Complex Formation with Rev1 Enhances the Proficiency of Saccharomyces cerevisiae DNA Polymerase ζ for Mismatch Extension and for Extension Opposite from DNA Lesions

Narottam Acharya; Robert E Johnson; Satya Prakash; Louise Prakash

doi:10.1128/MCB.01671-06

. 2006 Oct 9;26(24):9555–9563. doi: 10.1128/MCB.01671-06

Complex Formation with Rev1 Enhances the Proficiency of Saccharomyces cerevisiae DNA Polymerase ζ for Mismatch Extension and for Extension Opposite from DNA Lesions^▿

Narottam Acharya ¹, Robert E Johnson ¹, Satya Prakash ¹, Louise Prakash ^1,^*

PMCID: PMC1698531 PMID: 17030609

Abstract

Rev1, a Y family DNA polymerase (Pol) functions together with Polζ, 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 Polζ and Rev1 for damage-induced mutagenesis, implicating their involvement in mutagenic TLS. Polζ 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 Polζ-dependent TLS in a nonenzymatic way. Here, we provide evidence for the physical association of Rev1 with Polζ 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 rev1Δ mutation. We propose that Rev1 binding to Polζ is indispensable for the targeting of Polζ to the replication fork stalled at a DNA lesion. In addition to this structural role, Rev1 binding enhances the proficiency of Polζ for the extension of mismatched primer termini on undamaged DNAs and for the extension of primer termini opposite DNA lesions.

DNA lesions in the template strand block the progression of the replication fork. In Saccharomyces cerevisiae, the Rad6-Rad18 ubiquitin-conjugating enzyme complex (2, 3) promotes replication through DNA lesions via at least three different pathways that include translesion synthesis (TLS) by DNA polymerases (Pols) η and ζ and a Rad5-Mms2-Ubc13-dependent postreplication repair pathway (36).

Polη is unique among eukaryotic TLS Pols in its proficiency for replication through UV-induced cyclobutane pyrimidine dimers (CPDs) in a relatively error-free manner (17, 20, 38, 40). Inactivation of Polη in both yeast and humans confers enhanced UV-induced mutagenesis (35, 37, 41, 42) and, in humans, causes a cancer-prone syndrome, the variant form of xeroderma pigmentosum (XPV) (15, 28). Although proficient replication through certain DNA lesions, such as CPDs, can be accomplished by one Pol, replication through many DNA lesions requires the concerted action of two Pols in which one Pol carries out the nucleotide insertion reaction opposite the lesion site and the other Pol performs the subsequent extension reaction (33, 34). Polζ, comprised of the Rev3 catalytic subunit and the Rev7 accessory subunit (32), is highly specialized for performing the extension step of TLS (33, 34).

Extensive genetic studies with yeast have indicated a requirement of Polζ in mutagenesis induced by DNA-damaging agents (18, 22, 25, 26). For example, mutations in REV3 or REV7 cause a large reduction in the incidence of mutagenesis induced by UV radiation or by other DNA-damaging agents. The requirement of Polζ for damage-induced mutagenesis has indicated a role for this Pol in contributing to the mutagenic mode of TLS at many of the lesions (33). However, in spite of the fact that Polζ function leads to an enhanced rate of UV-induced mutagenesis and Polη promotes error-free TLS through the UV-induced CPDs, Polζ exhibits a higher fidelity for deoxynucleoside triphosphate (dNTP) incorporation on undamaged DNAs than Polη. Thus, Polζ's rate of misincorporation is ∼10⁻⁴ (10, 12, 19), compared to Polη, which misincorporates dNTPs with a frequency of ∼10⁻² (39). Polζ, however, differs strikingly from other DNA Pols in its proficiency for extending from mispaired primer termini, which it accomplishes with an efficiency of approximately 10⁻¹ to 10⁻² relative to the efficiency of extension from the correct base pairs (10, 12, 19). Also, Polζ is highly inefficient at incorporating dNTPs opposite various DNA lesions; for example, its dNTP incorporation opposite the 3′ T of a cis-syn T-T dimer or a (6-4) T-T photoproduct (19) or opposite an abasic site (12) is strongly inhibited. Polζ, however, is very efficient at extending from the nucleotide inserted opposite these lesion sites by another DNA Pol (12, 19, 21, 29).

Like Polζ, Rev1 is also required for mutagenesis induced by DNA damaging agents (18, 23, 26); but unlike Polζ, which incorporates dNTPs opposite the four template bases with very similar efficiencies and fidelities, Rev1 is a highly specialized Pol that predominantly incorporates a C opposite template G (11, 31). In Rev1, the templating G and the incoming dCTP do not pair with each other; instead, template G is evicted from the DNA helix and it hydrogen bonds with a segment of Rev1, whereas dCTP pairs with an arginine residue in Rev1 rather than with template G (30). Hence, the specificity for both the templating G and the incoming dCTP in Rev1 is provided by the Rev1 protein itself.

Although Rev1 is needed for UV-induced mutagenesis, its DNA polymerase activity makes no contribution to it, as a C is rarely incorporated opposite UV-induced lesions (6, 7); furthermore, the inactivation of Rev1 synthetic activity has no effect upon UV-induced mutagenesis. For many other DNA lesions also, although the Rev1 protein is necessary for Polζ-dependent TLS, its DNA polymerase activity is dispensable (4, 12). Thus, Rev1 plays an indispensable structural role in Polζ-dependent TLS.

Here we determined whether evidence can be adduced for a direct physical interaction of Rev1 with Polζ, and we examined whether Rev1 binding affects Polζ's fidelity or mismatch extension ability on undamaged DNAs and whether Polζ's inability to incorporate dNTP opposite DNA lesions and its proficiency for the extension of primer termini opposite DNA lesions are altered upon this association. We found that Rev1 physically associates with Polζ through its Rev3 catalytic subunit, and we provide evidence that Rev1 binding stimulates the proficiency of Polζ for mismatch extension on undamaged DNAs and enhances also its proficiency for extension opposite DNA lesions.

