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Full-Text Articles in Biochemistry, Biophysics, and Structural Biology

Evaluating The Effects Of Enhanced Processivity And Metal Ions On Translesion Dna Replication Catalyzed By The Bacteriophage T4 Dna Polymerase, Edmunds Z. Reineks, Anthony J. Berdis May 2003

Evaluating The Effects Of Enhanced Processivity And Metal Ions On Translesion Dna Replication Catalyzed By The Bacteriophage T4 Dna Polymerase, Edmunds Z. Reineks, Anthony J. Berdis

Chemistry Faculty Publications

The fidelity of DNA replication is achieved in a multiplicative process encompassing nucleobase selection and insertion, removal of misinserted nucleotides by exonuclease activity, and enzyme dissociation from primer/templates that are misaligned due to mispairing. In this study, we have evaluated the effect of altering these kinetic processes on the dynamics of translesion DNA replication using the bacteriophage T4 replication apparatus as a model system. The effect of enhancing the processivity of the T4 DNA polymerase, gp43, on translesion DNA replication was evaluated using a defined in vitro assay system. While the T4 replicase (gp43 in complex with gp45) can ...


Examination Of The Role Of The Clamp-Loader And Atp Hydrolysis In The Formation Of The Bacteriophage T4 Polymerase Holoenzyme, Michael A. Trakselis, Anthony J. Berdis, Stephen J. Benkovic Feb 2003

Examination Of The Role Of The Clamp-Loader And Atp Hydrolysis In The Formation Of The Bacteriophage T4 Polymerase Holoenzyme, Michael A. Trakselis, Anthony J. Berdis, Stephen J. Benkovic

Chemistry Faculty Publications

Transient kinetic analyses further support the role of the clamp-loader in bacteriophage T4 as a catalyst which loads the clamp onto DNA through the sequential hydrolysis of two molecules of ATP before and after addition of DNA. Additional rapid-quench and pulse-chase experiments have documented this stoichiometry. The events of ATP hydrolysis have been related to the opening/closing of the clamp protein through fluorescence resonance energy transfer (FRET). In the absence of a hydrolysable form of ATP, the distance across the subunit interface of the clamp does not increase as measured by intramolecular FRET, suggesting gp45 cannot be loaded onto ...