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Molecular and Cellular Biology, October 2007, p. 7266-7272, Vol. 27, No. 20
0270-7306/07/$08.00+0     doi:10.1128/MCB.01196-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Mutations in the Ubiquitin Binding UBZ Motif of DNA Polymerase Do Not Impair Its Function in Translesion Synthesis during Replication

Narottam Acharya,¹ Amrita Brahma,¹ Lajos Haracska,² Louise Prakash,¹ and Satya Prakash^1*

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1061,¹ Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary²

Received 5 July 2007/ Returned for modification 23 July 2007/ Accepted 7 August 2007

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Treatment of Saccharomyces cerevisiae cells with DNA-damaging agents elicits lysine 164-linked PCNA monoubiquitination by Rad6-Rad18. Recently, a number of ubiquitin (Ub) binding domains (UBDs) have been identified in translesion synthesis (TLS) DNA polymerases and it has been proposed that the UBD in a TLS polymerase affects its binding to Ub on PCNA and that this binding mode is indispensable for a TLS polymerase to access PCNA at the site of a stalled replication fork. To evaluate the contribution of the binding of UBDs to the Ub moiety on PCNA in TLS, we have examined the effects of mutations in the C₂H₂ zinc binding motif and in the conserved D570 residue that lies in the -helix portion of the UBZ domain of yeast Pol. We find that mutations in the C₂H₂ motif have no perceptible effect on UV sensitivity or UV mutagenesis, whereas a mutation of the D570 residue adversely affects Pol function. The stimulation of DNA synthesis by Pol with PCNA or Ub-PCNA was not affected by mutations in the C₂H₂ motif or the D570 residue. These observations lead us to suggest that the binding of Ub on PCNA via its UBZ domain is not a necessary requirement for the ability of polymerase to function in TLS during replication.

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Genetic and biochemical studies of Saccharomyces cerevisiae have indicated the requirement of the Rad6-Rad18 ubiquitin (Ub)-conjugating enzyme complex (1, 2) for promoting replication through DNA lesions which block synthesis by the replicative DNA polymerases (Pols). In yeast, Rad6-Rad18-dependent lesion bypass could occur via translesion DNA synthesis (TLS) by the action of Pol or Pol (20, 22, 25), or it may involve the Rad5-Mms2-Ubc13-dependent postreplicational repair of discontinuities that form in the newly synthesized DNA strand opposite from DNA lesions (27).

Both in yeast and humans, PCNA plays a key role in modulating the various Rad6-Rad18-dependent lesion bypass processes. The TLS Pols, such as Pol from yeast and Pols , , and from humans, interact physically and functionally with PCNA, and PCNA binding is stimulatory to the catalytic activity of these Pols on undamaged and damaged DNAs (9-11, 14). In addition, genetic studies of yeast have shown that PCNA binding is indispensable for the ability of Pol to function in TLS in vivo, since mutations in the PCNA binding PIP motif of Pol render cells as UV sensitive as those lacking Pol (11).

Treatment of yeast or human cells with DNA-damaging agents elicits the monoubiquitination of PCNA at its lysine 164 residue, and subsequently, this residue becomes polyubiquitinated via a lysine 63-linked chain (16). Genetic studies have shown that PCNA monoubiquitination is mediated by the Rad6-Rad18 enzyme and that polyubiquitination requires the additional action of the Mms2-Ubc13-Rad5 enzyme complex (16), in which Rad5 provides the ubiquitin ligase function (28) and the Mms2-Ubc13 complex specifically promotes lysine 63-linked polyubiquitin chain formation (18). Also, genetic observations of yeast have indicated that PCNA monoubiquitination modulates the TLS process and that PCNA polyubiquitination is necessary for Rad5-dependent postreplicational repair (12, 16, 26).

The various yeast and human TLS Pols such as , , and interact with PCNA via their consensus PCNA binding PIP motif, which binds the interdomain connector loop of PCNA, and genetic and biochemical studies have shown that this PCNA binding mode is indispensable for their function (9-11, 14). It has also been suggested that the TLS Pols bind to the ubiquitin moiety that becomes attached on PCNA via Rad6-Rad18 action and that this PCNA binding mode is indispensable for the recruitment of TLS Pols to PCNA. The idea that TLS Pols bind PCNA at the interdomain connector loop motif and to the lysine 164-attached Ub moiety and that both binding modes are indispensable for their function has received further support from the recent identification of ubiquitin binding motifs UBZ in Pols and and UBM in Pol and Rev1 and the demonstration that mutations in these motifs inactivate their function in TLS in vivo (5, 24).

The Rad6-Rad18-dependent PCNA monoubiquitination reaction at lysine 164 has been reconstituted from purified yeast proteins in two separate studies (7, 15). Whereas in one study, weak stimulation of Pol synthetic activity was reported with Ub-PCNA compared to that seen with unmodified PCNA (7), we have been unable to confirm this observation (15), and our published studies as well as subsequent unpublished studies have yielded no evidence for the additional stimulation of synthesis by Ub-PCNA over that seen with unmodified PCNA for any of the yeast or human TLS Pols.

