Monitoring the activity of the DinB protein in vivo

Abstract

The Escherichia coli protein DinB plays a vital role in the SOS response to DNA damage. DinB is a member of the Y-family of polymerases, which serve to copy DNA from a damaged bulky template in a process known as translesion synthesis (TLS). The wide active site of DinB allows it to perform this function that the more stringent replicative DNA polymerase is unsuited for. More specifically, DinB is the only Y-family polymerase that is conserved throughout all domains of life. In addition to synthesizing DNA opposite lesions, DinB is also associated with -1 frameshift mutations that are particularly lethal. RecA and UmuD have already been shown to modulate DinB activity preventing the -1 frameshift mutagenesis during TLS in vitro and increasing catalytic activity. However, no such study has been carried out in vivo. By constructing DinB, RecA, and UmuD fluorescent fusion proteins, the prevalence and localization of these proteins will be able to be tracked, thus proving or disproving the in vitro findings that DinB, RecA and UmuD form a multi-protein complex modulating DinB replication fidelity during TLS. æImportantly, the mutagenesis resulting from the Y-family of DNA polymerases has been implicated in antibiotic resistance acquisition in bacteria and likely plays a role in cancer in humans.