MATERIALS AND METHODS

Yeast strains and plasmids.

Saccharomyces cerevisiae strain BJ5464 and its isogenic rev7Δ derivative were used for protein purification. For in vitro binding assays, REV3, REV1, and various truncated forms of these genes were inserted into the vector pBJ842 (16) to produce an amino-terminal glutathione S-transferase (GST) fusion protein.

Purification of proteins.

For in vitro binding studies, wild-type GST-Rev1 and GST-Rev3 and their truncated forms were purified from the wild-type yeast strain BJ5464 or from its rev7Δ derivative on glutathione Sepharose beads by using a protocol described previously (12). To purify Polζ, GST-Rev3 protein was coexpressed with Rev7 in yeast strain BJ5464 by using plasmids pREV3.30 and pREV7.35. To obtain untagged proteins, GST fusion proteins bound to glutathione Sepharose beads were treated overnight at 4°C with PreScission protease, which cleaved between the GST tag and Rev1 or Rev3.

In vitro interactions of Rev1 with Polζ.

The physical interaction of Rev1 with Polζ was examined by using a protocol similar to that described previously (1). Briefly, GST-Rev1 or its truncated forms (2 μg) were incubated with Polζ (2 μg) in buffer I (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM dithiothreitol, 0.01% NP-40, 10% glycerol) in a 20-μl reaction mixture at 4°C for 30 min, followed by 10 min at 25°C. To such a mixture, 20 μl glutathione Sepharose beads were added and further incubated for 2 h with constant rocking at 4°C. The beads were spun down, and the unbound protein was collected. Next, the beads were washed thoroughly three times with 10 volumes of buffer I, and the bound proteins were then eluted with 20 μl of sodium dodecyl sulfate (SDS) loading buffer. The various fractions were resolved on a 12% denaturing polyacrylamide gel followed by Coomassie blue R-250 staining. A similar approach was taken to examine the interaction of GST-Polζ, GST-Rev3, or a truncated form of one of these proteins with Rev1.

DNA substrates and DNA polymerase assays.

Oligonucleotides were synthesized by Midland Certified Reagent Co. (Midland, TX). A standard primer extension reaction mixture (10 μl) contained 40 mM Tris-HCl (pH 7.5), 8 mM MgCl₂, 1 mM dithiothreitol, 100 μg/ml bovine serum albumin, 10% glycerol, 10 nM of 5′ ³²P-labeled oligonucleotide primer annealed to an oligonucleotide template, and increasing concentrations of a single dNTP and Polζ or Rev1*-Polζ complex, where Rev1 is catalytically inactive because of the change of aspartate 467 and glutamate 468 to alanines. For both Polζ and the Rev1*-Polζ complex, the Polζ concentration used was 0.5 nM. The Rev1*-Polζ complex was made by incubating equimolar concentrations of Rev1* and Polζ overnight at 4°C. Both Polζ and the Rev1*-Polζ complex were treated in an identical manner, and under these conditions, the catalytic activity of Polζ alone was not affected. Assays were assembled on ice, incubated at 30°C for 5 to 10 min, and stopped by the addition of 40 μl loading buffer containing 20 mM EDTA, 95% formamide, 0.3% bromphenol blue, and 0.3% cyanol blue. The reaction products were resolved on a 12% polyacrylamide gel containing 8 M urea. Gel band intensities of the substrates and products were quantitated by PhosphorImager, and the observed rate of deoxynucleotide incorporation was plotted as a function of dNTP concentration. The data were fit by nonlinear regression using SigmaPlot 5.0 to the Michaelis-Menten equation describing a hyperbola, v = (k_cat[E] × [dNTP]/(K_m + [dNTP]), where [E] refers to enzyme concentration. Apparent K_m and k_cat steady-state parameters were obtained from the fit and were used to calculate the efficiency of deoxynucleotide incorporation (k_cat/K_m). DNA substrates described below (see Table 1) were generated by annealing a 75-nucleotide template, 5′-AGC AAG TCA CCA ATG TCT AAG AGT TCG TAT XAT GCC TAC ACT GGA GTA CCG GAG CAT CGT CGT GAC TGG GAA AAC-3′, in which there was a T or a DNA lesion at the position indicated by X, to the following 5′ ³²P-labeled oligonucleotide primers: 5′-GTT TTC CCA GTC ACG ACG ATG CTC CGG TAC TCC AGT GTA G-3′ (40-mer), 5′-GTT TTC CCA GTC ACG ACG ATG CTC CGG TAC TCC AGT GTA GG-3′ (41-mer), 5′-GTT TTC CCA GTC ACG ACG ATG CTC CGG TAC TCC AGT GTA GGC-3′ (42-mer), and 5′-GTT TTC CCA GTC ACG ACG ATG CTC CGG TAC TCC AGT GTA GGC A-3′ (43-mer). DNA substrates described below (see Table 2) were generated by annealing the 53-nucleotide oligonucleotide template 5′-AT GCC TGC ACG AAG AGT TCC TAG TGC CTA CAC TGG AGT ACC GGA GCA TCG TCG-3′ to the 31-mer primer 5′-CGA CGA TGC TCC GGT ACT CCA GTG TAG GCA X-3′, in which there was a G, an A, a T, or a C at position X at the 3′ end. For DNA substrates used for experiments whose results are described below (see Tables 3, 4 and 5), we used the same 75-mer template that was described in Table 1, in which the consecutive 3′-XT-5′ has either a tetrahydrofuran moiety for the abasic site at position X, or a cis-syn T-T dimer or a (6-4) T-T photoproduct at this position. The 75-mer template bearing the respective DNA lesion was annealed to the 45-mer primer 5′-GTT TTC CCA GTC ACG ACG ATG CTC CGG TAC TCC AGT GTA GGC ATX-3′, in which there was an A, a T, a G, or a C at position X at the 3′ end. For the examination of nucleotide insertion at the 3′ T site of a T-T dimer or a (6-4) T-T photoproduct or opposite from the abasic site, we annealed the lesion-bearing 75-mer template to the 44-mer primer 5′-GTT TTC CCA GTC ACG ACG ATG CTC CGG TAC TCC AGT GTA GGC AT-3′.

