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Mol Cell Biol. Oct 2006; 26(20): 7783–7790.
Published online Aug 14, 2006. doi:  10.1128/MCB.01260-06
PMCID: PMC1636848

Mms2-Ubc13-Dependent and -Independent Roles of Rad5 Ubiquitin Ligase in Postreplication Repair and Translesion DNA Synthesis in Saccharomyces cerevisiae[down-pointing small open triangle]

Abstract

The Rad6-Rad18 ubiquitin-conjugating enzyme complex of Saccharomyces cerevisiae promotes replication through DNA lesions via three separate pathways that include translesion synthesis (TLS) by DNA polymerases η and ζ and postreplicational repair (PRR) of discontinuities that form in the newly synthesized DNA opposite from DNA lesions, mediated by the Mms2-Ubc13 ubiquitin-conjugating enzyme and Rad5. Rad5 is an SWI/SNF family ATPase, and additionally, it functions as a ubiquitin ligase in the ubiquitin conjugation reaction. To decipher the roles of these Rad5 activities in lesion bypass, here we examine the effects of mutations in the Rad5 ATPase and ubiquitin ligase domains on the PRR of UV-damaged DNA and on UV-induced mutagenesis. Even though the ATPase-defective mutation confers only a modest degree of UV sensitivity whereas the ubiquitin ligase mutation causes a high degree of UV sensitivity, we find that both of these mutations produce the same high level of PRR defect as that conferred by the highly UV-sensitive rad5Δ mutation. From these studies, we infer a requirement of the Rad5 ATPase and ubiquitin ligase activities in PRR, and based upon the effects of different rad5 mutations on UV mutagenesis, we suggest a role for Rad5 in affecting the efficiency of lesion bypass by the TLS polymerases. In contrast to the role of Rad5 in PRR, however, where its function is coupled with that of Mms2-Ubc13, Rad5 function in TLS would be largely independent of this ubiquitin-conjugating enzyme complex.

DNA lesions in the template strand block the progression of the replication fork. In eukaryotes, the Rad6 and Rad18 proteins play a crucial role in regulating the various lesion bypass processes; Rad6, a ubiquitin-conjugating (UBC) enzyme, exists in vivo in a tight complex with Rad18, a DNA binding protein, and the DNA binding activity of Rad18 presumably targets Rad6 to the regions of stalled replication at a lesion site (1, 2). In the yeast Saccharomyces cerevisiae, Rad6-Rad18-mediated ubiquitin conjugation promotes replication through DNA lesions via at least three different pathways. DNA polymerase η (Polη) and Polζ provide two of the means by which synthesis through DNA lesions can be achieved by these highly specialized polymerases (19, 30, 31). A third pathway, controlled by the Mms2-Ubc13 ubiquitin-conjugating enzyme complex and by Rad5, promotes the repair of discontinuities which form in the DNA synthesized from damaged templates (34).

UV light induces the formation of cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts in DNA. Polη provides an important pathway for promoting error-free replication through the CPDs. Both in yeast and in humans, inactivation of Polη confers an increase in the incidence of UV mutagenesis (20, 29, 32, 38), and in humans, it causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum (18, 28). Although replication through certain DNA lesions, for example, CPDs, can be handled by a single DNA polymerase, such as Polη, replication through many of the lesions entails the sequential action of two DNA Pols, in which one Pol inserts the nucleotide opposite the DNA lesion and a second Pol carries out the subsequent extension reaction (31). For most DNA lesions that have been examined thus far, Polζ functions at the extension step (10, 16, 22, 23, 37). Translesion DNA synthesis (TLS) through the (6-4) TT photoproducts, for example, involves the function of Polη at the insertion step and of Polζ at the extension step (16).

