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Previous Article | Next Article 
Molecular and Cellular Biology, December 2006, p. 9555-9563, Vol. 26, No. 24
0270-7306/06/$08.00+0 doi:10.1128/MCB.01671-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
for Mismatch Extension and for Extension Opposite from DNA Lesions
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1061
Received 6 September 2006/ Returned for modification 24 September 2006/ Accepted 27 September 2006
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ABSTRACT |
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, 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 Pol and Rev1 for damage-induced mutagenesis, implicating their involvement in mutagenic TLS. Pol 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 Pol-dependent TLS in a nonenzymatic way. Here, we provide evidence for the physical association of Rev1 with Pol 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 rev1
mutation. We propose that Rev1 binding to Pol is indispensable for the targeting of Pol to the replication fork stalled at a DNA lesion. In addition to this structural role, Rev1 binding enhances the proficiency of Pol for the extension of mismatched primer termini on undamaged DNAs and for the extension of primer termini opposite DNA lesions. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and and a Rad5-Mms2-Ubc13-dependent postreplication repair pathway (36).
Pol 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 Pol 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). Pol, 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 Pol 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 Pol 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 Pol function leads to an enhanced rate of UV-induced mutagenesis and Pol promotes error-free TLS through the UV-induced CPDs, Pol exhibits a higher fidelity for deoxynucleoside triphosphate (dNTP) incorporation on undamaged DNAs than Pol. Thus, Pol's rate of misincorporation is 
10–4 (10, 12, 19), compared to Pol, which misincorporates dNTPs with a frequency of 10–2 (39). Pol, 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, Pol 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. Pol, however, is very efficient at extending from the nucleotide inserted opposite these lesion sites by another DNA Pol (12, 19, 21, 29).
Like Pol, Rev1 is also required for mutagenesis induced by DNA damaging agents (18, 23, 26); but unlike Pol, 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 Pol-dependent TLS, its DNA polymerase activity is dispensable (4, 12). Thus, Rev1 plays an indispensable structural role in Pol-dependent TLS.
Here we determined whether evidence can be adduced for a direct physical interaction of Rev1 with Pol, and we examined whether Rev1 binding affects Pol's fidelity or mismatch extension ability on undamaged DNAs and whether Pol'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 Pol through its Rev3 catalytic subunit, and we provide evidence that Rev1 binding stimulates the proficiency of Pol for mismatch extension on undamaged DNAs and enhances also its proficiency for extension opposite DNA lesions.
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MATERIALS AND METHODS |
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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 rev7 derivative on glutathione Sepharose beads by using a protocol described previously (12). To purify Pol, 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 Pol.
The physical interaction of Rev1 with Pol was examined by using a protocol similar to that described previously (1). Briefly, GST-Rev1 or its truncated forms (2 µg) were incubated with Pol (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-Pol, 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 Pol or Rev1*-Pol complex, where Rev1 is catalytically inactive because of the change of aspartate 467 and glutamate 468 to alanines. For both Pol and the Rev1*-Pol complex, the Pol concentration used was 0.5 nM. The Rev1*-Pol complex was made by incubating equimolar concentrations of Rev1* and Pol overnight at 4°C. Both Pol and the Rev1*-Pol complex were treated in an identical manner, and under these conditions, the catalytic activity of Pol 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 = (kcat[E] x [dNTP]/(Km + [dNTP]), where [E] refers to enzyme concentration. Apparent Km and kcat steady-state parameters were obtained from the fit and were used to calculate the efficiency of deoxynucleotide incorporation (kcat/Km). 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'.
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derivative were grown in synthetic complete medium (SC), and the rev1 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 x 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 |
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occurs through the Rev3 catalytic subunit.
Pol, 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 Pol 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 Pol/Rev1 for TLS.
To check for the physical interaction of Rev1 with Pol, 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 Pol or Rev1 protein was incubated with GST-Rev1 or GST-Pol, 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, Pol bound Rev1 regardless of whether GST-Rev1 was incubated with Pol or whether GST-Pol was incubated with Rev1 (lanes 1 to 8). No nonspecific binding of Pol or Rev1 to GST was seen in control experiments (lanes 9 to 16). Next, we examined if the interaction of Rev1 with Pol was mediated by Rev3. For this purpose, we purified the GST-Rev1, Rev1, GST-Rev3, and Rev3 proteins from a rev7 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).
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Mapping of the regions in Rev1 and Rev3 involved in interaction.
To map the region of Rev1 involved in binding to Pol, 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 rev7 yeast strain. The GST-Rev1 or Rev1 protein was incubated with Pol or with GST-Pol, 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 Pol (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 Pol (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 Pol (Fig. 2B, lanes 17 to 24). From these observations, we conclude that at minimum, the carboxyl-terminal 72 residues of Rev1 are necessary for Pol binding and that the carboxyl-terminal 200 residues of Rev1 are sufficient for interaction with Pol.
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Rev1 binding enhances the mismatch extension efficiency of Pol.