TABLE 1.

Steady-state kinetic parameters for correct nucleotide incorporation opposite template nucleotides by Polζ and the Rev1*-Polζ complex

Template nucleotide	dNTP added	Enzyme^a	k_cat (min⁻¹)	K_m (μM)	k_cat/K_m	Relative efficiency
A	dTTP	Polζ	0.84 ± 0.03	0.03 ± 0.008	28	1
		Rev1*-Polζ	0.93 ± 0.03	0.02 ± 0.007	46.5	1.7
T	dATP	Polζ	0.93 ± 0.07	0.048 ± 0.01	19.37	1
		Rev1*-Polζ	0.95 ± 0.24	0.068 ± 0.02	14	0.72
G	dCTP	Polζ	0.45 ± 0.03	0.04 ± 0.01	11.25	1
		Rev1*-Polζ	0.4 ± 0.018	0.034 ± 0.003	11.76	1.04
C	dGTP	Polζ	1 ± 0.03	0.018 ± 0.002	55.55	1
		Rev1*-Polζ	0.85 ± 0.05	0.03 ± 0.001	28.33	0.5

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An asterisk indicates catalytically inactive Rev1 protein resulting from a change of D467 and E468 to alanines.

TABLE 2.

Steady-state kinetic parameters of extension reactions on undamaged DNAs catalyzed by Polζ and the Rev1*-Polζ complex

Site^a	dNTP added	Enzyme^b	k_cat (min⁻¹)	K_m (μM)	k_cat/K_m	Relative efficiency
5′-GCAG	dTTP	Polζ	0.73 ± 0.006	3.2 ± 0.04	0.23	1
---CGTGA--		Rev1*-Polζ	0.57 ± 0.007	0.18 ± 0.01	3.2	13.77
5′-GCAA	dTTP	Polζ	0.85 ± 0.009	0.77 ± 0.15	1.1	1
---CGTGA--		Rev1*-Polζ	0.43 ± 0.005	0.07 ± 0.008	6.14	4.4
5′-GCAT	dTTP	Polζ	0.65 ± 0.002	1.42 ± 0.1	0.46	1
---CGTGA--		Rev1*-Polζ	0.43 ± 0.04	0.08 ± 0.002	5.37	11.7
5′-GCAC	dTTP	Polζ	0.87 ± 0.009	0.03 ± 0.015	28.33	1
---CGTGA--		Rev1*-Polζ	0.43 ± 0.05	0.04 ± 0.008	10.75	0.4

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Hyphens indicate continuation of the sequence in either direction.

An asterisk indicates catalytically inactive Rev1 protein resulting from a change of D467 and E468 to alanines.

TABLE 3.

Steady-state kinetic parameters of extension reactions opposite the abasic site catalyzed by Polζ and the Rev1*-Polζ complex

Site (abasic)^a	dNTP added	Enzyme^b	k_cat (min⁻¹)	K_m (μM)	k_cat/K_m	Relative efficiency
5′-CATA	dATP	Polζ	0.14 ± 0.012	0.145 ± 0.06	0.97	1
---GTA0T--		Rev1*-Polζ	0.1 ± 0.01	0.105 ± 0.09	0.95	0.98
5′-CATT	dATP	Polζ	0.23 ± 0.01	0.41 ± 0.09	0.56	1
---GTA0T--		Rev1*-Polζ	0.16 ± 0.01	0.07 ± 0.03	2.28	4.08
5′-CATG	dATP	Polζ	0.18 ± 0.013	0.095 ± 0.03	1.9	1
---GTA0T--		Rev1*-Polζ	0.17 ± 0.016	0.026 ± 0.01	6.54	3.44
5′-CATC	dATP	Polζ	0.25 ± 0.01	3.16 ± 1	0.08	1
---GTA0T--		Rev1*-Polζ	0.14 ± 0.007	0.25 ± 0.08	0.56	7

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A zero in the sequences indicates an abasic site in the template. Hyphens indicate continuation of the sequence in either direction.

An asterisk indicates catalytically inactive Rev1 protein resulting from a change of D467 and E468 to alanines.

TABLE 4.

Steady-state kinetic parameters of extension reactions catalyzed opposite from the 3′ T of cis-syn T-T dimer by Polζ and Rev1*-Polζ complex

Site (cis-syn T-T dimer)^a	dNTP added	Enzyme^b	k_cat (min⁻¹)	K_m (μM)	k_cat/K_m	Relative efficiency
5′-CATA	dATP	Polζ	0.3 ± 0.01	0.06 ± 0.01	5	1
---GTAT<>T--		Rev1*-Polζ	0.22 ± 0.01	0.05 ± 0.02	4.4	0.88
5′-CATT	dATP	Polζ	0.3 ± 0.012	0.4 ± 0.08	0.75	1
---GTAT<>T--		Rev1*-Polζ	0.22 ± 0.017	0.07 ± 0.02	3.14	4.2
5′-CATG	dATP	Polζ	0.36 ± 0.023	0.046 ± 0.01	7.83	1
---GTAT<>T--		Rev1*-Polζ	0.26 ± 0.014	0.01 ± 0.004	26	3.3
5′-CATC	dATP	Polζ	0.19 ± 0.01	1.53 ± 0.1	0.12	1
---GTAT<>T--		Rev1*-Polζ	0.13 ± 0.006	0.46 ± 0.08	0.55	4.6

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T<>T indicates a cis-syn T-T dimer in the template. Hyphens indicate continuation of the sequence in either direction.

