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

ABSTRACT 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
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    • "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+ . "
    Computational Approaches to Protein Dynamics From Quantum to Coarse-Grained Methods, Edited by Monika Fuxreiter, 01/2015: pages 59-126; , ISBN: 978-1-4822-9786-7
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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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    ABSTRACT: N7-Methyl-2'-deoxyguanosine (m7dG) is the predominant lesion formed by methylating agents. A systematic investigation on the effect of m7dG on DNA replication has been difficult due to the chemical instability of m7dG. To gain insights into the m7dG effect, we employed a 2'-fluorine-mediated transition-state destabilzation strategy. Specifically, we determined kinetic parameters for dCTP insertion opposite a chemically stable m7dG analogue, 2'-fluoro-m7dG (Fm7dG), by human DNA polymerase β (polβ) and solved three X-ray structures of polβ in complex with the templating Fm7dG paired with incoming dCTP or dTTP analogues. The kinetic studies reveal that the templating Fm7dG slows polβ catalysis ∼300-fold, suggesting that m7dG in genomic DNA may impede replication by some DNA polymerases. The structural analysis reveals that Fm7dG forms a canonical Watson-Crick base pair with dCTP, but metal ion coordination is suboptimal for catalysis in the polβ-Fm7dG:dCTP complex, which partially explains the slow insertion of dCTP opposite Fm7dG by polβ. In addition, the polβ-Fm7dG:dTTP structure shows open protein conformations and staggered base pair conformations, indicating that N7-methylation of dG does not promote a promutagenic replication. Overall, the first systematic studies on the effect of m7dG on DNA replication reveal that polβ catalysis across m7dG is slow, yet highly accurate.
    Nucleic Acids Research 06/2014; 42(13). DOI:10.1093/nar/gku554 · 9.11 Impact Factor
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    • "Unlike EcDpo4 and the archaeal homolog ssDpo4, the ability of MsDpo4 to promote mismatches appears to be restricted mostly to G:dTTP and T:dGTP mismatches. Structures of ternary complexes of DNA polymerases with DNA and nucleotides that lead to the presence of a mismatch in the active site have been determined earlier [8, 33–38]. These studies show that the manner in which a mismatch is accommodated in the active site appears to be unique for each polymerase and also depends on which nucleotide is present at the templating and incoming positions. "
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    ABSTRACT: Error-prone DNA synthesis in prokaryotes imparts plasticity to the genome to allow for evolution in unfavorable environmental conditions, and this phenomenon is termed adaptive mutagenesis. At a molecular level, adaptive mutagenesis is mediated by upregulating the expression of specialized error-prone DNA polymerases that generally belong to the Y-family, such as the polypeptide product of the dinB gene in case of E. coli. However, unlike E. coli, it has been seen that expression of the homologs of dinB in Mycobacterium tuberculosis are not upregulated under conditions of stress. These studies suggest that DinB homologs in Mycobacteria might not be able to promote mismatches and participate in adaptive mutagenesis. We show that a representative homolog from Mycobacterium smegmatis (MsDpo4) can carry out template-dependent nucleotide incorporation and therefore is a DNA polymerase. In addition, it is seen that MsDpo4 is also capable of misincorporation with a significant ability to promote G:T and T:G mismatches. The frequency of misincorporation for these two mismatches is similar to that exhibited by archaeal and prokaryotic homologs. Overall, our data show that MsDpo4 has the capacity to facilitate transition mutations and can potentially impart plasticity to the genome.
    Journal of nucleic acids 03/2012; 2012(6):285481. DOI:10.1155/2012/285481
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