The third lesion bypass pathway, dependent upon the MMS2, UBC13, and RAD5 genes, promotes the repair of discontinuities that form in the newly synthesized strand opposite from DNA lesions. Although the mechanism by which this pathway operates is not known, presumably it mediates replication through the DNA lesions by a template-switching mechanism, wherein the newly synthesized daughter strand of the undamaged complementary sequence is used as the template for bypassing the lesion. Mms2 forms a specific complex with Ubc13, a ubiquitin-conjugating enzyme, and the Mms2-Ubc13 complex promotes the assembly of polyubiquitin chains linked through lysine 63 of ubiquitin (14). Rad5, a member of the SWI/SNF family of ATPases (17), exhibits a DNA-dependent ATPase activity (21), and it has a C3HC4 RING motif characteristic of ubiquitin ligase (E3) proteins (5, 15, 27, 39). E3 proteins promote the ubiquitin conjugation reaction by binding the UBC enzyme complex as well as the protein substrate, thereby increasing the access of the protein substrate to the UBC enzyme (42). In yeast, Rad5 physically associates with the Mms2-Ubc13 complex via Ubc13, and this association requires the C3HC4 motif of Rad5. Additionally, Rad5 interacts with Rad18 in the Rad6-Rad18 complex (36).

Proliferating cell nuclear antigen (PCNA) plays a key role in the modulation of different lesion bypass processes. The Rad6-Rad18-dependent postreplicational repair (PRR) of discontinuities formed in the DNA synthesized from UV-irradiated templates is abolished by a mutation in the POL30 gene encoding PCNA (35), and PCNA provides the central scaffold to which the various TLS polymerases bind (6-8, 11). The Rad6-Rad18-dependent ubiquitylation of PCNA at its lysine 164 residue is a key regulatory event that modulates the access of lesion bypass proteins to the replication ensemble stalled at the lesion site (9, 13, 33). In DNA-damaged yeast cells, PCNA becomes monoubiquitinated at lysine 164 by Rad6-Rad18; subsequently, this lysine residue is polyubiquitylated via a lysine 63-linked ubiquitin chain in an Mms2-Ubc13-Rad5-dependent manner. Rad5, which binds to both Ubc13 and PCNA, promotes this reaction via its role as a ubiquitin ligase by bringing the Mms2-Ubc13 ubiquitin-conjugating activity into close contact with PCNA (13). Since PRR is greatly impaired in the rad5Δ and mms2Δ mutants and also in the pol30-119 mutant, where the lysine 164 residue of PCNA has been changed to arginine, these observations have implicated the requirement of Mms2-Ubc13-Rad5-dependent PCNA polyubiquitylation in PRR. PCNA monoubiquitylation, on the other hand, activates Polη- and Polζ-dependent TLS.

Although there exists considerable genetic and biochemical evidence for the contributions of Polη and Polζ to lesion bypass, little is known about the mechanisms that modulate Rad5-dependent PRR. To begin to delineate the role of Rad5 in lesion bypass, we have examined the effects of mutations in the ATPase and ubiquitin ligase functions of Rad5 on PRR and damage-induced mutagenesis. We find that even though the ATPase-defective mutant exhibits a very modest level of UV sensitivity, PRR is impaired in this mutant to the same degree as in the highly UV-sensitive ubiquitin ligase mutant. From these observations, we infer the requirement of both the Rad5 ATPase and ubiquitin ligase activities in PRR.

In addition to its role in PRR, where it functions as a ubiquitin ligase in Mms2-Ubc13-dependent polyubiquitylation of PCNA, Rad5 is also involved in generating UV-induced mutations at certain sites and in promoting TLS through abasic sites. Here, we have examined the effects of rad5, mms2, and ubc13 mutations on UV-induced reversion of arg4-17. We show that even though Rad5 is required for arg4-17 reversion, its ATPase or ubiquitin ligase activities are not needed for this role, and neither is Mms2 or Ubc13. These observations implicate a role for Rad5 in the modulation of TLS; however, in contrast to its role in PRR, where its function is coupled with that of Mms2-Ubc13, Rad5's function in TLS would be largely independent of this ubiquitin-conjugating complex.

MATERIALS AND METHODS

Strains.