An asterisk indicates catalytically inactive Rev1 protein resulting from a change of D467 and E468 to As.

TABLE 5.

Steady-state kinetic parameters of extension reactions opposite the 3′ T of a (6-4) T-T photoproduct catalyzed by Polζ and the Rev1*-Polζ complex

Site [(6-4) T-T dimer]^a	dNTP added	Enzyme^b	k_cat (min⁻¹)	K_m (μM)	k_cat/K_m	Relative efficiency
5′-CATA	dATP	Polζ	0.2 ± 0.01	0.05 ± 0.1	0.4	1
---GTAT=T--		Rev1*-Polζ	0.14 ± 0.01	0.06 ± 0.2	0.23	0.6
5′-CATT	dATP	Polζ	0.1 ± 0.01	0.94 ± 0.06	0.1	1
---GTAT=T--		Rev1*-Polζ	0.06 ± 0.007	0.13 ± 0.09	0.46	4.6
5′-CATG	dATP	Polζ	0.17 ± 0.008	0.07 ± 0.01	2.42	1
---GTAT=T--		Rev1*-Polζ	0.16 ± 0.005	0.04 ± 0.007	4	1.7
5′-CATC	dATP	Polζ	0.16 ± 0.008	0.6 ± 0.1	0.26	1
---GTAT=T--		Rev1*-Polζ	0.14 ± 0.007	0.16 ± 0.08	0.88	3.36

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T=T indicates a (6-4) T-T photoproduct in the template. Hyphens indicate continuation of the sequence in either direction.

An asterisk indicates catalytically inactive Rev1 protein resulting from a change of D467 and E468 to alanines.

Sensitivity and mutagenesis in response to UV irradiation.

Wild-type Saccharomyces cerevisiae strain EMY74.7 and its isogenic rev1Δ derivative were grown in synthetic complete medium (SC), and the rev1Δ strain carrying the rev1-1(1-913) mutant gene (encoding amino acids [aa] 1 to 913) on a CEN ARS URA3 plasmid was grown in synthetic complete medium lacking uracil to maintain selection for the plasmid. When cultures had reached mid-logarithmic phase, they were washed by centrifugation, subjected to sonication to disperse cell clumps, pelleted by centrifugation, and resuspended at a density of 2 × 10⁸ cells per ml. Appropriate dilutions of cells were spread onto the surface of plates containing SC or SC lacking uracil for viability determinations and onto SC lacking arginine but containing canavanine for mutagenesis assays. UV irradiation was done at a dose rate of 1 J/m²/s. Following UV irradiation, plates were incubated in the dark, and colonies were counted after 3 to 5 days.

RESULTS

Physical interaction of Rev1 with Polζ occurs through the Rev3 catalytic subunit.

Polζ, a heterodimer consisting of the Rev3 catalytic subunit and Rev7 accessory subunit, functionally interacts with the Rev1 protein, as mutations in the genes encoding them display epistasis for sensitivity to DNA damaging agents and a defect in mutagenesis induced by them. In both yeast and humans, the induction of mutations by UV is dependent upon Polζ and Rev1 (8, 24, 27), and the high incidence of UV-induced mutagenesis in individuals with XPV, the underlying cause of sunlight-induced skin cancers, presumably results from the sole dependence of XPV cells upon Polζ/Rev1 for TLS.

To check for the physical interaction of Rev1 with Polζ, we made use of a GST pull-down assay. In such an assay, the GST fusion protein binds tightly to the glutathione Sepharose affinity beads and the interacting protein is pulled down only if it forms a stable complex with the GST fusion protein. Purified Polζ or Rev1 protein was incubated with GST-Rev1 or GST-Polζ, respectively, and then bound to glutathione-Sepharose affinity beads. After extensive washings with 150 mM NaCl-containing buffer, the proteins were eluted by SDS-containing buffer. As shown in Fig. 1, Polζ bound Rev1 regardless of whether GST-Rev1 was incubated with Polζ or whether GST-Polζ was incubated with Rev1 (lanes 1 to 8). No nonspecific binding of Polζ or Rev1 to GST was seen in control experiments (lanes 9 to 16). Next, we examined if the interaction of Rev1 with Polζ was mediated by Rev3. For this purpose, we purified the GST-Rev1, Rev1, GST-Rev3, and Rev3 proteins from a rev7Δ yeast strain to ensure that the purified Rev1 or Rev3 protein was free of any contaminating Rev7, as they can both form heterodimers with Rev7. Since both the GST-Rev1 and GST-Rev3 proteins could bind to Rev3 and Rev1, respectively, a direct interaction of Rev1 with Rev3 is indicated (lanes 17 to 24).

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).

The Rev1-Rev7 complex does not interact with Rev3.

Since Rev1 forms a stable complex with Rev7 (1), we next examined whether the Rev1-Rev7 complex could also bind Rev3. For this purpose, the GST-Rev1-Rev7 complex was incubated with Rev3 or the GST-Rev3 protein was incubated with the Rev1-Rev7 complex; however, we found no evidence for the binding of Rev3 with Rev1-Rev7 (Fig. 1, lanes 25 to 32).

Mapping of the regions in Rev1 and Rev3 involved in interaction.

