Cell biology and genetic studies have demonstrated that DNA double strand break (DSB) repair can be performed using an RNA transcript that spans the site of the DNA break as a template for repair. This type of DSB repair requires a reverse transcriptase to convert an RNA sequence into DNA to facilitate repair of the break, rather than copying from a DNA template as in canonical DSB repair. Translesion synthesis (TLS) DNA polymerases (Pol) are often more promiscuous than DNA Pols, raising the notion that reverse transcription could be performed by a TLS Pol. Indeed, several studies have demonstrated that human Pol eta has reverse transcriptase activity, while others have suggested that the yeast TLS Pol zeta is involved. Here, we purify all seven known nuclear DNA Pols of Saccharomyces cerevisiae and compare their reverse transcriptase activities. The comparison shows that Pol zeta far surpasses Pol eta and all other DNA Pols in reverse transcriptase activity. We find that Pol zeta reverse transcriptase activity is not affected by RPA or RFC/PCNA and acts distributively to make DNA complementary to an RNA template strand. Consistent with prior S. cerevisiae studies performed in vivo, we propose that Pol zeta is the major DNA Pol that functions in the RNA templated DSB repair pathway.

• PMID: 39454951
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Journal Article

Authors

Mayle R, Holloman WK, O'Donnell ME

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