In E. coli, exposure to ultra-violet light, as well as certain antibiotics, triggers a cellular survival mechanism known as the SOS response. A consequence of this is the up-regulation of three specialised DNA polymerases; pols II, IV and V. These proteins participate in DNA damage tolerance mechanism known as translesion DNA synthesis (TLS), which allows DNA synthesis to continue on damaged templates. However, TLS polymerases often make mistakes, producing mutations in the DNA sequence. On one hand mutations introduced by TLS polymerases can be seen as an unfortunate, but necessary, consequence of DNA damage tolerance. On the other hand, the increase in mutation rate caused by TLS may fuel rapid evolution and is likely to be involved in the development of antibiotic resistance.
We use single-molecule fluorescence microscopy to visualise pol IV as it carries out TLS in live E. coli cells. With this approach it is possible to extract detailed information on regulation processes, cellular localisation and binding kinetics of pol IV in cells treated with different DNA damaging agents. In response to DNA damage, pol IV expression is increased and foci form as it binds to DNA. Interestingly, we observe that only 10 % of pol IV molecules bound to DNA are in the vicinity of replisomes, indicating that replication rescue may represent a small proportion of pol IV’s total activities in cells. Even more interestingly, we found pol IV is only allowed access to replication forks for a window of just a few minutes. We find that access is licenced by RecA nucleoprotein filaments and is inhibited by the pol V protein UmuD, as well as its cleaved form UmuD′. These observations indicate that regulation of TLS at replication forks goes far beyond the existing text-book model of mass action-driven polymerase competition at stalled replication forks.