Abstract
High fidelity replicative DNA polymerases are unable to synthesize past DNA adducts that result from diverse chemicals, reactive oxygen species or UV light. To bypass these replication blocks, cells utilize specialized translesion DNA polymerases that are intrinsically error prone and associated with mutagenesis, drug resistance, and cancer. How untimely access of translesion polymerases to DNA is prevented is poorly understood. Here we use co-localization single-molecule spectroscopy (CoSMoS) to follow the exchange of the E. coli replicative DNA polymerase Pol IIIcore with the translesion polymerases Pol II and Pol IV. We find that in contrast to the toolbelt model, the replicative and translesion polymerases do not form a stable complex on one clamp but alternate their binding. Furthermore, while the loading of clamp and Pol IIIcore is highly organized, the exchange with the translesion polymerases is stochastic and is not determined by lesion-recognition but instead a concentration-dependent competition between the polymerases.
Highlights
To ensure faithful replication of the genomic DNA, replicative DNA polymerases have a narrow active site that limits the incorporation of incorrect nucleotides
When the polymerase encounters a lesion on the template strand in the form of a modified base caused by diverse chemicals, reactive oxygen species, or UV light (Liu et al, 2016; Lindahl, 1996), the high-fidelity replicative DNA polymerases are stalled
We find that the sequential activities of the replication proteins clamp loader, clamp, and Pol IIIa are highly organized while the exchange with the translesion polymerases Pol II and Pol IV is disordered and determined by mass action through concentration-dependent competition for the hydrophobic groove on the surface of the b-clamp
Summary
To ensure faithful replication of the genomic DNA, replicative DNA polymerases have a narrow active site that limits the incorporation of incorrect nucleotides. When the polymerase encounters a lesion on the template strand in the form of a modified base caused by diverse chemicals, reactive oxygen species, or UV light (Liu et al, 2016; Lindahl, 1996), the high-fidelity replicative DNA polymerases are stalled. To bypass these replication blocks, all cells harbor multiple specialized translesion DNA polymerases (Goodman and Woodgate, 2013) that have more open active sites and are able to accommodate bulky DNA adducts and continue DNA synthesis. The access of the translesion polymerases to DNA needs to be tightly controlled, but how this is achieved has been the subject of debate
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