Linda B. Bloom

Learn More
The fidelity of Escherichia coli DNA polymerase III (pol III) is measured and the effects of beta, gamma processivity and epsilon proofreading subunits are evaluated using a gel kinetic assay. Pol III holoenzyme synthesizes DNA with extremely high fidelity, misincorporating dTMP, dAMP, and dGMP opposite a template G target with efficiencies finc = 5.6 x(More)
DNA polymerase is the critical enzyme maintaining genetic integrity during DNA replication. Individual steps in the replication process that contribute to DNA synthesis fidelity include nucleotide insertion, exonucleolytic proofreading, and binding to and elongation of matched and mismatched primer termini. Each process has been investigated using(More)
Sliding clamps and clamp loaders were initially identified as DNA polymerase processivity factors. Sliding clamps are ring-shaped protein complexes that encircle and slide along duplex DNA, and clamp loaders are enzymes that load these clamps onto DNA. When bound to a sliding clamp, DNA polymerases remain tightly associated with the template being copied,(More)
Gemcitabine [2',2'-difluorodeoxycytidine (dFdCyd)], a potent antitumor agent, inhibits DNA synthesis and is incorporated internally into DNA. The effect of a template-incorporated dFdCyd molecule (dFdCyd-) on DNA polymerase function was examined. Two 25-base deoxyoligonucleotides were synthesized with either a single dFdCyd- or template-incorporated(More)
The beta sliding clamp encircles DNA and enables processive replication of the Escherichia coli genome by DNA polymerase III holoenzyme. The clamp loader, gamma complex, assembles beta around DNA in an ATP-fueled reaction. Previous studies have shown that gamma complex opens the beta ring and also interacts with DNA on binding ATP. Here, a rapid kinetic(More)
Crystal structures of an Escherichia coli clamp loader have provided insight into the mechanism by which this molecular machine assembles ring-shaped sliding clamps onto DNA. The contributions made to the clamp loading reaction by two subunits, chi and psi, which are not present in the crystal structures, were determined by measuring the activities of three(More)
The effects of nearest neighbor interactions between a nucleotide base at the primer 3'-terminus and an incoming deoxyribonucleoside triphosphate on DNA polymerase catalyzed insertion were examined. Kinetics of inserting the fluorescent nucleotide analog 2-aminopurine deoxyribonucleotide (dAPMP) and dAMP opposite a template T by 3'-->5'(More)
The Escherichia coli DNA polymerase III gamma complex loads the beta clamp onto DNA, and the clamp tethers the core polymerase to DNA to increase the processivity of synthesis. ATP binding and hydrolysis promote conformational changes within the gamma complex that modulate its affinity for the clamp and DNA, allowing it to accomplish the mechanical task of(More)
Efficiencies of insertion and extension at a single site-directed abasic lesion, X, were measured while varying 5'- and 3'-template bases adjacent to X. The preference for insertion was found to be A > G > T approximately C, with the "upstream" (3'-neighboring) template base perturbing insertion efficiencies by an order of magnitude or more. Efficiencies of(More)