vitro system was developed that uses human uracil DNA glycosylase (UDG), apyrimidinic͞apurinic endonuclease (APE), and DNA polymerase ␤ (pol ␤) on homogeneously damaged, rotationally positioned DNA in nucleosomes. We find that UDG and APE carry out their combined catalytic activities with reduced efficiency on nucleosome substrates (Ϸ10% of that on naked DNA). Furthermore, these enzymes distinguish between two different rotational settings of the lesion on the histone surface, showing a 2-to 3-fold difference in activity between uracil facing ''toward'' and ''away from'' the histones. However, UDG and APE will digest such substrates to completion in a concentration-dependent manner. Conversely, the synthesis activity of pol ␤ is inhibited completely by nucleosome substrates and is independent of enzyme concentration. These results suggest that the first two steps of BER, UDG and APE, may occur ''unassisted'' in chromatin, whereas downstream factors in this pathway (i.e., pol ␤) may require nucleosome remodeling for efficient DNA BER in at least some regions of chromatin in eukaryotic cells.chromatin ͉ glycosylase ͉ DNA polymerase ␤ ͉ apyrmidinic͞apurinic endonuclease