Natural tetracyclic diterpenoid aphidicolin is a potent and specific inhibitor of B-family DNA polymerases, haltering replication possessing strong antimitotic activity in human cancer cell lines. Clinical trials revealed limitations as an antitumor drug because its low solubility fast clearance from plasma. The absence structural information hampered the improvement aphidicolin-like inhibitors: more than 50 modifications have been generated so far, but all lost inhibitory properties. Here...

Using iterative database searches, we identified a new subfamily of the AID/APOBEC family RNA/DNA editing cytidine deaminases. The subfamily, which is represented by readily identifiable orthologs in mammals, chicken, and frog, but not fishes, was designated APOBEC4. zinc-coordinating motifs involved catalysis secondary structure APOBEC4 deaminase domain are evolutionarily conserved, suggesting that proteins active polynucleotide (deoxy)cytidine In reconstructed maximum likelihood...

Abstract The base analog 6-N-hydroxylaminopurine (HAP) induces bidirectional GC → AT and transitions that are enhanced in DNA polymerase ϵ δ 3′ 5′ exonuclease-deficient yeast mutants, pol2-4 pol3-01, respectively. We have constructed a set of isogenic strains to determine whether the polymerases contribute equally proofreading replication errors provoked by HAP during leading lagging strand synthesis. Site-specific Pol→, or pol3-01 genetic background were scored as reversions ura3 missense...

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDNA Replication Fidelity with 8-Oxodeoxyguanosine TriphosphateYouri I. Pavlov, Dana T. Minnick, Shunji Izuta, and Thomas A. KunkelCite this: Biochemistry 1994, 33, 15, 4695–4701Publication Date (Print):April 19, 1994Publication History Published online1 May 2002Published inissue 19 April 1994https://pubs.acs.org/doi/10.1021/bi00181a029https://doi.org/10.1021/bi00181a029research-articleACS PublicationsRequest reuse permissionsArticle...

Eukaryotic DNA polymerase δ (Pol δ) plays an essential role in replicating large nuclear genomes, a process that must be accurate to maintain stability over many generations. Based on kinetic studies of insertion individual dNTPs opposite template guanine, Pol is believed have high selectivity for inserting correct nucleotides. This selectivity, conjunction with intrinsic 3′-exonuclease activity, implies should base substitution fidelity. Here we demonstrate the wild type Saccharomyces...

Evolution of DNA polymerases, the key enzymes replication and repair, is central to any reconstruction history cellular life. However, details evolutionary relationships between polymerases archaea eukaryotes remain unresolved. We performed a comparative analysis archaeal, eukaryotic, bacterial B-family which are main replicative in eukaryotes, combined with an domain architectures. Surprisingly, we found that eukaryotic Polymerase ε consists two tandem exonuclease-polymerase modules, active...

Abstract Clusters of localized hypermutation in human breast cancer genomes, named “kataegis” (from the Greek for thunderstorm), are hypothesized to result from multiple cytosine deaminations catalyzed by AID/APOBEC proteins. However, a direct link between APOBECs and kataegis is still lacking. We have sequenced genomes yeast mutants induced diploids expression gene PmCDA1, hypermutagenic deaminase sea lamprey. Analysis distribution 5,138 mutations revealed clusters very similar those found...

DNA replication in almost all organisms depends on the activity of primase, a DNA-dependent RNA polymerase that synthesizes short primers defined size for polymerases. Eukaryotic and archaeal primases are heterodimers consisting small catalytic large accessory subunits, both which necessary activity. The mode interaction primase subunits with substrates during various steps primer synthesis results counting length is not clear. Here we show C-terminal domain subunit (p58C) plays major role...

We have purified wild type and exonuclease-deficient four-subunit DNA polymerase ϵ (Pol ϵ) complex from Saccharomyces cerevisiae analyzed the fidelity of synthesis by two enzymes. Wild Pol synthesizes accurately, generating single-base substitutions deletions at average error rates ≤2 × 10-5 ≤5 10-7, respectively. lacking 3′ → 5′ exonuclease activity is less accurate to a degree suggesting that proofreads least 92% base substitution errors 99% frameshift made polymerase. Surprisingly...

Abstract Several amino acids in the active site of family A DNA polymerases contribute to accurate synthesis. For two these residues, B have conserved tyrosine residues regions II and III that are suggested similar functions. Here we replaced each with alanine catalytic subunits yeast α, δ, ε, ζ examined consequences vivo. Strains substitution SL/MYPS/N motif region Polδ or Polε inviable. same Rev3, subunit Polζ, nearly UV immutable, suggesting severe loss function. strain this Polα...

To test the hypothesis that inaccurate DNA synthesis by mammalian polymerase eta (pol eta) contributes to somatic hypermutation (SHM) of Ig genes, we measured error specificity mouse pol during each strand a kappa light chain transgene. We then compared results base substitution SHM this same gene in mouse. The vitro and vivo spectra shared number common features. A highly significant correlation was observed for overall substitutions at A-T pairs but not G-C pairs. Sixteen mutational...

AbstractThe eukaryotic DNA polymerase delta (Pol delta) participates in genome replication, homologous recombination, repair and damage tolerance. Regulation of the plethora Pol functions depends on interaction between second (p50) third (p66) non-catalytic subunits. We report crystal structure p50p66N complex featuring oligonucleotide binding phosphodiesterase domains p50 winged helix-turn-helix N-terminal domain p66. Disruption yeast orthologs p66 by strategic amino acid changes leads to...

Yeast DNA polymerase ε (Pol ε) is a highly accurate and processive enzyme that participates in nuclear replication of the leading strand template. In addition to large subunit (Pol2) harboring proofreading exonuclease active sites, Pol also has one essential (Dpb2) two smaller, non-essential subunits (Dpb3 Dpb4) whose functions are not fully understood. To probe Dpb3 Dpb4, here we investigate consequences their absence on biochemical properties vitro genome stability vivo. The fidelity...