Metal-Dependent Conformational Activation Explains Highly Promutagenic Replication across O6-Methylguanine by Human DNA Polymerase β

Abstract

Human DNA polymerase β (polβ) inserts, albeit slowly, T opposite the carcinogenic lesion O6-methylguanine (O6MeG) ∼30-fold more frequently than C. To gain insight into this promutagenic process, we solved four ternary structures of polβ with an incoming dCTP or dTTP analogue base-paired with O6MeG in the presence of active-site Mg2+ or Mn2+. The Mg2+-bound structures show that both the O6MeG·dCTP/dTTP–Mg2+ complexes adopt an open protein conformation, staggered base pair, and one active-site metal ion. The Mn2+-bound structures reveal that, whereas the O6Me·dCTP–Mn2+ complex assumes the similar altered conformation, the O6MeG·dTTP–Mn2+ complex adopts a catalytically competent state with a closed protein conformation and pseudo-Watson–Crick base pair. On the basis of these observations, we conclude that polβ slows nucleotide incorporation opposite O6MeG by inducing an altered conformation suboptimal for catalysis and promotes mutagenic replication by allowing Watson–Crick-mode for O6MeG·T but not for O6MeG·C in the enzyme active site. The O6MeG·dTTP–Mn2+ ternary structure, which represents the first structure of mismatched polβ ternary complex with a closed protein conformation and coplanar base pair, the first structure of pseudo-Watson–Crick O6MeG·T formed in the active site of a DNA polymerase, and a rare, if not the first, example of metal-dependent conformational activation of a DNA polymerase, indicate that catalytic metal-ion coordination is utilized as a kinetic checkpoint by polβ and is crucial for the conformational activation of polβ. Overall, our structural studies not only explain the promutagenic polβ catalysis across O6MeG but also provide new insights into the replication fidelity of polβ.

Insights into the effect of minor groove interactions and metal cofactors on mutagenic replication by human DNA polymerase β

“…We chose O6MeG because it is a close analog of the enol tautomeric species of guanine and because an analog of the tautomeric species of dTTP was not commercially available. Active-site Mn 2+ was used because this metal ion is known to facilitate the formation of a closed polβ conformation [20]. We expected O6MeG and dTTP to form a Watson-Crick-like base pair in the absence of the minor groove contact with dTTP.…”

“…The steady-state kinetic parameters for insertion opposite the templating guanine by polβ were measured as described previously [20]. Briefly, the DNA oligonucleotides for the kinetic assays (upstream primer, 5′-FAM-CTGCAGCTGATGCG-3′, downstream primer, 5′-phosphate/CGTACGGATCCCCGGGTAC-3′, and template, 5′-GTACCCGGGGATCCGTACGGCGCATCAGCTGCAG-3′) were synthesized by Midland Certified Reagent company (Midland, TX) and Integrated DNA Technologies (Coralville, IA).…”

“…Recently, we have reported that the X-family DNA polymerase β (polβ) induces the formation of a staggered dG:dTTP base pair in the presence of active-site Mg 2+ and a Watson-Crick-like dG:dTTP base pair in the presence of Mn 2+ (Figure 1) [19]. Unlike BF, polβ accommodates a staggered dA:dCTP base pair in the presence of either Mg 2+ or Mn 2+ [20]. …”

Structural Basis for the Inefficient Nucleotide Incorporation Opposite Cisplatin-DNA Lesion by Human DNA Polymerase β

“…The Pt-GG⅐dCTP*-Mg 2ϩ structure with a semiopen protein conformation most likely represents a ground state that requires a further conformational change to adopt a catalytically favorable conformation. To gain further insight into how pol␤ inserts dCTP opposite the 5Ј-dG of the Pt-GG lesion, we determined a ternary structure of the Pt-GG⅐dCTP* complex in the presence of the active site Mn 2ϩ , which has been shown to increase the insertion efficiency for various DNA polymerases (23,31,36). The Pt-GG⅐dCTP*-Mn 2ϩ structure, refined to 2.4-Å resolution, is substantially different from the Pt-GG⅐ dCTP*-Mg 2ϩ structure (r.m.s.d.…”

“…Steady-state Kinetics of Single Nucleotide Incorporation Opposite the Templating Pt-GG by Pol␤-Steady-state kinetic parameters were determined using the conditions described previously (23). Oligonucleotides used for kinetic assays (upstream primer, 5Ј-FAM (fluorescein amidite)-ATGGGGTTGATGTGC-3Ј; downstream primer, 5Ј-phosphate-GTAGGGATGTTTGGG-TAG-3Ј; and template, 5Ј-CTACCCAAACATCCCTACGGCA-CATCAACTCCAT-3Ј where GG denotes the site of cisplatin modification) were purchased from Integrated DNA Technologies.…”