Abstract
Polymerase eta (Polη) is a low fidelity translesion synthesis DNA polymerase that rescues damage-stalled replication by inserting deoxy-ribonucleotides opposite DNA damage sites resulting in error-free or mutagenic damage bypass. In this study we identify a new specific RNA extension activity of Polη of Saccharomyces cerevisiae. We show that Polη is able to extend RNA primers in the presence of ribonucleotides (rNTPs), and that these reactions are an order of magnitude more efficient than the misinsertion of rNTPs into DNA. Moreover, during RNA extension Polη performs error-free bypass of the 8-oxoguanine and thymine dimer DNA lesions, though with a 103 and 102–fold lower efficiency, respectively, than it synthesizes opposite undamaged nucleotides. Furthermore, in vivo experiments demonstrate that the transcription of several genes is affected by the lack of Polη, and that Polη is enriched over actively transcribed regions. Moreover, inactivation of its polymerase activity causes similar transcription inhibition as the absence of Polη. In summary, these results suggest that the new RNA synthetic activity of Polη can have in vivo relevance.
Highlights
Essential for the in vivo function of Polη, as mutations disrupting this interaction cause the same phenotype in yeast as the complete lack of Polη[11]
The results presented in this report uncover a specific RNA synthesis activity for the TLS DNA polymerase Polη
By measuring mRNA levels from inducible and constitutive promoters, we showed that transcription was generally diminished in the absence of Polη
Summary
Essential for the in vivo function of Polη, as mutations disrupting this interaction cause the same phenotype in yeast as the complete lack of Polη[11]. The exclusion is not complete and even the major replicative DNA polymerases have been shown to insert rNTPs during DNA synthesis with varying low frequency[16,17,18,19,20]. In this study we show that Polη is inefficient in inserting rNTPs during DNA synthesis, but unexpectedly, it has the specific activity to extend RNA strands with ribonucleotides. Polη can insert dNTPs into RNA, and it does so with similar efficiencies as with rNTPs at nucleotide concentrations estimating the in vivo conditions. Polη is required for the efficient transcription of several genes in vivo and is physically associated with the open reading frame of the actively transcribed GAL1 gene. We demonstrate that the polymerase activity of Polη is required for its transcription-associated function in vivo. Based on our findings we propose a role for the discovered new RNA synthetic activity of Polη during transcription
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