To map the region of Rev1 involved in binding to Polζ, the wild-type Rev1 and the Rev1 proteins with different portions deleted (Fig. 2A) were purified with the amino-terminal GST fusion, or without it, from a rev7Δ yeast strain. The GST-Rev1 or Rev1 protein was incubated with Polζ or with GST-Polζ, respectively, and pull-down assays performed on glutathione Sepharose affinity beads (Fig. 2B). While the deletion of the carboxyl-terminal 72 amino acids in Rev1-1 (aa 1 to 913) abolished interaction with Polζ (Fig. 2B, lanes 1 to 8), removal of the N-terminal BRCT domain in Rev1-2 (aa 297 to 985) did not affect interaction with Polζ (Fig. 2B, lanes 9 to 16). We also observed the binding of the Rev1-3 peptide, which contains only the carboxyl-terminal 200 residues of Rev1, with Polζ (Fig. 2B, lanes 17 to 24). From these observations, we conclude that at minimum, the carboxyl-terminal 72 residues of Rev1 are necessary for Polζ binding and that the carboxyl-terminal 200 residues of Rev1 are sufficient for interaction with Polζ.

To map the region in Rev3 involved in binding to Rev1, we purified two truncated Rev3 proteins, Rev3-1 and Rev3-2. In Rev3-1, the N-terminal region and also the C-terminal region containing the zinc finger domain have been deleted, and in Rev3-2, the region involved in Rev7 binding has been additionally deleted (Fig. 2A). Thus, whereas Rev3-1 retains the Rev7 binding region as well as the polymerase domain, the Rev3-2 protein has only the polymerase domain left in it. As expected from the presence of the Rev7 binding site in it, the Rev3-1 protein copurified with Rev7, whereas the Rev3-2 protein had no Rev7 associated with it. Since both the Rev3-1 and Rev3-2 proteins could interact with Rev1 (Fig. 2C), Rev1 binding to Rev3 occurs through its catalytic domain.

Rev1 binding enhances the mismatch extension efficiency of Polζ.

The binding of Rev1 to Rev3 in its polymerase domain raised the possibility that this interaction might affect the catalytic properties of Polζ. To examine this, we first determined whether interaction with Rev1 modifies the catalytic efficiency of Polζ for correct nucleotide incorporation opposite the template nucleotides G, A, T, and C. Since Rev1 can also incorporate the various dNTPs with various efficiencies opposite these template nucleotides, we opted to form a complex of Polζ with a catalytically inactive Rev1* protein in which the catalytic Asp467 and Glu468 residues involved in Mg²⁺ binding and present in the conserved motif III (SIDE motif) have both been altered to alanine to rule out any Rev1 contribution to synthesis by the Rev1*-Polζ complex. The Rev1*-Polζ complex was formed by incubating equimolar concentrations of Polζ and catalytically inactive Rev1 overnight at 4°C, and we expect Rev1* and Polζ to form a 1:1 complex under these conditions. The efficiencies (k_cat/K_m) of dNTP incorporation by Polζ and by Rev1*-Polζ were determined as a function of deoxynucleotide concentration under steady-state conditions. Care was taken so that the amount of Polζ when used alone and the amount used in the complex were the same. From the kinetics of deoxynucleotide incorporation, the steady-state apparent K_m and k_cat values were obtained from the curve fitted to the Michaelis-Menten equation. As shown in Table 1, the Rev1*-Polζ complex incorporated the correct dNTPs opposite templates A, T, G, and C with about the same efficiency as did Polζ alone. Thus, interaction with Rev1 does not significantly alter the efficiency of correct dNTP incorporation opposite undamaged nucleotides by Polζ.

As Polζ is an efficient extender of mispaired primer termini, we next compared the efficiency of mismatch extension by Polζ to the efficiency of mismatch extension by Rev1*-Polζ. The efficiencies of incorporation of the correct nucleotide dTTP opposite template nucleotide A situated subsequent to various mismatched pairs were determined, and as shown in Table 2, the Rev1*-Polζ complex extended from a G · G or a G · T mismatch approximately 12- to 14-fold more efficiently than did Polζ, and an ∼4-fold increase in extension efficiency was seen for the G · A mismatched pair. No such enhancement in extension efficiency occurred for the correct G · C base pair; rather, the extension efficiency declined by more than twofold for the Rev1*-Polζ complex. Overall, and interestingly, the Rev1*-Polζ complex extends from the mismatched primer termini only two- to threefold less efficiently than it extends from the correct base pair. The mismatch extension proficiency of Polζ, thus, is greatly enhanced upon Rev1 binding.

Enhanced proficiency of extension opposite from abasic sites by the Rev1*-Polζ complex.

Polζ inserts nucleotides opposite abasic sites poorly, with a frequency of 10⁻⁵, but it extends from the primer end situated opposite the abasic site quite efficiently, with a frequency of approximately 10⁻¹ to 10⁻² (12). As Rev1 binding facilitates mismatch extension by Polζ on undamaged DNAs (Table 2), we next determined if Rev1 also stimulates the efficiency of extension opposite abasic sites by Polζ. Although the efficiency of nucleotide incorporation opposite an abasic site for the Rev1*-Polζ complex remained the same as that for Polζ alone (data not shown), the extension from a G, T, or C nucleotide opposite an abasic site was enhanced approximately three- to sevenfold for the Rev1*-Polζ complex compared to Polζ alone. However, the extension from an A opposite the abasic site for Rev1*-Polζ remained the same as that for Polζ (Table 3). That may be because an A opposite the abasic site is the least distorting and retains all aspects of B form DNA.

Enhanced efficiency of extension opposite the 3′ T of a cis-syn T-T dimer or a (6-4) T-T photoproduct by the Rev1*-Polζ complex.

