DNA polymerase ζ has robust reverse transcriptase activity relative to other cellular DNA polymerases

Abstract

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 η has reverse transcriptase activity, while others have suggested that the yeast TLS Pol ζ 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 ζ far surpasses Pol η and all other DNA Pols in reverse transcriptase activity. We find that Pol ζ 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 ζ is the major DNA Pol that functions in the RNA-templated DSB repair pathway.

Keywords: DNA polymerase zeta; DNA synthesis; double-strand DNA break repair; reverse transcriptase; translesion DNA polymerase.

Figure 1

Model of RT activity for RNA mediated double strand DNA break repair.A, dsDNA break may occur after transcription has produced an mRNA transcript. In this event, any resection and consequent loss of DNA information can be “rescued” by the mRNA transcript as follows. Following (A) end resection, (B) the mRNA can bridge the DSB via annealing and Rad52-mediated strand exchange. After this, (C) the gap created by resection can be filled in by a reverse transcriptase. After (D) the RNA dissociates from the break site, (E) normal DNA templated gap filling and nick ligation restore an intact duplex.

Figure 2

Pol ζ is the only yeast Pol having significant Reverse Transcriptase activity.A, comparative DNA synthesis activity using 20 nM of either Pol ε, δ, ⍺, ζ, or η. Elevated concentrations were used for Rev1 (60 nM) and Pol IV (100 nM) in order to observe any extension activity. B, RT activity of each Pol, using the same fmol of Pol as in panel A.

Figure 3

Pol ζ is distributive during RT activity.A, titration of Pol ζ into extension assays with DNA primed RNA or DNA templates. RNA template reactions were stopped at 1 min and DNA template reactions at 10 s, and products were analyzed by denaturing PAGE. B and C, titration of competitor primed DNA into extension assays with DNA primed RNA (B) or DNA (C) templates. Aliquots of reactions were terminated at the indicated times followed by denaturing PAGE.

Figure 4

Pol ζ has similar affinity for both RNA and DNA template strands. Gel shift assay following incubation of Pol ζ with DNA primed DNA or RNA templates for 5 min at 30 °C.