For postreplication repair studies, the yeast strains were treated with ethidium bromide to obtain [rho0] derivatives lacking mitochondrial DNA. The following yeast strains used in these studies are all derived from EMY74.7, MATa his3-Δ1 leu2-3,112 trp1Δ ura3-52: YR1-65, rad1Δ [rho0]; YR1-118, rad1Δ rad5Δ [rho0]; YR1-307, rad1Δ rad5Δ [rho0] carrying the Cys914, 917→Ala Ala rad5 mutant gene on a CEN plasmid; and YR1-322, rad1Δ rad5Δ [rho0] carrying the Asp681 Glu682→Ala Ala rad5 mutant gene on a CEN plasmid. The rad5Δ strain YR5-179, the mms2Δ strain YMMS2.70, and the ubc13Δ strain YRP774 used for UV mutagenesis studies were derived from CL1265-7C, MATα arg4-17 leu2-3,112 his3-Δ1 trp1 ura3-52. YR5-187, the rad5Δ strain carrying the Asp681 Glu682→Ala Ala rad5 mutant gene on a CEN URA3 plasmid, and YR5-213, the rad5Δ strain carrying the Cys914, 917→Ala Ala rad5 mutant gene on a CEN URA3 plasmid, were constructed by transforming the arg4-17 rad5Δ strain to Ura+ with plasmids pR5.30 and pR5.16, respectively.

Proteins.

The RAD5 cDNA was cloned in frame with the glutathione S-transferase (GST) gene, which is under the control of a galactose-inducible phosphoglycerate promoter, using the plasmid pBJ842. The GST-Rad5 protein was expressed in a protease-deficient yeast strain, BJ5464. Yeast cells were grown at 30°C to stationary phase in synthetic complete medium (SC) lacking leucine to select for the plasmid. The culture was then diluted 10-fold in fresh medium lacking dextrose but containing 2% glycerol and 2% lactic acid, followed by overnight incubation. Expression of recombinant GST-Rad5 protein was induced by adding 2% galactose, followed by propagation for 7 h at 30°C. Cells were harvested by centrifugation and disrupted by a bead beater in buffer A (40 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 0.01% Nonidet P-40, 10% glycerol, 5 mM EDTA) supplemented with 500 mM NaCl and protease inhibitor mixture (Mini-Complete; Roche, Indianapolis, IN). After clarification of the crude extract by centrifugation, the supernatant was loaded onto a glutathione-Sepharose column (Amersham Pharmacia Biotech). First, the column was washed with buffer A plus 1,000 mM NaCl, followed by washing with buffer A plus 100 mM NaCl. The Rad5 proteins were eluted in buffer A plus 100 mM NaCl by PreScission protease, which cleaved in the spacer region between GST and Rad5 protein and left a 7-amino-acid-long leader peptide attached to the N-terminal end of the purified proteins. Purified Rad5 proteins were frozen in aliquots under liquid nitrogen and kept at −70°C. For producing the mutant Rad5 DE681,682AA and Rad5 CC914,917AA proteins, the RAD5 cDNA was mutated by PCR using a QuikChange site-directed mutagenesis kit (Stratagene Corporation, La Jolla, CA). The Rad5 DE681,682AA and Rad5 CC914,917AA proteins were overexpressed and purified in parallel with wild-type Rad5.

ATPase assay.

Purified wild-type and mutant Rad5 proteins (50 ng) were incubated in 10-μl reaction mixtures containing 20 mM Tris-HCl, pH 7.0, 20 mM KCl, 2 mM MgCl2, 100 μg/ml bovine serum albumin, 1 mM dithiothreitol, and 0.5 mM [γ-32P]ATP in the presence or absence of 200 ng of single-stranded M13 DNA for 5 min at 30°C. ATPase activity was monitored by thin-layer chromatography on polyethyleneimine-cellulose and visualized by autoradiography.

Alkaline sucrose gradients.

