Complex formation with Rev1 enhances the proficiency of Saccharomyces cerevisiae DNA polymerase zeta for mismatch extension and for extension opposite from DNA lesions

Abstract

Rev1, a Y family DNA polymerase (Pol) functions together with Polzeta, a B family Pol comprised of the Rev3 catalytic subunit and Rev7 accessory subunit, in promoting translesion DNA synthesis (TLS). Extensive genetic studies with Saccharomyces cerevisiae have indicated a requirement of both Polzeta and Rev1 for damage-induced mutagenesis, implicating their involvement in mutagenic TLS. Polzeta is specifically adapted to promote the extension step of lesion bypass, as it proficiently extends primer termini opposite DNA lesions, and it is also a proficient extender of mismatched primer termini on undamaged DNAs. Since TLS through UV-induced lesions and various other DNA lesions does not depend upon the DNA-synthetic activity of Rev1, Rev1 must contribute to Polzeta-dependent TLS in a nonenzymatic way. Here, we provide evidence for the physical association of Rev1 with Polzeta and show that this binding is mediated through the C terminus of Rev1 and the polymerase domain of Rev3. Importantly, a rev1 mutant that lacks the C-terminal 72 residues which inactivate interaction with Rev3 exhibits the same high degree of UV sensitivity and defectiveness in UV-induced mutagenesis as that conferred by the rev1Delta mutation. We propose that Rev1 binding to Polzeta is indispensable for the targeting of Polzeta to the replication fork stalled at a DNA lesion. In addition to this structural role, Rev1 binding enhances the proficiency of Polzeta for the extension of mismatched primer termini on undamaged DNAs and for the extension of primer termini opposite DNA lesions.

FIG. 1.

Physical interaction of Rev1 with Polζ. Yeast Polζ (Rev3-Rev7) was mixed with GST-Rev1 (lanes 1 to 4), and Rev1 was mixed with GST-Polζ (lanes 5 to 8). Two micrograms of each protein was used in the study. After incubation, samples were bound to glutathione Sepharose beads, followed by multiple washings with buffer I containing 150 mM NaCl and elution of the bound proteins by SDS sample buffer. Aliquots of each sample before addition to the beads (L), the flowthrough fraction (F), last washing fraction (W), and the eluted proteins (E) were analyzed on a SDS-12% polyacrylamide gel developed with Coomassie blue. A control experiment was also done for GST with Polζ (lanes 9 to 12) and with Rev1 (lanes 13 to 16). Other experiments were performed using GST-Rev1 with Rev3 (lanes 17 to 20), GST-Rev3 with Rev1 (lanes 21 to 24), the GST-Rev1-Rev7 complex with Rev3 (lanes 25 to 28), and GST-Rev3 with the Rev1-Rev7 complex (lanes 29 to 32).

FIG. 2.

Mapping of regions involved in Rev1 interaction with Rev3. (A) Schematic representation of wild-type Rev1 and Rev3 proteins and their truncated forms. (Panel i) Yeast Rev1 protein is comprised of 985 amino acid residues, and it has the conserved motifs I through V characteristic of Y family polymerases. Motifs I and II contribute to the Fingers domain, motifs III and IV form the Palm, and motif V makes the Thumb and is followed by the polymerase-associated domain (PAD). Rev1 also contains an amino-terminal BRCT domain and a carboxyl-terminal domain (CTD). (Panel ii) Rev3, the catalytic subunit of Polζ, is comprised of 1,504 amino acids, and it has an N-terminal region (N-term), a Rev7 binding domain, an exonuclease-like domain (Exo), and the polymerase domain characteristic of B family Pols. Towards the C terminus, Rev3 has a putative C₄ zinc finger motif. For both Rev1 and Rev3, the various deletion mutations are shown and the results of their interactions summarized. Amino acids that remain in the various proteins are indicated in parentheses. (B) C terminus of Rev1 mediates interaction with Rev3. Yeast Polζ was mixed with GST-Rev1-1 (lanes 1 to 4), GST-Rev1-2 (lanes 9 to 12), or GST-Rev1-3 (lanes 17 to 20), and Rev1-1 (lanes 5 to 8), Rev1-2 (lanes 13 to 16), or Rev1-3 (lanes 21 to 24) was mixed with GST-Polζ. Two micrograms of each protein was used for the study. (C) The polymerase domain of Rev3 mediates interaction with Rev1. The GST-Rev3-1-Rev7 complex was mixed with Rev1 (lanes 1 to 4) or GST-Rev1 was mixed with Rev3-2 protein (lanes 5 to 8). One microgram of each protein was used for this study. (B and C) After incubation, samples were bound to glutathione Sepharose beads, followed by washings and elution of the bound proteins by SDS sample buffer. Aliquots of each sample before addition to the beads (L), the flowthrough fraction (F), last washing fraction (W), and the eluted proteins (E) were analyzed on a SDS-12% polyacrylamide gel stained with Coomassie blue.

FIG. 3.

Effects of Rev1 protein lacking the last 72 amino acids on UV sensitivity and UV-induced mutagenesis. (A) Survival after UV irradiation of wild-type strain EMY74.7 (•), its isogenic rev1Δ strain (○), and the rev1Δ strain carrying the rev1-1(1-913) mutation (▪). (B) UV-induced can1^r mutations in the wild-type strain EMY74.7 (•), the rev1Δ strain (○), and the rev1Δ strain carrying the rev1-1(1-913) mutation (▪). Each point on the curve represents the average of results of at least two experiments.