During Translesion Synthesis, Escherichia coli DinB89 (T120P) Alters Interactions of DinB (Pol IV) with Pol III Subunit Assemblies and SSB, but Not with the β Clamp.

Michelle K Scotland, Caleb Homiski, Mark D Sutton

Journal of bacteriology 2022 Apr 19

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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.

Citation

Michelle K Scotland, Caleb Homiski, Mark D Sutton. During Translesion Synthesis, Escherichia coli DinB89 (T120P) Alters Interactions of DinB (Pol IV) with Pol III Subunit Assemblies and SSB, but Not with the β Clamp. Journal of bacteriology. 2022 Apr 19;204(4):e0061121