Complex formation of yeast Rev1 and Rev7 proteins: a novel role for the polymerase-associated domain

Abstract

The Rev1 protein of Saccharomyces cerevisiae functions in translesion synthesis (TLS) together with DNA polymerase (Pol) zeta, which is comprised of the Rev3 catalytic and the Rev7 accessory subunits. Rev1, a member of the Y family of Pols, differs from other members in its high degree of specificity for incorporating a C opposite template G as well as opposite an abasic site. Although Rev1 is indispensable for Polzeta-dependent TLS, its DNA synthetic activity is not required for many of the Polzeta-dependent lesion bypass events. This observation has suggested a structural role for Rev1 in this process. Here we show that in yeast, Rev1 forms a stable complex with Rev7, and the two proteins copurify. Importantly, the polymerase-associated domain (PAD) of Rev1 mediates its binding to Rev7. These observations reveal a novel role for the PAD region of Rev1 in protein-protein interactions, and they raise the possibility of a similar involvement of the PAD of other Y family Pols in protein-protein interactions. We discuss the possible roles of Rev1 versus the Rev1-Rev7 complex in TLS.

FIG. 1.

Purification of Rev1-Rev7 complex. (A) Rev1 copurifies with the Rev7 protein. N-terminal GST-Rev1 fusion protein was overexpressed in a wild-type yeast strain and was purified first on glutathione-Sepharose beads and then on an S-Sepharose column. (i) The GST-Rev1-containing protein sample was analyzed on a 10% denaturing polyacrylamide gel and stained with Coomassie blue. The positions of Rev7 present in the purified Rev1 sample (lane 1) and of purified Rev7 alone (lane 2) are indicated. The positions of molecular weight standards are indicated. (ii) Western blot analysis with anti-Rev7 antibodies to purified GST Rev1-Rev7 (lane 1) and to purified Rev7 (lane 2). (B) In vitro reconstitution of the Rev1-Rev7 complex. Purified Rev7 (9 μg) was added to the GST-Rev1-containing yeast extract (100 μg). After incubation, the reaction mixture was bound to glutathione-Sepharose beads; this was followed by washings and elution of the bound proteins with PreScission protease (2 units), which cleaves the GST tag. Fractions enriched for the Rev1-Rev7 complex were pooled, concentrated, and analyzed on an SDS-12% polyacrylamide gel and developed by Coomassie blue staining. Lane 2, Rev1 (200 ng); lane 3, Rev1-Rev7 complex (200 ng); lane 4, Rev7 (200 ng). *, unidentified but possibly heat shock proteins.

FIG. 2.

Mapping the Rev1 region needed for interaction with Rev7. (A) Schematic representation of wild-type Rev1 and various Rev1 mutant proteins. The yeast Rev1 protein is comprised of 985 amino acids, and it shares conserved motifs I to V with the other Y family DNA polymerases. Although not as conserved as these five motifs, all Y family polymerases have another domain, the PAD, which follows after motif V. Motifs I and II constitute the fingers, motifs III and IV form the palm, and motif V forms the thumb. The PAD is joined to the thumb via a flexible linker. In addition to motifs I to V and PAD, which are present in all Y family polymerases, Rev1 also contains an amino-terminal BRCT domain and a carboxyl-terminal domain (CTD). For the Rev1 proteins with different portions deleted, the amino acids that remain in the protein are shown, and the results of direct physical interaction with Rev7 are summarized. (B) The linker and PAD regions of Rev1 are necessary and sufficient for interaction with Rev7. Yeast Rev7 or Rev1 protein was mixed with GST Rev1 or GST Rev7 proteins, respectively. About 3 μg of each protein was used for this study. After incubation, samples were bound to glutathione-Sepharose beads; this was followed by washings and elution of the bound proteins by SDS-sample buffer. Aliquots of each sample, taken before addition to the beads (L), from the flowthrough fraction (F), from the last washing fraction (W), and from the eluted proteins (E), were analyzed on an SDS-12% polyacrylamide gel stained with Coomassie blue.

FIG. 3.

Nucleotide incorporation by Rev1 and Rev1-Rev7. (A) Specificity of deoxynucleotide incorporation by Rev1 and Rev1-Rev7 enzymes. The nucleotide sequence adjacent to the primer-template junction is shown for the DNA substrate. Rev1 (5 nM) or Rev1-Rev7 complex (5 nM) were incubated with the DNA substrate (20 nM) in the absence (−) or presence of a single nucleotide (G, A, T, or C) or all four deoxynucleotides (N) (100 μM each) for 10 min at 30°C. The reaction products were resolved on 10% polyacrylamide gels containing 8 M urea; this was followed by autoradiography. (B) Steady-state kinetic analysis of dCTP incorporation opposite an undamaged G by Rev1 and Rev1-Rev7. Rev1 (2 nM) and Rev1-Rev7 complex (2 nM) were incubated with the primer template DNA (20 nM) and with the indicated concentrations of dCTP for 10 min at 30°C. The rate of incorporation as a function of dCTP concentration was measured, and the data were fitted to the Michaelis-Menten equation. The V_max and K_m parameters were obtained from the fit, and the efficiency (V_max/K_m) of dCTP incorporation was calculated.