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A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode.

ABSTRACT: Processive DNA synthesis by the ??? core of the Escherichia coli Pol III replicase requires it to be bound to the ?2 clamp via a site in the ? polymerase subunit. How the ? proofreading exonuclease subunit influences DNA synthesis by ? was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of ?. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel ?-binding site in ? that, in conjunction with the site in ?, maintains a closed state of the ???-?2 replicase in the polymerization mode of DNA synthesis. The ?-? interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states.

SUBMITTER: Jergic S

PROVIDER: S-EPMC3642676 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode.

Jergic Slobodan S Horan Nicholas P NP Elshenawy Mohamed M MM Mason Claire E CE Urathamakul Thitima T Ozawa Kiyoshi K Robinson Andrew A Goudsmits Joris M H JM Wang Yao Y Pan Xuefeng X Beck Jennifer L JL van Oijen Antoine M AM Huber Thomas T Hamdan Samir M SM Dixon Nicholas E NE

The EMBO journal 20130222 9

Processive DNA synthesis by the αεθ core of the Escherichia coli Pol III replicase requires it to be bound to the β2 clamp via a site in the α polymerase subunit. How the ε proofreading exonuclease subunit influences DNA synthesis by α was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of ε. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a n ...[more]

PMID: 23435564

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Project description:Translesion synthesis (TLS) by specialized DNA polymerases (Pols) is an evolutionarily conserved mechanism for tolerating replication-blocking DNA lesions. Using the Escherichia coli dinB-encoded Pol IV as a model to understand how TLS is coordinated with the actions of the high-fidelity Pol III replicase, we previously described a novel Pol IV mutant containing a threonine 120-to-proline mutation (Pol IV-T120P) that failed to exchange places with Pol III at the replication fork in vitro as part of a Pol III-Pol IV switch. This in vitro defect correlated with the inability of Pol IV-T120P to support TLS in vivo, suggesting Pol IV gains access to the DNA, at least in part, via a Pol III-Pol IV switch. Interaction of Pol IV with the β sliding clamp and the single-stranded DNA binding protein (SSB) significantly stimulates Pol IV replication and facilitates its access to the DNA. In this work, we demonstrate that Pol IV interacts physically with Pol III. We further show that Pol IV-T120P interacts normally with the β clamp, but is impaired in interactions with the α catalytic and εθ proofreading subunits of Pol III, as well as SSB. Taken together with published work, these results provide strong support for the model in which Pol IV-Pol III and Pol IV-SSB interactions help to regulate the access of Pol IV to the DNA. Finally, we describe several additional E. coli Pol-Pol interactions, suggesting Pol-Pol interactions play fundamental roles in coordinating bacterial DNA replication, DNA repair, and TLS. IMPORTANCE Specialized DNA polymerases (Pols) capable of catalyzing translesion synthesis (TLS) generate mutations that contribute to bacterial virulence, pathoadaptation, and antimicrobial resistance. One mechanism by which the bacterial TLS Pol IV gains access to the DNA to generate mutations is by exchanging places with the bacterial Pol III replicase via a Pol III-Pol IV switch. Here, we describe multiple Pol III-Pol IV interactions and discuss evidence that these interactions are required for the Pol III-Pol IV switch. Furthermore, we describe several additional E. coli Pol-Pol interactions that may play fundamental roles in managing the actions of the different bacterial Pols in DNA replication, DNA repair, and TLS.

Dataset Information

A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode.

Publications

A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode.

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