Asynchronously growing yeast cells were UV irradiated in logarithmic phase at a density of 0.5 × 107 to 1.0 × 107 cells per ml of synthetic complete medium lacking uracil but containing 5 μg of uridine/ml at room temperature with constant stirring in 150- by 20-mm petri dishes at a dose rate of 0.1 J/m2/s. All operations after UV irradiation were performed in yellow light to avoid photoreactivation. After UV irradiation, cells were collected by filtration and resuspended in fresh uridine medium at a density of 1 × 108 to 2 × 108 cells per ml. Pulse-labeling was achieved by the addition of 100 μCi of 6′-[3H]uracil (20 to 25 Ci/mmol, 1 mCi/ml; Moravek Biochemicals and Radiochemicals, Brea, Calif.) to 1 ml of cells, followed by vigorous shaking for 15 min at 30°C. Cells were then washed, resuspended in synthetic complete medium containing 1.67 mg of uracil/ml (high-uracil medium), and incubated for an additional 30 min or 6 h prior to conversion to spheroplasts. An aliquot of 0.3 ml of the spheroplast suspension was layered directly onto a 0.2-ml lysing layer consisting of 0.79 M sorbitol, 0.66 M EDTA, 2.5% sarcosyl, 0.3 M NaCl on top of a 15 to 30% (wt/vol) linear alkaline sucrose solution gradient made in 0.3 M NaOH, 0.7 M NaCl, 40 mM EDTA, and 1% sarcosyl (pH 12.5). Centrifugation and processing of samples were performed as described previously (35), except that alkaline sucrose gradients were centrifuged at 21,000 rpm for 15.5 h and acid precipitation of alkaline-hydrolyzed samples was carried out with 1 N HCl containing 0.1 M sodium pyrophosphate.

UV sensitivity and UV mutagenesis.

Cells of the RAD+ strain and its isogenic derivatives containing rad5, mms2, or ubc13 genomic deletions were grown to mid-logarithmic phase in SC, while rad5Δ strains carrying mutant rad5 genes on CEN URA3 plasmids were grown in SC lacking uracil to maintain selection for the plasmid. Cultures were washed by centrifugation, followed by sonication to disperse cell clumps. After sonication, the cell suspension was centrifuged again and resuspended to a density of 2 × 108 cells per ml. Cells were diluted and spread onto plates containing SC or SC lacking uracil for viability determinations and onto plates containing SC lacking arginine for mutagenesis determinations, followed by UV irradiation at a dose rate of 1 J/m2/s. Plates were incubated in the dark, and colonies were counted after 3 to 5 days.

RESULTS

ATPase and ubiquitin ligase activities of Rad5 protein.

Rad5 exhibits a DNA-dependent ATPase activity (21), and genetic studies have indicated a ubiquitin ligase function for Rad5 (36). PRR of newly synthesized DNA in UV-irradiated yeast cells is greatly diminished in the rad5Δ strain (34); however, the contribution of these two activities to PRR is not known. We sought to characterize Rad5 further by determining the effect of mutations in both of these activities on PRR.

As shown in Fig. Fig.1A,1A, Rad5 has several interesting structural features. Of particular note is the presence of a C3HC4 RING domain which is a characteristic of ubiquitin ligases. The RING domain plays a central role in the ubiquitin ligase function, as its deletion or disruption inactivates this function (27, 36). The X-ray crystal structure of c-Cbl ubiquitin ligase bound to the UBC enzyme and to a peptide substrate has indicated that the RING modules of ubiquitin ligases function as scaffolds that position the UBC enzyme and the protein substrate optimally for the ubiquitin conjugation reaction to occur (42).

FIG. 1.
Purity and ATPase activity of wild-type and mutant Rad5 proteins. (A) Schematic representation of Rad5. The highly conserved DE residues at positions 681 and 682 in Rad5 that correspond to conserved ATPase active-site residues and the CC residues at positions ...

In addition, Rad5 contains the seven motifs characteristic of SWI/SNF ATPases, and the RING domain is located between ATPase motifs III and IV. The DE residues in Rad5 at positions 681 and 682, respectively, in motif II correspond to the residues that constitute the active site of Rad5-related ATPases. The C residues present at positions 914 and 917 are the key residues of the RING domain, and the corresponding residues in other RING-type ubiquitin ligases have been shown to be indispensable for their function. To determine whether the ATPase and the ubiquitin ligase activities of Rad5 have a role in postreplication repair, we mutated both the 681D and 682E residues to A's and the 914C and 917C residues to A's.

The Rad5 DE681,682AA protein has no ATPase activity.

