Abstract:

Error-prone DNA polymerases (pols) are conserved in all three kingdoms of life to promote the potentially mutagenic process of translesion DNA synthesis (TLS) following DNA damage. Due to their reduced fidelity, organisms must manage the actions of their error-prone polymerases to balance mutagenic TLS with replication. Using E. coli, I have utilized genetic and biochemical approaches to understand the roles of polymerase concentration, RecA nucleo-protein filament activation, and specific protein-protein contacts with the ?-clamp in polymerase switching. My findings suggest that access to the replication fork of DNA pol IV is regulated largely by its concentration. Furthermore, RecA appears to exclude access of pol IV while increasing access of DNA pol II and pol V. Biochemical characterization of pol-? clamp interactions in vitro indicate that the different pols bind overlapping yet discrete regions on the clamp, suggesting that unique pol-clamp interactions underlie the switching mechanism. To test this, pol III[arrow right]IV switching experiments were performed utilizing the ?R mutant, which contained mutations to the rim region of the clamp. This mutant displays near wild-type pol III and IV replication but is inhibited for pol III[arrow right]IV switching in vitro. Additionally, a second ? mutant ?+/? C , which contains only one functional hydrophobic cleft, does not inhibit a pol III[arrow right]IV switch. Furthermore, this switching mechanism is unique to pol IV as both pol II and the ? subunit are unaffected by the ?R mutation or do not exhibit switching respectively. Taken together, pol IV has a concerted switching mechanism with pol III which utilizes a pol IV unique binding region (?R ) for switching with pol III.