Polζ plays a pivotal role in promoting replication through UV-induced DNA lesions, and it is indispensable for UV-induced mutagenesis along with Rev1. Polζ is highly inefficient at inserting nucleotides opposite the 3′ T of either the cis-syn T-T dimer or the (6-4) T-T photoproduct; however, it can extend from nucleotides inserted opposite the 3′ T of either lesion (19). Next, we determined whether Rev1 interaction with Polζ alters the efficiency of extension from the nucleotide inserted opposite the 3′ T of the lesion by another DNA Pol. Again, although Rev1 had no stimulatory effect on the efficiency of nucleotide incorporation by Polζ opposite the 3′ T of either lesion (data not shown), Rev1 enhanced the efficiency of extension from a G, a T, or a C opposite the 3′ T of either lesion by approximately two- to fivefold (Tables 4 and 5). In particular, even though Polζ extended from a G opposite the 3′ T of a cis-syn T-T dimer quite efficiently (k_cat/K_m, ∼8), still a >3-fold enhancement of this efficiency occurred with Rev1*-Polζ (Table 4); consequently, the extension from a G opposite the 3′ T of a T-T dimer became as efficient as the extension from correctly base-paired termini (Tables 1 and 2). We also note that the efficiency of Polζ for extension from an A opposite the 3′ T of a T-T dimer or a (6-4) T-T photoproduct is not enhanced upon Rev1 binding; and also, Polζ, either with or without Rev1, is more adept at performing extension opposite from the T-T dimer than from the (6-4) T-T photoproduct (Tables 4 and 5).

The Rev1 C-terminal region involved in interaction with Polζ is indispensable for Polζ's role in TLS.

Since the deletion of the C-terminal 72 amino acid residues of Rev1 abrogates its physical association with Polζ, we next examined whether the inability to complex with Rev1 adversely affects Polζ's ability to promote mutagenic TLS through UV-induced DNA lesions. Similar to rev3Δ and rev7Δ strains, the rev1Δ strain exhibits an increased sensitivity to UV, and the incidence of UV-induced mutations in all these strains is lowered to about the same extent. As is shown in Fig. 3, the C-terminal deletion mutant rev1-1(1-913) displays the same UV sensitivity as the rev1Δ strain, and the generation of UV-induced can1^r mutations in the two mutants is affected to the same degree. From these observations, we infer that Rev1 binding is indispensable for Polζ function in TLS.

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 *rev1*Δ strain (○), and the *rev1*Δ strain carrying the *rev1-1*(*1-913*) mutation (▪). (B) UV-induced *can1*^r mutations in the wild-type strain EMY74.7 (•), the *rev1*Δ strain (○), and the *rev1*Δ strain carrying the *rev1-1*(*1-913*) mutation (▪). Each point on the curve represents the average of results of at least two experiments.

DISCUSSION

Even though the DNA synthetic activity of Rev1 is not needed for TLS through many lesions, such as cyclobutane pyrimidine dimers and (6-4) photoproducts, Rev1 plays an indispensable role in Polζ-dependent TLS and mutagenesis. Here we provide evidence for a direct physical interaction of Rev1 with Polζ and show that this interaction occurs through the binding of Rev1 with Rev3 in Polζ.

The Rev1-Polζ association involves the C terminus of Rev1 and the region of Rev3 that has the conserved polymerase domains characteristic of B family Pols. In particular, we show that deletion of the C-terminal 72 residues of Rev1 inactivates this interaction. Since all the conserved motifs required for Rev1 DNA polymerase activity are contained within the first 746 residues and Rev1 protein with the C-terminal region beyond it deleted is catalytically as active as the full-length protein (30), we assume that the primary role of the Rev1 C terminus is to modulate the physical interaction of Rev1 with Polζ. Our observation that deletion of the Rev1 C-terminal 72 residues elicits the same degree of UV sensitivity and the same level of reduced UV mutability as that conferred by the rev1Δ mutation implies that Rev1 binding is a prerequisite for Polζ function in TLS and mutagenesis.

How might Rev1 binding contribute to Polζ function in TLS? In this regard, it is of interest that even though Rev1-Polζ and Polη both function in TLS in yeast cells in a manner dependent upon Rad6-Rad18 and upon PCNA ubiquitylation, they differ in important ways. First, Polη binds directly to PCNA that has been loaded on the DNA by replication factor C (9). This interaction is mediated through the conserved PCNA binding motif present in the C terminus of Polη, and PCNA binding stimulates the DNA synthetic activity of Polη. Furthermore, PCNA binding is essential for Polη function in TLS since mutations in the PCNA binding motif render Polη nonfunctional in vivo (9). In striking contrast, we found no evidence for PCNA binding by Rev1, or Polζ, or the Rev1-Polζ complex (13; N. Acharya, S. Prakash, and L. Prakash, unpublished observations). These observations lead us to suggest that the Rev1-Polζ complex gains access to PCNA at the replication fork in a more indirect manner rather than the direct PCNA binding mode adopted by Polη. Second, in addition to its requirement for Rev1, the function of Polζ in TLS depends upon Pol32, a nonessential subunit of Polδ (5, 12, 14). Also, even though Pol32 is indispensable for Polζ-mediated TLS and mutagenesis, it is not required for Polη function in TLS. In view of these considerations, we raise the possibility that Polζ's targeting to the replication fork stalled at a lesion site is mediated via the interaction of Rev1-Polζ with Pol32.

In undamaged yeast cells, Rev1 exists in a stable complex with Rev7, as the two proteins copurify under stringent conditions and a Rev1-Rev7 complex can be formed in vitro from the purified proteins (1). However, Rev7 binding has no effect on Rev1 synthetic activity (1). Here we have examined whether the Rev1-Rev7 complex can associate with Rev3 and found that, unlike Rev1, the Rev1-Rev7 complex does not bind Rev3. Since the Rev7-bound form of Rev1 is unable to participate in complex formation with Polζ, the Rev1-Rev7 complex may play no role in Polζ-mediated TLS.