To facilitate the purification of wild-type and mutant Rad5 proteins, their cDNAs were fused downstream of the GST gene, and they were overexpressed in a protease-deficient yeast strain. The wild-type GST-Rad5 and mutant GST-Rad5 DE681,682AA proteins were purified on glutathione beads, followed by elution of Rad5 with PreScission protease, which cleaved in the linker between the GST and Rad5 portion, leaving a 7-amino-acid-long leader sequence at the N terminus of Rad5. On a denaturing polyacrylamide gel, the wild-type and mutant Rad5 proteins migrate at the same position, at ~139 kDa, which is consistent with the calculated molecular mass of 134 kDa for Rad5. Coomassie staining of the gel revealed that the wild-type and the mutant Rad5 proteins were purified to ~95% homogeneity (Fig. (Fig.1B1B).

Next, we ascertained that the mutation of DE at positions 681 and 682 in Rad5 to AA inactivates its ATPase activity. ATPase assays were done by incubating the Rad5 protein with [γ-32P]ATP in the absence or presence of single-stranded M13 DNA (Fig. (Fig.1C).1C). As expected, without the addition of M13 DNA, Rad5 exhibited little ATPase activity (Fig. (Fig.1C,1C, lane 2), whereas in the presence of DNA, Rad5 showed robust ATPase activity (Fig. (Fig.1C,1C, lane 3). Importantly, the Rad5 DE681,682AA mutant protein did not have any ATPase activity (Fig. (Fig.1C,1C, lane 4).

UV sensitivity of rad5 mutants defective in ATPase and ubiquitin ligase functions.

Although the RAD5, MMS2, and UBC13 genes function in the Rad6-Rad18-dependent postreplication repair of UV-damaged DNA, deletions of these genes produce different effects on UV sensitivity (42). As shown in Fig. Fig.2,2, the rad5Δ mutant displays a much higher level of UV sensitivity than the mms2Δ or the ubc13Δ mutant, and the ATPase-defective rad5 DE681,682AA mutant exhibits the same moderate level of UV sensitivity as the mms2Δ or ubc13Δ mutant. The rad5 CC914,917AA mutant defective in the ubiquitin ligase function generates a higher level of UV sensitivity than the mms2Δ or ubc13Δ mutant; its sensitivity, however, is not as severe as that of the rad5Δ mutant.

FIG. 2.
UV sensitivity of rad5 mutations. Survival curves after UV irradiation of wild-type strain EMY74.7 (•), its isogenic rad5Δ (○), mms2Δ ([open triangle]), and ubc13Δ ([filled triangle]) derivatives, and the rad5Δ strain ...

Effect of mutations in the ATPase and ubiquitin ligase functions of Rad5 on postreplication repair.

To determine the effect of rad5 mutations on the PRR of UV-damaged DNA, we examined the size of newly synthesized DNA in UV-irradiated rad1Δ, rad1Δ rad5Δ, rad1Δ rad5 DE681,682AA, and rad1Δ rad5 CC914,917AA yeast cells. Because of the lack of nucleotide excision repair in the rad1Δ strain, UV damage persists in DNA and replication of such DNA requires the various lesion bypass processes. The rad1Δ and rad1Δ rad5Δ cells were UV irradiated at 3.5 J/m2, and the size of newly synthesized DNA formed from the UV-damaged templates was examined by pulse-labeling of DNA with [3H]uracil for 15 min, followed by a chase for 30 min. DNA from rad1Δ cells obtained following this treatment sediments towards the top of the alkaline sucrose gradient, indicating the presence of discontinuities in the newly synthesized DNA (Fig. (Fig.3A).3A). In unirradiated rad1Δ cells, the size of newly synthesized DNA following the 15-min pulse and 6-h chase periods is the same as in uniformly labeled cells. In fact, in unirradiated rad1Δ cells, even a 30-min chase following the 15-min pulse is sufficient to restore the DNA to the same size as in uniformly labeled cells (data not shown). In rad1Δ cells that were UV irradiated and incubated for 6 h following the 15-min pulse, daughter strands attained the same size as in unirradiated cells, indicating that the postreplicative gap-filling process has restored normal size to the daughter strands (Fig. (Fig.3A).3A). The rad1Δ rad5Δ strain, however, is unable to restore normal-sized DNA in UV-irradiated cells following the 6-h incubation period (Fig. (Fig.3B).3B). Since PRR is inhibited to about the same degree in the rad1Δ rad5 DE681,682AA and the rad1Δ rad5 CC914,917AA mutant strains as in the rad1Δ rad5Δ strain (Fig. 3C and D), a requirement for both Rad5 ATPase and Rad5 ubiquitin ligase activities is indicated in this process.

