Plasmid-based lacZalpha assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase

The preparation of a gapped pUC18 derivative, containing the lacZalpha reporter gene in the single-stranded region, is described. Gapping is achieved by flanking the lacZalpha gene with sites for two related nicking endonucleases, enabling the excision of either the coding or non-coding strand. However, the excised strand remains annealed to the plasmid through non-covalent Watson-Crick base-pairing; its removal, therefore, requires a heat-cool cycle in the presence of an exactly complementary competitor DNA. The gapped plasmids can be used to assess DNA polymerase fidelity using in vitro replication, followed by transformation into Escherichia coli and scoring the blue/white colony ratio. Results found with plasmids are similar to the well established method based on gapped M13, in terms of background ( approximately 0.08% in both cases) and the mutation frequencies observed with a number of DNA polymerases, providing validation for this straightforward and technically uncomplicated approach. Several error prone variants of the archaeal family-B DNA polymerase from Pyrococcus furiosus have been investigated, illuminating the potential of the method

Substrate-induced DNA strand misalignment during catalytic cycling by DNA polymerase λ

The simple deletion of nucleotides is common in many organisms. It can be advantageous when it activates genes beneficial to microbial survival in adverse environments, and deleterious when it mutates genes relevant to survival, cancer or degenerative diseases. The classical idea is that simple deletions arise by strand slippage. A prime opportunity for slippage occurs during DNA synthesis, but it remains unclear how slippage is controlled during a polymerization cycle. Here, we report crystal structures and molecular dynamics simulations of mutant derivatives of DNA polymerase λ bound to a primer–template during strand slippage. Relative to the primer strand, the template strand is in multiple conformations, indicating intermediates on the pathway to deletion mutagenesis. Consistent with these intermediates, the mutant polymerases generate single-base deletions at high rates. The results indicate that dNTP-induced template strand repositioning during conformational rearrangements in the catalytic cycle is crucial to controlling the rate of strand slippage

Requirement for PCNA in DNA Mismatch Repair at a Step Preceding DNA Resynthesis

Abstractid system was used to screen yeast and human expression libraries for proteins that interact with mismatch repair proteins. PCNA was recovered from both libraries and shown in the case of yeast to interact with both MLH1 and MSH2. A yeast strain containing a mutation in the PCNA gene had a strongly elevated mutation rate in a dinucleotide repeat, and the rate was not further elevated in a strain also containing a mutation in MLH1. Mismatch repair activity was examined in human cell extracts using an assay that does not require DNA repair synthesis. Activity was inhibited by p21 WAF1 or a p21 peptide, both of which bind to PCNA, and activity was restored to inhibited reactions by addition of PCNA. The data suggest a PCNA requirement in mismatch repair at a step preceding DNA resynthesis. The ability of PCNA to bind to MLH1 and MSH2 may reflect linkage between mismatch repair and replication and may be relevant to the roles of mismatch repair proteins in other DNA transactions

Loop 1 modulates the fidelity of DNA polymerase λ

Differences in the substrate specificity of mammalian family X DNA polymerases are proposed to partly depend on a loop (loop 1) upstream of the polymerase active site. To examine if this is the case in DNA polymerase λ (pol λ), here we characterize a variant of the human polymerase in which nine residues of loop 1 are replaced with four residues from the equivalent position in pol β. Crystal structures of the mutant enzyme bound to gapped DNA with and without a correct dNTP reveal that the change in loop 1 does not affect the overall structure of the protein. Consistent with these structural data, the mutant enzyme has relatively normal catalytic efficiency for correct incorporation, and it efficiently participates in non-homologous end joining of double-strand DNA breaks. However, DNA junctions recovered from end-joining reactions are more diverse than normal, and the mutant enzyme is substantially less accurate than wild-type pol λ in three different biochemical assays. Comparisons of the binary and ternary complex crystal structures of mutant and wild-type pol λ suggest that loop 1 modulates pol λ’s fidelity by controlling dNTP-induced movements of the template strand and the primer-terminal 3′-OH as the enzyme transitions from an inactive to an active conformation

Trace amounts of 8-oxo-dGTP in mitochondrial dNTP pools reduce DNA polymerase γ replication fidelity

Replication of the mitochondrial genome by DNA polymerase γ requires dNTP precursors that are subject to oxidation by reactive oxygen species generated by the mitochondrial respiratory chain. One such oxidation product is 8-oxo-dGTP, which can compete with dTTP for incorporation opposite template adenine to yield A-T to C-G transversions. Recent reports indicate that the ratio of undamaged dGTP to dTTP in mitochondrial dNTP pools from rodent tissues varies from ∼1:1 to >100:1. Within this wide range, we report here the proportion of 8-oxo-dGTP in the dNTP pool that would be needed to reduce the replication fidelity of human DNA polymerase γ. When various in vivo mitochondrial dNTP pools reported previously were used here in reactions performed in vitro, 8-oxo-dGTP was readily incorporated opposite template A and the resulting 8-oxo-G-A mismatch was not proofread efficiently by the intrinsic 3′ exonuclease activity of pol γ. At the dNTP ratios reported in rodent tissues, whether highly imbalanced or relatively balanced, the amount of 8-oxo-dGTP needed to reduce fidelity was <1% of dGTP. Moreover, direct measurements reveal that 8-oxo-dGTP is present at such concentrations in the mitochondrial dNTP pools of several rat tissues. The results suggest that oxidized dNTP precursors may contribute to mitochondrial mutagenesis in vivo, which could contribute to mitochondrial dysfunction and disease