In addition to the possible role of Rev1 binding in promoting the targeting of Polζ to the replication fork, interestingly, we find that Rev1 binding enhances the mismatch extension proficiency of Polζ on undamaged DNAs but has no effect on the efficiency of dNTP incorporation on DNAs with matched primer termini. Likewise, on damaged DNAs, Rev1 stimulates the ability of Polζ for extension opposite from DNA lesions but has no effect on dNTP incorporation opposite them. Thus, Rev1 further stimulates Polζ's proficiency for mismatch extension and for extension opposite DNA lesions, in spite of the fact that even on its own, Polζ is quite proficient at performing these roles.

How might Rev1 binding enhance Polζ's ability to extend from mismatched and damaged primer termini? Since Rev1 binds Rev3 in its polymerase domain, this binding could modulate the active site of Rev3 in a way that affects any of the steps of dNTP incorporation; for example, it could effect a more efficient binding of Polζ to the mismatched or damaged primer terminus or it could enable a more optimal alignment of the 3′ OH of the nucleotide at the primer terminus for nucleophilic attack by the incoming dNTP.

In summary, we show here that Rev1 physically associates with Polζ and this interaction is mediated via the C terminus of Rev1 and the polymerase domain of Rev3, and importantly, these studies reveal that Rev1 binding is indispensable for Polζ function in TLS. Furthermore, we provide evidence that Rev1 binding enhances the proficiency of Polζ for mismatch extension and for extension from the DNA lesion-bearing primer termini.

Acknowledgments

This work was supported from National Institutes of Health grant CA107650.

Footnotes

^▿

Published ahead of print on 9 October 2006.

REFERENCES

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[r1] 1.Acharya, N., L. Haracksa, R. E. Johnson, I. Unk, S. Prakash, and L. Prakash. 2005. Complex formation of yeast Rev1 and Rev7 proteins: a novel role for the polymerase-associated domain. Mol. Cell. Biol. 25:9734-9740. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r2] 2.Bailly, V., J. Lamb, P. Sung, S. Prakash, and L. Prakash. 1994. Specific complex formation between yeast RAD6 and RAD18 proteins: a potential mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA damage sites. Genes Dev. 8:811-820. [DOI] [PubMed] [Google Scholar]

[r3] 3.Bailly, V., S. Lauder, S. Prakash, and L. Prakash. 1997. Yeast DNA repair proteins Rad6 and Rad18 form a heterodimer that has ubiquitin conjugating, DNA binding, and ATP hydrolytic activities. J. Biol. Chem. 272:23360-23365. [DOI] [PubMed] [Google Scholar]

[r4] 4.Baynton, K., A. Bresson-Roy, and R. P. P. Fuchs. 1999. Distinct roles for Rev1p and Rev7p during translesion synthesis in Saccharomyces cerevisiae. Mol. Microbiol. 34:124-133. [DOI] [PubMed] [Google Scholar]

[r5] 5.Gerik, K. J., X. Li, A. Pautz, and P. M. J. Burgers. 1998. Characterization of the two small subunits of Saccharomyces cerevisiae DNA polymerase δ. J. Biol. Chem. 273:19747-19755. [DOI] [PubMed] [Google Scholar]

[r6] 6.Gibbs, P. E. M., A. Borden, and C. W. Lawrence. 1995. The T-T pyrimidine (6-4) pyrimidinone UV photoproduct is much less mutagenic in yeast than in Escherichia coli. Nucleic Acids Res. 23:1919-1922. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r7] 7.Gibbs, P. E. M., B. J. Kilbey, S. K. Banerjee, and C. W. Lawrence. 1993. The frequency and accuracy of replication past a thymine-thymine cyclobutane dimer are very different in Saccharomyces cerevisiae and Escherichia coli. J. Bacteriol. 175:2607-2612. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r8] 8.Gibbs, P. E. M., X.-D. Wang, Z. Li, T. P. McManus, W. G. McGregor, C. W. Lawrence, and V. M. Maher. 2000. The function of the human homolog of Saccharomyces cerevisiae REV1 is required for mutagenesis induced by UV light. Proc. Natl. Acad. Sci. USA 97:4186-4191. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r9] 9.Haracska, L., C. M. Kondratick, I. Unk, S. Prakash, and L. Prakash. 2001. Interaction with PCNA is essential for yeast DNA polymerase η function. Mol. Cell 8:407-415. [DOI] [PubMed] [Google Scholar]

[r10] 10.Haracska, L., S. Prakash, and L. Prakash. 2003. Yeast DNA polymerase ζ is an efficient extender of primer ends opposite from 7,8-dihydro-8-oxogunaine and O⁶-methylguanine. Mol. Cell. Biol. 23:1453-1459. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r11] 11.Haracska, L., S. Prakash, and L. Prakash. 2002. Yeast Rev1 protein is a G template-specific DNA polymerase. J. Biol. Chem. 277:15546-15551. [DOI] [PubMed] [Google Scholar]

[r12] 12.Haracska, L., I. Unk, R. E. Johnson, E. Johansson, P. M. J. Burgers, S. Prakash, and L. Prakash. 2001. Roles of yeast DNA polymerases δ and ζ and of Rev1 in the bypass of abasic sites. Genes Dev. 15:945-954. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r13] 13.Haracska, L., I. Unk, L. Prakash, and S. Prakash. 2006. Ubiquitylation of yeast proliferating cell nuclear antigen and its implications for translesion DNA synthesis. Proc. Natl. Acad. Sci. USA 103:6477-6482. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r14] 14.Huang, M.-E., A. de Calignon, A. Nicolas, and F. Galibert. 2000. POL32, a subunit of the Saccharomyces cerevisiae DNA polymerase δ, defines a link between DNA replication and the mutagenic bypass repair pathway. Curr. Genet. 38:178-187. [DOI] [PubMed] [Google Scholar]

