Interactions Between the E. Coli Sos Response Protein Umud and DNA Polymerase III Alpha Subunit Have Implications for Regulating Replication in Response to DNA Damage

Abstract

Faithful and efficient DNA replication is critical for the survival of all organisms; however, DNA is constantly subject to damage from endogenous and exogenous sources. Attempts to replicate damaged DNA can result in harmful mutations. The bacterial SOS response system regulates DNA repair and other pathways responsible for the cellular response to DNA damage. Among genes upregulated during the SOS response is the umuD gene, encoding the manager protein UmuD that subsequently undergoes self-cleavage to remove its N-terminal 24 amino acids to form UmuD'. The umuD gene products are involved in managing the cellular response to DNA damage through their interactions with damage-bypass DNA polymerases DNA pol IV and pol V. UmuD and UmuD' also interact with the alpha DNA polymerase and beta processivity clamp subunits of the replicative DNA polymerase III. We probed the interactions between UmuD or UmuD' and the alpha polymerase subunit and determined that the umuD gene products interact with alpha at two sites: the N-terminal PHP domain, which is responsible for interactions with the epsilon proofreading subunit, and the C-terminal domain, which harbors interaction sites for the beta clamp, the tau subunit of the clamp loader, and single-stranded DNA (ssDNA). The C-terminal domain of alpha preferentially binds to full-length UmuD. With FRET experiments, we subsequently showed that UmuD but not UmuD' inhibits the interaction between alpha and beta, suggesting that early in the SOS response to DNA damage, UmuD could displace alpha from the beta processivity clamp. Furthermore, optical tweezers experiments revealed that UmuD specifically inhibits the interaction between alpha and ssDNA. Together, these observations suggest that UmuD plays a key role in regulating the replication fork after DNA damage.