The ability of wild type and mutant T4 DNA polymerases to discriminate in the utilization of the base analog 2-aminopurine (2AP) and the fluorescence of 2AP were used to determine how DNA polymerases distinguish between correct and incorrect nucleotides. Because T4 DNA polymerase incorporates dTMP opposite 2AP under single-turnover conditions, it was possible to compare directly the kinetic parameters for incorporation of dTMP opposite template 2AP to the parameters for incorporation of dTMP opposite template A without the complication of enzyme dissociation. The most significant difference detected was in the K d for dTTP, which was 10-fold higher for incorporation of dTMP opposite template 2AP (ϳ367 M) than for incorporation of dTMP opposite template A (ϳ31 M). In contrast, the dTMP incorporation rate was reduced only about 2-fold from about 318 s ؊1 with template A to about 165 s ؊1 for template 2AP. Discrimination is due to the high selectivity in the initial nucleotide-binding step. T4 DNA polymerase binding to DNA with 2AP in the template position induces formation of a nucleotide binding pocket that is preshaped to bind dTTP and to exclude other nucleotides. If nucleotide binding is hindered, initiation of the proofreading pathway acts as an error avoidance mechanism to prevent incorporation of incorrect nucleotides.High fidelity DNA polymerases are responsible for replicating the genomic DNA of most organisms. Replication fidelity is achieved by accurate nucleotide incorporation, which has an error frequency of about 1 ϫ 10 Ϫ5 (1), and by exonucleolytic proofreading, which preferentially removes incorrect versus correct nucleotides from the primer terminus (2, 3) and improves fidelity another 100-fold or more. Many organisms also have additional mismatch correction pathways that further improve fidelity another 100-fold. However, the largest single contributor to accurate DNA replication is the ability of the DNA polymerase to insert the correct nucleotide with high precision.How do DNA polymerases discriminate between correct and incorrect nucleotides? Although hydrogen bonding appears to be an obvious mechanism for determining base pair specificity, this mechanism alone is not adequate. In the presence of water, free energy differences in hydrogen bonding between complementary and noncomplementary bases are not sufficient to produce the observed low nucleotide error frequency of 1 ϫ 10 Ϫ5 (4). Instead, results from numerous studies are consistent with the proposal that DNA polymerases discriminate between inserting correct or incorrect nucleotides based on the geometry of the base pair. For example, the Klenow fragment of Escherichia coli DNA polymerase I was shown to incorporate an isosteric analog of thymidylate with accuracy, even though the base analog could not form any hydrogen bonds with the template base (5). Structural studies of nucleotide pre-incorporation complexes formed with several DNA polymerases suggest that DNA polymerases may assess the geometry of the newly forming base pair by "clo...

The fluorescence of the base analogue 2-aminopurine (2AP) was used to detect physical changes in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase. Fluorescent enzyme-DNA complexes were formed with 2AP placed in the template strand opposite the primer terminus (the n position) and placed one template position 5' to the primer terminus (the n + 1 position). The fluorescence enhancement for 2AP at the n position was shown to be due to formation of the editing complex, which indicates that the 2AP-T terminal base pair is recognized primarily as a mismatch. 2AP fluorescence at the n + 1 position, however, was a reporter for DNA interactions in the polymerase active center that induce intrastrand base unstacking. T4 DNA polymerase produced base unstacking at the n + 1 position following formation of the phosphodiester bond. Thus, the increase in fluorescence intensity for 2AP at the n + 1 position could be used to measure the nucleotide incorporation rate in primer extension reactions in which 2AP was placed initially at the n + 2 position. Primer extension occurred at the rate of about 314 s(-1). The amount of base unstacking at the template n + 1 position was sensitive to the local DNA sequence. More base unstacking was detected for DNA substrates with an A-T base pair at the primer terminus compared to C-G or G-C base pairs. Since proofreading is also increased by A-T base pairs compared to G-C base pairs at the primer terminus, we propose that base unstacking may provide an opportunity for the DNA polymerase to reexamine the primer terminus.

The heterogeneity of brain cancers, as most solid tumors, complicates diagnosis and treatment. Identifying and targeting populations of cells driving tumorigenesis is a top priority for the cancer biology field. This is not a trivial task; considerable variance exists in the driving mutations, identifying markers, and evolutionary pressures influencing initiating cells in different individual tumors. Despite this, the ability to self-renew and differentiate must be conserved to reseed a heterogeneous tumor mass. Focusing on one example of a tumor-initiating cell population, we demonstrate that the atypical cyclin-like protein Spy1 plays a role in balancing the division properties of glioma cells with stemness properties. This mechanistic insight may provide new opportunities for therapeutic intervention of brain cancer.

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DNA polymerases achieve high-fidelity DNA replication in part by checking the accuracy of each nucleotide that is incorporated and, if a mistake is made, the incorrect nucleotide is removed before further primer extension takes place. In order to proofread, the primer-end must be separated from the template strand and transferred from the polymerase to the exonuclease active center where the excision reaction takes place; then the trimmed primer-end is returned to the polymerase active center. Thus, proofreading requires polymerase-to-exonuclease and exonuclease-to-polymerase active site switching. We have used a fluorescence assay that uses differences in the fluorescence intensity of 2-aminopurine (2AP) to measure the rates of active site switching for the bacteriophage T4 DNA polymerase. There are three findings: (i) the rate of return of the trimmed primer-end from the exonuclease to the polymerase active center is rapid, >500 s−1; (ii) T4 DNA polymerase can remove two incorrect nucleotides under single turnover conditions, which includes presumed exonuclease-to-polymerase and polymerase-to-exonuclease active site switching steps and (iii) proofreading reactions that initiate in the polymerase active center are not intrinsically processive.