FIG. 3.
Requirement of Rad5 ATPase and ubiquitin ligase activities for postreplication repair of UV-damaged DNA. Sedimentation in alkaline sucrose gradients of nuclear DNA from cells incubated for different periods following UV irradiation is shown. The rad1 ...

Involvement of Rad5 in damage-induced mutagenesis.

Since the frequency of UV-induced mutations at many loci is not lowered by the rad5Δ mutation, Rad5 has been thought to affect primarily the error-free component of Rad6-Rad18-dependent lesion bypass. For example, the incidence of UV-induced forward mutations at the CAN1 locus is not affected in the rad5Δ strain (17), and the rate of UV-induced mutations increases in the rad5Δ rad30Δ double mutant compared to that in either single mutant (20). Based on such observations (20, 29), the Rad5-dependent PRR pathway and the RAD30-encoded Polη have been proposed to promote replication through UV lesions by two separate and competing means that are predominantly error free.

The role of Rad5 in lesion bypass, however, is likely to be more complex than just the contribution to error-free PRR. Even though Rad5, Mms2, and Ubc13 function in the same PRR pathway, the much higher UV sensitivity of the rad5Δ mutant than of the mms2Δ and ubc13Δ mutants would suggest that Rad5 functions in lesion bypass in roles that are in addition to PRR. Although the inactivation of Rad5 does not affect the incidence of UV-induced mutations at many sites, at some sites, the rate of UV-induced reversion is lowered (17, 20, 24-26, 29). For example, the reversion of arg4-17 to Arg+ is reduced ~10-fold in the rad5Δ strain (17). Sequence analyses of UV-induced arg4-17 to ARG4+ revertants has indicated the reversion to be predominantly from a T→C transition of T127 that would constitute the 3′ T of a potential TT photoproduct (41). It has been suggested that this reversion results from the insertion of a G opposite the 3′ T of a (6-4) TT photoproduct by Polη, as inactivation of Polη lowers its incidence (41). Also, experiments with double-stranded plasmids carrying a site-specific (6-4) TT lesion have shown that in yeast cells, replication through this lesion depends upon Polη (4). Biochemical studies have indicated that replication through a (6-4) TT photoproduct can be mediated by the sequential action of Polη and Polζ, in which Polη inserts a G opposite the 3′ T of the lesion and Polζ then extends from it (16). The possible requirement of Rad5 in promoting replication through a (6-4) TT photoproduct might then suggest a role for this protein in coordinating the action of the two polymerases involved in its bypass. Such a role for Rad5 is also supported from our observation that this protein is indispensable for TLS through the abasic sites. The yeast apn1Δ apn2Δ strain exhibits a high degree of CAN1S-to-can1r mutagenesis following methyl methanesulfonate (MMS) treatment (10), resulting from a defect in the removal of abasic sites because of the lack of these apurinic/apyrimidinic endonucleases. The frequency of MMS-induced can1r mutations, however, is severely curtailed in the apn1Δ apn2Δ rad5Δ strain, indicative of a requirement of Rad5 for mutagenic TLS through the abasic sites (data not shown). Replication through an abasic site requires the consecutive action of two polymerases in which one Pol inserts a nucleotide opposite the abasic site and Polζ carries out the extension reaction (10).

