DNA polymerase β (pol β) fills single nucleotide gaps in DNA during base excision repair and nonhomologous end-joining. Pol β must select the correct nucleotide fromamong a pool of four nucleotides with similar structures and properties in order to maintain genomic stability during DNA repair. Here, we use a combination of X-ray crystallography, fluorescence resonance energy transfer and nuclear magnetic resonance to show that pol β's ability to access the appropriate conformations both before and upon binding to nucleotide substrates is integral to its fidelity. Importantly, we also demonstrate that the inability of the I260Q mutator variant of pol β to properly navigate this conformational landscape results in error-prone DNA synthesis. Our work reveals that precatalytic conformational rearrangements themselves are an important underlying mechanism of substrate selection by DNA pol β.
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