The binding of Rev1 to Rev3 in its polymerase domain raised the possibility that this interaction might affect the catalytic properties of Pol. To examine this, we first determined whether interaction with Rev1 modifies the catalytic efficiency of Pol 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 Pol 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*-Pol complex. The Rev1*-Pol complex was formed by incubating equimolar concentrations of Pol and catalytically inactive Rev1 overnight at 4°C, and we expect Rev1* and Pol to form a 1:1 complex under these conditions. The efficiencies (kcat/Km) of dNTP incorporation by Pol and by Rev1*-Pol were determined as a function of deoxynucleotide concentration under steady-state conditions. Care was taken so that the amount of Pol when used alone and the amount used in the complex were the same. From the kinetics of deoxynucleotide incorporation, the steady-state apparent Km and kcat values were obtained from the curve fitted to the Michaelis-Menten equation. As shown in Table 1, the Rev1*-Pol complex incorporated the correct dNTPs opposite templates A, T, G, and C with about the same efficiency as did Pol alone. Thus, interaction with Rev1 does not significantly alter the efficiency of correct dNTP incorporation opposite undamaged nucleotides by Pol.
As Pol is an efficient extender of mispaired primer termini, we next compared the efficiency of mismatch extension by Pol to the efficiency of mismatch extension by Rev1*-Pol. 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*-Pol complex extended from a G · G or a G · T mismatch approximately 12- to 14-fold more efficiently than did Pol, 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*-Pol complex. Overall, and interestingly, the Rev1*-Pol 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 Pol, thus, is greatly enhanced upon Rev1 binding.
Enhanced proficiency of extension opposite from abasic sites by the Rev1*-Pol complex.
Pol 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 Pol on undamaged DNAs (Table 2), we next determined if Rev1 also stimulates the efficiency of extension opposite abasic sites by Pol. Although the efficiency of nucleotide incorporation opposite an abasic site for the Rev1*-Pol complex remained the same as that for Pol 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*-Pol complex compared to Pol alone. However, the extension from an A opposite the abasic site for Rev1*-Pol remained the same as that for Pol (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*-Pol complex.
Pol plays a pivotal role in promoting replication through UV-induced DNA lesions, and it is indispensable for UV-induced mutagenesis along with Rev1. Pol 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 Pol 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 Pol 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 Pol extended from a G opposite the 3' T of a cis-syn T-T dimer quite efficiently (kcat/Km, 8), still a >3-fold enhancement of this efficiency occurred with Rev1*-Pol (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 Pol 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, Pol, 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 Pol is indispensable for Pol's role in TLS.
Since the deletion of the C-terminal 72 amino acid residues of Rev1 abrogates its physical association with Pol, we next examined whether the inability to complex with Rev1 adversely affects Pol's ability to promote mutagenic TLS through UV-induced DNA lesions. Similar to rev3 and rev7 strains, the rev1 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 rev1 strain, and the generation of UV-induced can1r mutations in the two mutants is affected to the same degree. From these observations, we infer that Rev1 binding is indispensable for Pol function in TLS.
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DISCUSSION |
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-dependent TLS and mutagenesis. Here we provide evidence for a direct physical interaction of Rev1 with Pol and show that this interaction occurs through the binding of Rev1 with Rev3 in Pol.
The Rev1-Pol 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 Pol. 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 rev1 mutation implies that Rev1 binding is a prerequisite for Pol function in TLS and mutagenesis.
How might Rev1 binding contribute to Pol function in TLS? In this regard, it is of interest that even though Rev1-Pol and Pol both function in TLS in yeast cells in a manner dependent upon Rad6-Rad18 and upon PCNA ubiquitylation, they differ in important ways. First, Pol 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 Pol, and PCNA binding stimulates the DNA synthetic activity of Pol. Furthermore, PCNA binding is essential for Pol function in TLS since mutations in the PCNA binding motif render Pol nonfunctional in vivo (9). In striking contrast, we found no evidence for PCNA binding by Rev1, or Pol, or the Rev1-Pol complex (13; N. Acharya, S. Prakash, and L. Prakash, unpublished observations). These observations lead us to suggest that the Rev1-Pol complex gains access to PCNA at the replication fork in a more indirect manner rather than the direct PCNA binding mode adopted by Pol. Second, in addition to its requirement for Rev1, the function of Pol in TLS depends upon Pol32, a nonessential subunit of Pol
(5, 12, 14). Also, even though Pol32 is indispensable for Pol-mediated TLS and mutagenesis, it is not required for Pol function in TLS. In view of these considerations, we raise the possibility that Pol's targeting to the replication fork stalled at a lesion site is mediated via the interaction of Rev1-Pol 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 Pol, the Rev1-Rev7 complex may play no role in Pol-mediated TLS.
In addition to the possible role of Rev1 binding in promoting the targeting of Pol to the replication fork, interestingly, we find that Rev1 binding enhances the mismatch extension proficiency of Pol 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 Pol for extension opposite from DNA lesions but has no effect on dNTP incorporation opposite them. Thus, Rev1 further stimulates Pol's proficiency for mismatch extension and for extension opposite DNA lesions, in spite of the fact that even on its own, Pol is quite proficient at performing these roles.
How might Rev1 binding enhance Pol'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 Pol 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 Pol 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 Pol function in TLS. Furthermore, we provide evidence that Rev1 binding enhances the proficiency of Pol for mismatch extension and for extension from the DNA lesion-bearing primer termini.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published ahead of print on 9 October 2006.
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), and the rev1
). (B) UV-induced can1r mutations in the wild-type strain EMY74.7 (•), the rev1