To examine whether the ability of Rad5 to promote TLS, such as by coordinating the function of two Pols, was shared by Mms2 and Ubc13, we determined the effects of mms2Δ and ubc13Δ mutations on the rate of UV-induced reversion of arg4-17. As shown in Fig. Fig.4,4, while the frequency of UV-induced Arg4+ revertants was reduced 10-fold or more in the rad5Δ mutant, the mms2Δ or ubc13Δ mutation lowered the rate of reversion at most 2-fold. Next, we examined whether the Rad5 ATPase and ubiquitin ligase activities affect arg4-17 mutagenesis. While the ATPase-defective mutation produces no appreciable effect, the ubiquitin ligase mutation does seem to lower the rate of Arg4+ revertants by ~50%. From these observations, we suggest that in promoting the TLS involving the sequential action of two Pols, Rad5 functions largely independently of the Mms2-Ubc13 enzyme, and this Rad5 function also does not involve its ATPase or ubiquitin ligase activities to any appreciable degree.

FIG. 4.
Requirement of Rad5 but not of its ATPase or ubiquitin ligase activities for UV reversion at arg4-17. Cells grown to mid-logarithmic phase in synthetic medium, to which the appropriate supplements for growth of the particular strain had been added, were ...

DISCUSSION

Requirement of Rad5 ATPase and ubiquitin ligase functions for postreplication repair.

Here, we show that the inactivation of Rad5 ATPase and ubiquitin ligase functions produces the same degree of PRR defect as that incurred by the rad5Δ mutation. The lysine 63-linked polyubiquitylation at lysine 164 of PCNA requires the Rad5 protein in addition to Mms2 and Ubc13. Since Rad5 interacts with Ubc13 in the Mms2-Ubc13 complex via its C3HC4 ubiquitin ligase motif (36) and since Rad5 binds PCNA (13), presumably, Rad5 is indispensable for the targeting of Mms2-Ubc13 to PCNA. The absence of PCNA polyubiquitylation in the ubiquitin ligase mutant of Rad5 could then be the basis of the defective PRR. Such an inference is supported by the observation that PRR is impaired to nearly the same degree in the rad5Δ and the rad5 ubiquitin ligase mutant as in the pol30-119 mutant, where the lysine 164 residue of PCNA has been changed to arginine.

Although it is not readily apparent how the Rad5 ATPase might function in PRR, the known role of SWI/SNF ATPases in affecting the disruption of protein assemblies also suggests the possibility of Rad5 ATPase modulating the dissociation of some protein(s) bound to the replication ensemble, which otherwise is inhibitory to the assembly of proteins involved in Rad5-dependent PRR.

Involvement of Rad5 in TLS.

The observation that the rad5Δ mutation confers a much higher level of UV sensitivity than the mms2Δ or ubc13Δ mutation implies that, in addition to its role in PRR, where it functions in conjunction with Mms2-Ubc13, Rad5 modulates some aspects of other Rad6-Rad18-dependent lesion bypass processes, where its function is largely independent of the Mms2-Ubc13 complex. Although Rad5 is not involved in the production of UV mutations at a number of loci, a limited role in mutagenesis, and hence in TLS, is suggested from the reduced rate of UV mutagenesis at certain loci, and our observation that Rad5 is indispensable for TLS through abasic sites also supports this idea. The inferred involvement of Rad5 in promoting TLS through the (6-4) TT photoproducts and the requirement of Rad5 for TLS through the abasic sites could suggest a role for Rad5 in coordinating the TLS process involving the sequential action of two Pols, in which one inserts the nucleotide opposite the lesion site and the other performs the subsequent extension reaction. The absence of appreciable impairment of UV mutagenesis at arg4-17 in the Rad5 ATPase or ubiquitin ligase mutants, however, implies that these Rad5 activities make little contribution to this TLS process, and that raises the possibility that Rad5 promotes the two-polymerase mode of TLS via a direct mechanistic involvement.

Our observation that inactivation of the ubiquitin ligase function of Rad5 or deletion of MMS2 or UBC13 lowers the rate of arg4-17 UV mutagenesis at most twofold implies that the Mms2-Ubc13-Rad5-dependent polyubiquitylation of PCNA, or of any other protein that might also be subject to this modification, does not make a significant contribution to modulating the two-Pol TLS events. However, the much higher level of UV sensitivity conferred by the rad5 ubiquitin ligase mutation than that by the rad5 ATPase mutation or by the mms2Δ and ubc13Δ mutations could be reflective of an additional role of Rad5, in which, as a ubiquitin ligase, it modulates the efficiency of TLS independent of the Mms2-Ubc13 complex (see below).