[r15] 15.Johnson, R. E., C. M. Kondratick, S. Prakash, and L. Prakash. 1999. hRAD30 mutations in the variant form of xeroderma pigmentosum. Science 285:263-265. [DOI] [PubMed] [Google Scholar]

[r16] 16.Johnson, R. E., L. Prakash, and S. Prakash. 2006. Yeast and human translesion DNA synthesis polymerases: expression, purification, and biochemical characterization. Methods Enzymol. 408:390-407. [DOI] [PubMed] [Google Scholar]

[r17] 17.Johnson, R. E., S. Prakash, and L. Prakash. 1999. Efficient bypass of a thymine-thymine dimer by yeast DNA polymerase, Polη. Science 283:1001-1004. [DOI] [PubMed] [Google Scholar]

[r18] 18.Johnson, R. E., C. A. Torres-Ramos, T. Izumi, S. Mitra, S. Prakash, and L. Prakash. 1998. Identification of APN2, the Saccharomyces cerevisiae homolog of the major human AP endonuclease HAP1, and its role in the repair of abasic sites. Genes Dev. 12:3137-3143. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r19] 19.Johnson, R. E., M. T. Washington, L. Haracska, S. Prakash, and L. Prakash. 2000. Eukaryotic polymerases ι and ζ act sequentially to bypass DNA lesions. Nature 406:1015-1019. [DOI] [PubMed] [Google Scholar]

[r20] 20.Johnson, R. E., M. T. Washington, S. Prakash, and L. Prakash. 2000. Fidelity of human DNA polymerase η. J. Biol. Chem. 275:7447-7450. [DOI] [PubMed] [Google Scholar]

[r21] 21.Johnson, R. E., S.-L. Yu, S. Prakash, and L. Prakash. 2003. Yeast DNA polymerase zeta (ζ) is essential for error-free replication past thymine glycol. Genes Dev. 17:77-87. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r22] 22.Lawrence, C. W., and R. B. Christensen. 1979. Ultraviolet-induced reversion of cyc1 alleles in radiation-sensitive strains of yeast. III. rev3 mutant strains. Genetics 92:397-408. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r23] 23.Lawrence, C. W., and R. B. Christensen. 1978. Ultraviolet-induced reversion of cyc1 alleles in radiation-sensitive strains of yeast. I. rev1 mutant strains. J. Mol. Biol. 122:1-21. [DOI] [PubMed] [Google Scholar]

[r24] 24.Lawrence, C. W., and V. M. Maher. 2001. Eukaryotic mutagenesis and translesion replication dependent on DNA polymerase ζ and Rev1 protein. Biochem. Soc. Trans. 29:187-191. [DOI] [PubMed] [Google Scholar]

[r25] 25.Lawrence, C. W., P. E. Nisson, and R. B. Christensen. 1985. UV and chemical mutagenesis in rev7 mutants of yeast. Mol. Gen. Genet. 200:86-91. [DOI] [PubMed] [Google Scholar]

[r26] 26.Lawrence, C. W., T. O'Brien, and J. Bond. 1984. UV-induced reversion of his4 frameshift mutations in rad6, rev1, and rev3 mutants of yeast. Mol. Gen. Genet. 195:487-490. [DOI] [PubMed] [Google Scholar]

[r27] 27.Li, Z., H. Zhang, T. P. McManus, J. J. McCormick, C. W. Lawrence, and V. M. Maher. 2002. hREV3 is essential for error-prone translesion synthesis past UV or benzo[a]pyrene diol epoxide-induced DNA lesions in human fibroblasts. Mutat. Res. 510:71-80. [DOI] [PubMed] [Google Scholar]

[r28] 28.Masutani, C., R. Kusumoto, A. Yamada, N. Dohmae, M. Yokoi, M. Yuasa, M. Araki, S. Iwai, K. Takio, and F. Hanaoka. 1999. The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase η. Nature 399:700-704. [DOI] [PubMed] [Google Scholar]

[r29] 29.Nair, D. T., R. E. Johnson, L. Prakash, S. Prakash, and A. K. Aggarwal. 2006. Hoogsteen base pair formation promotes synthesis opposite the 1,N⁶-ehthenodeoxyadenosine lesion by human DNA polymerase ι. Nat. Struct. Mol. Biol. 13:619-625. [DOI] [PubMed] [Google Scholar]

[r30] 30.Nair, D. T., R. E. Johnson, L. Prakash, S. Prakash, and A. K. Aggarwal. 2005. Rev1 employs a novel mechanism of DNA synthesis using a protein template. Science 309:2219-2222. [DOI] [PubMed] [Google Scholar]

[r31] 31.Nelson, J. R., C. W. Lawrence, and D. C. Hinkle. 1996. Deoxycytidyl transferase activity of yeast REV1 protein. Nature 382:729-731. [DOI] [PubMed] [Google Scholar]

[r32] 32.Nelson, J. R., C. W. Lawrence, and D. C. Hinkle. 1996. Thymine-thymine dimer bypass by yeast DNA polymerase ζ. Science 272:1646-1649. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Complex Formation with Rev1 Enhances the Proficiency of Saccharomyces cerevisiae DNA Polymerase ζ for Mismatch Extension and for Extension Opposite from DNA Lesions^▿

Narottam Acharya

Robert E Johnson

Satya Prakash

Louise Prakash

Abstract