Mechanisms Underlying Actions of Escherichia coli DNA Polymerase IV in Translesion Synthesis
Heltzel, Justin Mark Howard
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Cells posses multiple systems to maintain genomic integrity from constant exposure to both exogenous and endogenous DNA damage. Translesion synthesis [TLS] polymerases replicate over lesions in front of the fork, that the replicative polymerase [Pol III] is unable too, preventing fork stalling. TLS polymerases are several orders of magnitude lower in fidelity then replicative polymerases. Unregulated access of them would result in loss of genomic integrity. E. coli posses 3 TLS polymerases. One of them, Pol IV is the most conserved polymerase in the animal kingdom. Pol IV is expressed at 250 copies per cell and is up regulated 10 fold by the cell stress response SOS system. One mechanism of regulating the polymerases is through their required interaction with sliding clamp proteins. Sliding clamp are ring shaped proteins that encircle and slide along DNA. All polymerase share a required interaction site [cleft contact] on the clamp. We have found that Pol IV has an additional site of interaction [rim contact] that is required for it to switch with Pol III. Pol IV can switch with a stalled Pol III but not a moving one and a 5-fold overexpression of Pol IV to toxic in strains that have slower moving forks and are prone to stalling. Overexpression of the other polymerases is not toxic to these strains. This suggests that Pol IV is either regularly recruited to the fork or that it is a component of the replisome. Pol IV may have unique contacts with the fork that are involved in it recruitment. To understand further how Pol IV is regulated, we have utilized a genetic selection to identify mutations in Pol IV the impair it's function. Thirteen novel Pol IV mutations were identified characterized. Genetic and biochemical characterization of the Pol IV-T120P mutant suggest a model for how the functions of Pol IV are coordinated with those of Pol III.