Mark C. Young

Single Molecule Studies of the Effect of Template Tension on T7 DNA Polymerase Activity

T7 DNA polymerase catalyses DNA replication in vitro at rates of more than 100 bases per second and has a 3′→5′ exonuclease (nucleotide removing) activity at a separate active site. This enzyme possesses a ‘right hand’ shape which is common to most polymerases with fingers, palm and thumb domains. The rate-limiting step for replication is thought to involve a conformational change between an ‘open fingers’ state in which the active site samples nucleotides, and a ‘closed’ state in which nucleotide incorporation occurs. DNA polymerase must function as a molecular motor converting chemical energy into mechanical force as it moves over the template. Here we show, using a single-molecule assay based on the differential elasticity of single-stranded and double-stranded DNA, that mechanical force is generated during the rate-limiting step and that the motor can work against a maximum template tension of ∼34 pN. Estimates of the mechanical and entropic work done by the enzyme show that T7 DNA polymerase organizes two template bases in the polymerization site during each catalytic cycle. We also find a force-induced 100-fold increase in exonucleolysis above 40 pN.

Structural Analyses of gp45 Sliding Clamp Interactions during Assembly of the Bacteriophage T4 DNA Polymerase Holoenzyme I. CONFORMATIONAL CHANGES WITHIN THE gp44/62-gp45-ATP COMPLEX DURING CLAMP LOADING

A multisubunit ring-shaped protein complex is used to tether the polymerase to the DNA at the primer-template junction in most DNA replication systems. This “sliding clamp” interacts with the polymerase, completely encircles the DNA duplex, and is assembled onto the DNA by a specific clamp loading complex in an ATP-driven process. Site-specific mutagenesis has been used to introduce single cysteine residues as reactive sites for adduct formation within each of the three subunits of the bacteriophage T4-coded sliding clamp complex (gp45). Two such mutants, gp45S19C and gp45K81C, are reacted with the cysteine-specific photoactivable cross-linker TFPAM-3 and used to track the changes in the relative positioning of the gp45 subunits with one another and with the other components of the clamp loading complex (gp44/62) in the various stages of the loading process. Cross-linking interactions performed in the presence of nucleotide cofactors show that ATP binding and hydrolysis, interaction with primer-template DNA, and release of ADP all result in significant conformational changes within the clamp loading cycle. A structural model is presented to account for the observed rearrangements of intersubunit contacts within the complex during the loading process.

Mass Spectrometric Protocol for the Analysis of UV-Crosslinked Protein-Nucleic Acid Complexes

Publisher Summary This chapter describes the mass spectrometric protocol for the analysis of UV-crosslinked protein–nucleic acid complexes. It presents a protocol that combines developed methods for UV-light-induced photochemical crosslinking of proteins to nucleic acids with MALDI, and ESI mass spectrometry to locate and identify the particular amino acid and nucleotide residues that covalently bind to each other. The chapter explains the application of some of the stages in this analytical scheme to two different systems of protein–nucleic acid complexes, the phage T4 gene 32 protein UV-laser cross-linked to the oligonucleotide photoaffinity-labeled with 4-thiouridinediphosphate. In the first stage, the nucleic acid binding protein and its nucleic acid substrate or a photoactivatable analogue thereof are incubated under proper conditions to form the protein–nucleic acid complex. The sample is subsequently irradiated with UV light for a given period of time to create photochemically crosslinked protein–nucleic acid complexes.