Insights into the Conformation of Aminofluorene-Deoxyguanine Adduct in a DNA Polymerase Active Site

The active site conformation of the mutagenic fluoroaminofluorene-deoxyguanine adduct (dG-FAF, N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) has been investigated in the presence of Klenow fragment of Escherichia coli DNA polymerase I (Kfexo−) and DNA polymerase β (pol β) using 19F NMR, insertion assay, and surface plasmon resonance. In a single nucleotide gap, the dG-FAF adduct adopts both a major-groove- oriented and base-displaced stacked conformation, and this heterogeneity is retained upon binding pol β. The addition of a non-hydrolysable 2′-deoxycytosine-5′-[(α,β)-methyleno]triphosphate (dCMPcPP) nucleotide analog to the binary complex results in an increase of the major groove conformation of the adduct at the expense of the stacked conformation. Similar results were obtained with the addition of an incorrect dAMPcPP analog but with formation of the minor groove binding conformer. In contrast, dG-FAF adduct at the replication fork for the Kfexo− complex adopts a mix of the major and minor groove conformers with minimal effect upon the addition of non-hydrolysable nucleotides. For pol β, the insertion of dCTP was preferred opposite the dG-FAF adduct in a single nucleotide gap assay consistent with 19F NMR data. Surface plasmon resonance binding kinetics revealed that pol β binds tightly with DNA in the presence of correct dCTP, but the adduct weakens binding with no nucleotide specificity. These results provide molecular insights into the DNA binding characteristics of FAF in the active site of DNA polymerases and the role of DNA structure and sequence on its coding potential

Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse

DNA polymerases use a general two-metal ion mechanism for DNA synthesis. Recent time-lapse crystallographic studies identified additional adjunct metal ions in the polymerase active site. One of these ions correlates with appearance of pyrophosphate and was proposed to be involved in pyrophosphorolysis (reverse reaction of DNA synthesis). Because DNA polymerases can use pyrophosphorolysis to remove chain-terminating nucleotides during chemotherapies, a better understanding of this reaction is warranted. Through site-directed mutagenesis, pyrophosphorolysis measurements, and computational analysis, we examine the role of metal ions in the reverse reaction. The results indicate that the product-associated metal ion facilitates pyrophosphorolysis during the early stages of the reaction but deters the reaction at later stages, suggesting dynamic metal behavior that can modulate the chemical equilibrium

Amino Acid Substitution in the Active Site of DNA Polymerase β Explains the Energy Barrier of the Nucleotidyl Transfer Reaction

DNA polymerase β (pol β) is a bifunctional enzyme widely studied for its roles in base excision DNA repair where one key function is gap-filling DNA synthesis. In spite of significant progress in recent years, the atomic level mechanism of the DNA synthesis reaction has remained poorly understood. Based on crystal structures of pol β in complex with its substrates and theoretical considerations of amino acids and metals in the active site, we have proposed that a nearby carboxylate group of Asp256 enables the reaction by accepting a proton from the primer O3′ group, thus activating O3′ as the nucleophile in the reaction path. Here, we tested this proposal by altering the side chain of Asp256 to Glu and then exploring the impact of this conservative change on the reaction. The D256E enzyme is more than 1,000-fold less active than the wild-type enzyme, and the crystal structures are subtly different in the active sites of the D256E and wild-type enzymes. Theoretical analysis of DNA synthesis by the D256E enzyme shows that the O3′ proton still transfers to the nearby carboxylate of residue 256. However, the electrostatic stabilization and location of the O3′ proton transfer during the reaction path are dramatically altered compared with wild-type. Surprisingly, this is due to repositioning of the Arg254 side chain in the Glu256 enzyme active site, such that Arg254 is not in position to stabilize the proton transfer from O3′. The theoretical results with the wild-type enzyme indicate early charge reorganization associated with the O3′ proton transfer, and this does not occur in the D256E enzyme. The charge reorganization is mediated by the catalytic magnesium ion in the active site

Nucleotide-Induced DNA Polymerase Active Site Motions Accommodating a Mutagenic DNA Intermediate

SummaryDNA polymerases occasionally insert the wrong nucleotide. For this error to become a mutation, the mispair must be extended. We report a structure of DNA polymerase β (pol β) with a DNA mismatch at the boundary of the polymerase active site. The structure of this complex indicates that the templating adenine of the mispair stacks with the primer terminus adenine while the templating (coding) cytosine is flipped out of the DNA helix. Soaking the crystals of the binary complex with dGTP resulted in crystals of a ternary substrate complex. In this case, the templating cytosine is observed within the DNA helix and forms Watson-Crick hydrogen bonds with the incoming dGTP. The adenine at the primer terminus has rotated into a syn-conformation to interact with the opposite adenine in a planar configuration. Yet, the 3′-hydroxyl on the primer terminus is out of position for efficient nucleotide insertion

Normal Human Aging: The Baltimore Longitudinal Study on Aging

Normal Human Aging is an overview of the first 23 years of research findings about the natural course of human aging. The Baltimore Longitudinal Study of Aging was started in 1958 to "trace the effects of aging in humans." The BLSA recruited men aged 17 to 96 and women beginning in 1978 to participate in repeated assessments of health and physical and psychological performance. Visits were every two years over 2 1/2 days

α,β-Methylene-2′-deoxynucleoside 5′-triphosphates as noncleavable substrates for DNA polymerases: Isolation, characterization, and stability studies of novel 2′-deoxycyclonucleosides, 3,5′-cyclo-dG, and 2,5′-cyclo-dT

We report synthesis and characterization of a complete set of α,β-methylene-2′-dNTPs (α,β-m-dNTP; N = A, C, T, G, 12-15) in which the α,β-oxygen linkage of natural dNTP was replaced by a methylene group. These nucleotides were designed to be noncleavable substrates for DNA polymerases. Synthesis entails preparation of 2′-deoxynucleoside 5′-diphosphate precursors, followed by an enzymatic γ-phosphorylation. All four synthesized α,β-m-dNTPs were found to be potent inhibitors of polymerase β, with Ki values ranging 1-5 μM. During preparation of the dG and dT derivatives of α,β-methylene diphosphate, we also isolated significant amounts of 3,5′-cyclo-dG (16) and 2,5′-cyclo-dT (17), respectively. These novel 2′-deoxycyclonucleosides were formed via a base-catalyzed intramolecular cyclization (N3 → C5′ and O2 → C5′, respectively). In acidic solution, both 16 and 17 underwent glycolysis, followed by complete depurination. When exposed to alkaline conditions, 16 underwent an oxidative deamination to produce 3,5′-cyclo-2′-deoxyxanthosine (19), whereas 17 was hydrolyzed exclusively to dT. © 2008 American Chemical Society