Yeast DNA polymerase ζ maintains consistent activity and mutagenicity across a wide range of physiological dNTP concentrations

Abstract

In yeast, dNTP pools expand drastically during DNA damage response. We show that similar dNTP elevation occurs in strains, in which intrinsic replisome defects promote the participation of error-prone DNA polymerase ζ (Polζ) in replication of undamaged DNA. To understand the significance of dNTP pools increase for Polζ function, we studied the activity and fidelity of four-subunit Polζ (Polζ4) and Polζ4-Rev1 (Polζ5) complexes in vitro at ‘normal S-phase’ and ‘damage-response’ dNTP concentrations. The presence of Rev1 inhibited the activity of Polζ and greatly increased the rate of all three ‘X-dCTP’ mispairs, which Polζ4 alone made extremely inefficiently. Both Polζ4 and Polζ5 were most promiscuous at G nucleotides and frequently generated multiple closely spaced sequence changes. Surprisingly, the shift from ‘S-phase’ to ‘damage-response’ dNTP levels only minimally affected the activity, fidelity and error specificity of Polζ complexes. Moreover, Polζ-dependent mutagenesis triggered by replisome defects or UV irradiation in vivo was not decreased when dNTP synthesis was suppressed by hydroxyurea, indicating that Polζ function does not require high dNTP levels. The results support a model wherein dNTP elevation is needed to facilitate non-mutagenic tolerance pathways, while Polζ synthesis represents a unique mechanism of rescuing stalled replication when dNTP supply is low.

The CysB motif of Rev3p involved in the formation of the four‐subunit DNA polymerase ζ is required for defective‐replisome‐induced mutagenesis

“…Such analysis of mutation specificity should reveal whether the elimination of the Rev3p‐Pol31p interaction, thus lack of Pol ζ 4 , leads to the disappearance of some features attributable to Pol ζ. As shown by a recent in vitro study, base substitutions, most frequently G→T, predominate in Pol ζ 4 spectra and also a noticeable number of complex mutations is observed (Kochenova et al ., ). Additionally, a significant increase in the rates of X‐dCTP mispairs is observed in spectra of Pol ζ 5 (stoichiometric Rev3p, Rev7p, Pol31p, Pol32p and Rev1p complex), apparently as a result of Rev1p deoxycytidyl‐transferase activity (Kochenova et al ., ).…”

“…As shown by a recent in vitro study, base substitutions, most frequently G→T, predominate in Pol ζ 4 spectra and also a noticeable number of complex mutations is observed (Kochenova et al ., ). Additionally, a significant increase in the rates of X‐dCTP mispairs is observed in spectra of Pol ζ 5 (stoichiometric Rev3p, Rev7p, Pol31p, Pol32p and Rev1p complex), apparently as a result of Rev1p deoxycytidyl‐transferase activity (Kochenova et al ., ). These results are consistent with previous in vivo studies in which complex mutations and GC→CG transversions were considered as hallmarks of Pol ζ‐dependent mutagenesis (Harfe and Jinks‐Robertson, ; Kraszewska et al ., ; Grabowska et al ., ; Northam et al ., ).…”

Bypass of DNA Interstrand crosslinks by a Rev1-DNA polymerase ζ complex

“…We have previously shown that Pol z can be purified as a stable 5-subunit complex together with Rev1 (36). A complex with the identical activity can also be obtained by simply mixing Pol z together with Rev1, thereby reconstituting the 5-subunit complex.…”

“…The reaction was started by addition of the individual DNA polymerases. dNTPs were present at concentrations that prevail during the DNA damage response in yeast (36). Replication of the undamaged control DNA was carried out very efficiently, with the exception of replication by Rev1-Pol z (lane 5, discussed below).…”

“…All proteins are the Saccharomyces cerevisiae species and were purified as previously described. Pol z, Rev1-Pol z, Rev1 and Rev1 mutants, Pol h, Pol d and Pol d-DV (D520V) were purified from yeast overexpression systems (5,6,36,58). RPA, PCNA and RFC were purified from E. coli overexpression systems (59)(60)(61).…”