Human DNA polymerase beta (pol beta) inserts, albeit slowly, T opposite the carcinogenic lesion O6-methylguanine (O6MeG) similar to 30-fold more frequently than C. To gain insight into this promutagenic process, we solved four ternary structures of pol beta 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 center dot 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 center dot dCTP-Mn2+ complex assumes the similar altered conformation, the O6MeG center dot 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 pol beta slows nucleotide incorporation opposite O6MeG by inducing an altered conformation suboptimal for catalysis and promotes mutagenic replication by allowing Watson Crick-mode for O6MeG center dot T but not for O6MeG center dot C in the enzyme active site. The O6MeG center dot dTTP-Mn2+ ternary structure, which represents the first structure of mismatched pol beta ternary complex with a closed protein conformation and coplanar base pair, the first structure of pseudo-Watson-Crick O6MeG center dot 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 pol beta and is crucial for the conformational activation of pol beta. Overall, our structural studies not only explain the promutagenic pol beta catalysis across O6MeG but also provide new insights into the replication fidelity of pol beta.