Possible roles of Rad5 in lesion bypass. (i) Postreplication repair.

Although the mechanism by which the Rad5-dependent postreplicational repair of discontinuities formed in the newly synthesized DNA opposite from DNA lesions is accomplished is not known, recent genetic studies with plasmids have suggested that this pathway might utilize a copy choice type of synthesis (40). Rad5 could affect this process via its role as a ubiquitin ligase, wherein it mediates the polyubiquitylation of PCNA (Fig. (Fig.5).5). Previously, we have suggested a role for PCNA monoubiquitylation in disrupting the PCNA binding of some protein(s) which is inhibitory to the binding of TLS polymerases to PCNA (12). A similar role could be envisaged for the polyubiquitylated form of PCNA, as that also could be disruptive of the PCNA binding of a protein(s) whose removal promotes the PCNA binding of proteins that function in Rad5-controlled PRR. Another possibility, and one which we have suggested before, is that PCNA polyubiquitylation destabilizes the binding of the replicative polymerase and of other associated proteins from the replication fork stalled at the lesion site (9). In both of these scenarios, Rad5 function would be primarily regulatory, with its ubiquitin ligase function mediating PCNA polyubiquitylation and its ATPase activity, in conjunction with polyubiquitylated PCNA, mediating the disruption of the inhibitory protein(s) from the replication ensemble and thereby activating PRR (Fig. (Fig.55).

FIG. 5.
Mms2-Ubc13-dependent and -independent roles of Rad5 in lesion bypass. (Left) Rad5 functions in PRR as a ubiquitin (Ub) ligase in conjunction with Mms2-Ubc13. As a ubiquitin ligase, Rad5 promotes the polyubiquitylation of PCNA at lysine 164 by binding ...

(ii) Translesion synthesis.

Rad5 could contribute to TLS in a direct mechanistic manner by coordinating the action of two polymerases involved in promoting replication through the lesion site (Fig. (Fig.5).5). One possibility, and one which we are pursuing, is that Rad5 physically associates with the inserter and the extender polymerase and stimulates the efficiency of the two-polymerase TLS reaction.

The greater UV sensitivity of the rad5 ubiquitin ligase mutant than that of the mms2Δ or the ubc13Δ mutant raises the possibility that the ubiquitin ligase function of Rad5 contributes to increasing the efficiency of TLS Pols by promoting their ubiquitylation by the Rad6-Rad18 complex (Fig. (Fig.5).5). Recent evidence suggests that human Polη and Polι are ubiquitylated in vivo, but how that affects their function is not known (3). Although the Rad6-Rad18 complex may ubiquitylate the TLS Pols directly without the intervention of Rad5 as the ubiquitin ligase, there is the possibility that Rad5 contributes to the ubiquitylation of TLS Pols by binding the TLS Pols as well as the Rad6-Rad18 complex, to which it binds via Rad18 (36), thereby increasing the efficiency of ubiquitylation of the TLS polymerases. In such a reaction, the ubiquitylation of TLS polymerases would require the Rad5 ubiquitin ligase function in addition to the Rad6-Rad18 complex but not the Mms2-Ubc13 complex.

In summary, we make the following observations from these studies. (1) Both the ATPase and ubiquitin ligase activities of Rad5 are indispensable for its role in PRR, and in this role, Rad5 function depends upon the Mms2-Ubc13 enzyme complex (2). Rad5 functions additionally in TLS, but for this role, Rad5 does not require the Mms2-Ubc13 complex (3) and Rad5 could modulate TLS in two separate ways, one being dependent upon its ubiquitin ligase function and the other being independent of it (Fig. (Fig.55).

Acknowledgments

This work was supported by National Institutes of Health grant CA107650, a Wellcome Trust International Senior Research Fellowship, and Howard Hughes Medical Institute grant 55005612.

Footnotes

[down-pointing small open triangle]Published ahead of print on 14 August 2006.

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