Def1 Promotes the Degradation of Pol3 for Polymerase Exchange to Occur During DNA-Damage-Induced Mutagenesis in Saccharomyces cerevisiae

Daraba, Andreea and Gali, Vamsi K. and Halmai, Miklós and Haracska, Lajos and Unk, Ildikó (2014) Def1 Promotes the Degradation of Pol3 for Polymerase Exchange to Occur During DNA-Damage-Induced Mutagenesis in Saccharomyces cerevisiae. PLOS BIOLOGY, 12 (1). pp. 1-11. ISSN 1544-9173 (print); 1545-7885 (online)

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Abstract

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 delta, whereas Pol31 and Pol32, the other two subunits of polymerase delta, 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.

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