Structures of DNA Polymerase β with Active-Site Mismatches Suggest a Transient Abasic Site Intermediate during Misincorporation

Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709, USA.
Molecular cell (Impact Factor: 14.02). 06/2008; 30(3):315-24. DOI: 10.1016/j.molcel.2008.02.025
Source: PubMed


We report the crystallographic structures of DNA polymerase beta with dG-dAMPCPP and dC-dAMPCPP mismatches in the active site. These premutagenic structures were obtained with a nonhydrolyzable incoming nucleotide analog, dAMPCPP, and Mn(2+). Substituting Mn(2+) for Mg(2+) significantly decreases the fidelity of DNA synthesis. The structures reveal that the enzyme is in a closed conformation like that observed with a matched Watson-Crick base pair. The incorrect dAMPCPP binds in a conformation identical to that observed with the correct nucleotide. To accommodate the incorrect nucleotide and closed protein conformation, the template strand in the vicinity of the active site has shifted upstream over 3 A, removing the coding base from the active site and generating an abasic templating pocket. The primer terminus rotates as its complementary template base is repositioned. This rotation moves O3' of the primer terminus away from the alpha-phosphate of the incoming nucleotide, thereby deterring misincorporation.

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Available from: Samuel Wilson, Feb 17, 2014
    • "This work allowed for a direct analysis of proposals that induced fit play an important role in the fidelity, and interestingly , demonstrated that similar to the proposal put forward in the earlier study of T7 DNA polymerase (Florián et al. 2005), the W nucleotide is incorporated through a transition state in one (or more) partially open conformations of the polymerase, whereas the R nucleotide is incorporated in a closed conformation . This work could be taken to a much more detailed level with the recent elucidation of crystallographic structures that showed different binding sites for the for the R (Batra et al. 2006) and W (Batra et al. 2008) nucleotides in the closed conformation of Pol β. That is, in a recent study (Prasad and Warshel 2011), the W to R movement between binding sites in the closed conformation of Pol β was simulated using potential of mean force (PMF) calculations, which moved the template O3′ to the catalytic Mg 2+ . "

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    • "The perturbation of the 3′ end of the primer terminus has been also observed with published polβ structures with incorrect insertions, showing a longer distance between primer terminus 3′-OH and the catalytic metal ion (e.g. dC:dATP (PDB ID: 3C2L) and dA:dGTP (PDB ID: 3C2M), 4.4 and 4.5 Å, respectively (29)). Mismatched structures also show a staggered base pair conformation and an upstream shift (∼3 Å) of template strand. "
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