Jeffrey G. Bertram

A Model for Escherichia coli DNA Polymerase III Holoenzyme Assembly at Primer/Template Ends DNA TRIGGERS A CHANGE IN BINDING SPECIFICITY OF THE γ COMPLEX CLAMP LOADER

The γ complex of the Escherichia coli DNA polymerase III holoenzyme assembles the β sliding clamp onto DNA in an ATP hydrolysis-driven reaction. Interactions between γ complex and primer/template DNA are investigated using fluorescence depolarization to measure binding of γ complex to different DNA substrates under steady-state and presteady-state conditions. Surprisingly, γ complex has a much higher affinity for single-stranded DNA (K d in the nmrange) than for a primed template (K d in the μm range) under steady-state conditions. However, when examined on a millisecond time scale, we find that γ complex initially binds very rapidly and with high affinity to primer/template DNA but is converted subsequently to a much lower affinity DNA binding state. Presteady-state data reveals an effective dissociation constant of 1.5 nm for the initial binding of γ complex to DNA and a dissociation constant of 5.7 μm for the low affinity DNA binding state. Experiments using nonhydrolyzable ATPγS show that ATP binding converts γ complex from a low affinity “inactive” to high affinity “active” DNA binding state while ATP hydrolysis has the reverse effect, thus allowing cycling between active and inactive DNA binding forms at steady-state. We propose that a DNA-triggered switch between active and inactive states of γ complex provides a two-tiered mechanism enabling γ complex to recognize primed template sites and load β, while preventing γ complex from competing with DNA polymerase III core for binding a newly loaded β·DNA complex.

Pre-steady State Analysis of the Assembly of Wild Type and Mutant Circular Clamps of Escherichia coli DNA Polymerase III onto DNA

The β protein, a dimeric ring-shaped clamp essential for processive DNA replication by Escherichia coli DNA polymerase III holoenzyme, is assembled onto DNA by the γ complex. This study examines the clamp loading pathway in real time, using pre-steady state fluorescent depolarization measurements to investigate the loading reaction and ATP requirements for the assembly of β onto DNA. Two β dimer interface mutants, L273A and L108A, and a nonhydrolyzable ATP analog, adenosine 5′-O-(3-thiotriphosphate) (ATPγS), have been used to show that ATP binding is required for γ complex and β to associate with DNA, but that a γ complex-catalyzed ATP hydrolysis is required for γ complex to release the β·DNA complex and complete the reaction. In the presence of ATP and γ complex, the β mutants associate with DNA as efficiently as wild type β. However, completion of the reaction is much slower with the β mutants because of decreased ATP hydrolysis by the γ complex, resulting in a much slower release of the mutants onto DNA. The effects of mutations in the dimer interface were similar to the effects of replacing ATP with ATPγS in reactions using wild type β. Thus, the assembly of β around DNA is coupled tightly to the ATPase activity of the γ complex, and completion of the assembly process requires ATP hydrolysis for turnover of the catalytic clamp loader.

Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution

Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ∼5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer.

DNA polymerase V activity is autoregulated by a novel intrinsic DNA-dependent ATPase

Escherichia coli DNA polymerase V (pol V), a heterotrimeric complex composed of UmuD′2C, is marginally active. ATP and RecA play essential roles in the activation of pol V for DNA synthesis including translesion synthesis (TLS). We have established three features of the roles of ATP and RecA. (1) RecA-activated DNA polymerase V (pol V Mut), is a DNA-dependent ATPase; (2) bound ATP is required for DNA synthesis; (3) pol V Mut function is regulated by ATP, with ATP required to bind primer/template (p/t) DNA and ATP hydrolysis triggering dissociation from the DNA. Pol V Mut formed with an ATPase-deficient RecA E38K/K72R mutant hydrolyzes ATP rapidly, establishing the DNA-dependent ATPase as an intrinsic property of pol V Mut distinct from the ATP hydrolytic activity of RecA when bound to single-stranded (ss)DNA as a nucleoprotein filament (RecA*). No similar ATPase activity or autoregulatory mechanism has previously been found for a DNA polymerase. DOI: http://dx.doi.org/10.7554/eLife.02384.001