After DNA damage, Def1 triggers degradation of the catalytic subunit of the replicative DNA polymerase at stalled replication forks, allowing special polymerases to take over DNA synthesis.
DNA damages hinder the advance of replication forks because of the inability of the replicative polymerases to synthesize across most DNA lesions. Because stalled replication forks are prone to undergo DNA breakage and recombination that can lead to chromosomal rearrangements and cell death, cells possess different mechanisms to ensure the continuity of replication on damaged templates. Specialized, translesion synthesis (TLS) polymerases can take over synthesis at DNA damage sites. TLS polymerases synthesize DNA with a high error rate and are responsible for damage-induced mutagenesis, so their activity must be strictly regulated. However, the mechanism that allows their replacement of the replicative polymerase is unknown. Here, using protein complex purification and yeast genetic tools, we identify Def1 as a key factor for damage-induced mutagenesis in yeast. In in vivo experiments we demonstrate that upon DNA damage, Def1 promotes the ubiquitylation and subsequent proteasomal degradation of Pol3, the catalytic subunit of the replicative polymerase δ, whereas Pol31 and Pol32, the other two subunits of polymerase δ, are not affected. We also show that purified Pol31 and Pol32 can form a complex with the TLS polymerase Rev1. Our results imply that TLS polymerases carry out DNA lesion bypass only after the Def1-assisted removal of Pol3 from the stalled replication fork.
Author Summary DNA damages can lead to the stalling of the cellular replication machinery if not repaired on time, inducing DNA strand breaks, recombination that can result in gross chromosomal rearrangements, even cell death. In order to guard against this outcome, cells have evolved several precautionary mechanisms. One of these involves the activity of special DNA polymerases—known as translesion synthesis (TLS) polymerases. In contrast to the replicative polymerases responsible for faithfully duplicating the genome, these can carry out DNA synthesis even on a damaged template. For that to occur, they have to take over synthesis from the replicative polymerase that is stalled at a DNA lesion. Although this mechanism allows DNA synthesis to proceed, TLS polymerases work with a high error rate even on undamaged DNA, leading to alterations of the original sequence that can result in cancer. Consequently, the exchange between replicative and special polymerases has to be highly regulated, and the details of this are largely unknown. Here we identified Def1—a protein involved in the degradation of RNA polymerase II—as a prerequisite for error-prone DNA synthesis in yeast. We showed that after treating the cells with a DNA damaging agent, Def1 promoted the degradation of the catalytic subunit of the replicative DNA polymerase δ, without affecting the other two subunits of the polymerase. Our data suggest that the special polymerases can take over synthesis only after the catalytic subunit of the replicative polymerase is removed from the stalled fork in a regulated manner. We predict that the other two subunits remain at the fork and participate in TLS together with the special polymerases.