The mono-ubiquitination of proliferating cell nuclear antigen (PCNA) by the RAD6-RAD18 complex is involved in the regulation of translesion DNA synthesis, which is one of the sub-pathways of post-replication repair. Translesion DNA polymerases that belong to the Y-family of DNA polymerases have ubiquitin interacting domains, which are required for high affinity binding to mono-ubiquitinated PCNA. This suggests an attractive model for mediating polymerase switching in which PCNA is mono-ubiquitinated at the stalled 3′ end of the replication fork, and the mono-ubiquitinated PCNA recruits translesion DNA polymerases for bypass replication. However, the fate of the replicative DNA polymerases at the damage site, the regulatory mechanism(s) governing the ubiquitination and the mechanism(s) controlling the switch back to replicative DNA polymerase after translesion DNA synthesis, are still obscure. Biochemical analyses in general, and reconstitution systems with purified proteins, in particular, provide powerful tools for addressing such questions, and have provided insights into the unexpected nature of polymerase switching. The possibility of whether the biochemical events demonstrated in vitro mimic in vivo cellular events in response to DNA damage is discussed.