Since the requirement for the binding of the ubiquitin moiety on PCNA has been inferred from genetic observations that have been made with mutations in the ubiquitin binding domain (UBD) of TLS Pols (5, 24), we have examined this question with a more extensive mutational analysis of the UBZ domain of yeast Pol. To our surprise, we found that mutations of the conserved cysteine and histidine residues in the UBZ motif of yeast Pol have no functional consequence with respect to its biological role in TLS in vivo; overall, the genetic and biochemical studies we present here yield no support to the idea that the binding of the Ub moiety on PCNA by Pol via its UBZ domain is indispensable for its function in TLS during replication.

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UV sensitivity and UV mutagenesis. Wild-type yeast strain EMY74.7 was grown in synthetic complete (SC) medium, and its isogenic rad30 derivative carrying either the CEN ARS LEU2 plasmid YCplac111 (8) with no insert (strain YR30.35) or carrying YCplac111 with the HH568,572AA mutant rad30 gene (strain YR30.202), YCplac111 with the C552A mutant rad30 gene (strain YR30.240), YCplac111 with the CC552,553AA mutant rad30 gene (strain YR30.242), or YCplac111 with the D570A mutant rad30 gene (strain YR30.238) was grown in SC medium lacking leucine (SC-leu) to maintain selection for the plasmid. When cultures had reached the midlogarithmic phase, they were washed by centrifugation, subjected to sonication to disperse cell clumps, pelleted by centrifugation, and resuspended to a density of 2 x 10⁸ cells per ml. Appropriate dilutions of cells were spread onto the surface of plates containing SC or SC-leu medium 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.

Purification of Pol proteins. The wild-type and mutant Rad30 proteins were expressed as glutathione S-transferase fusion proteins in pBJ842 (19) to generate GAL-PGK-GST-RAD30 or mutant rad30 (2µm leu2-d) plasmids. Wild-type or mutant Rad30 protein was purified from yeast strain BJ5464 which had been transformed with the plasmid expressing either the wild-type or mutant Rad30 protein by use of a protocol described previously (13, 19).

Oligonucleotides and DNA polymerase assays. Oligonucleotides were synthesized by Midland Certified Reagent Co. (Midland, TX). DNA substrates were generated by annealing 75mer 5'-biotin-A GCA ACG TCA CCA ATG TCT AAG AGT TCG TAT TAT GCC TAC ACT GGA GTA CCG GAG CAT CGT CGT GAC TGG GAA AAC-biotin-3', which contained one biotin molecule attached at each end to 44mer 5' ³²P-labeled oligonucleotide primer 5'-GTT TTC CCA GTC ACG ACG ATG CTC CGG TAC TCC AGT GTA GGC AT-3'. To bind streptavidin to biotin present at the ends of the linear DNA substrate, the primer-template was preincubated with streptavidin (1:4) in 25 µl DNA polymerase buffer which contained no MgCl₂ for 10 min at 30°C before its addition to the DNA polymerase reaction mixtures.

The standard PCNA-dependent DNA polymerase stimulation reaction mixture (10 µl) contained 40 mM Tris-HCl (pH 7.5), 8 mM MgCl₂, 150 mM NaCl, 1 mM dithiothreitol, 10% glycerol, 100 µg/ml bovine serum albumin, 100 µM (each) deoxynucleoside triphosphates, and 100 µM ATP. The reactions were started with 0.5 nM concentrations of enzymes with or without PCNA or Ub-PCNA (50 ng), replication factor C (25 ng), and replication protein A (200 ng) and 10 nM linear primer-template DNA. Assays were assembled on ice, incubated at 30°C for 10 min, and stopped by the addition of loading buffer (40 µl) containing EDTA (20 mM), 95% formamide, 0.3% bromophenol blue, and 0.3% xylene cyanol blue. The reaction products were resolved on a 12% polyacrylamide gel containing 8 M urea.

Far UV-CD spectra. "Far UV-CD" (180 to 260 nm) spectra of wild-type or D570A Pol in buffer containing 5 mM dithiothreitol, 10% glycerol, and 150 mM NaCl at pH 7.5 were recorded in a 1 mm path length cell by use of an AVIV model 62DS circular dichroism (CD) spectropolarimeter. The spectra were recorded with a response time of 1 s and a scan speed of 30 nm/min. Each data point represented an average of five accumulations. The results are expressed as molar residue ellipticity ([]) values (in degrees per square centimeter per decimole) that were determined from the following equation:

where l is the light-path length in centimeters, [Pr] is the concentration of protein in milligrams/milliliter, Mr = 1/N_A (where N_A is number of residues in protein), and Mw is the molecular weight of the protein.

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Mutational analyses of the UBZ domain of Pol. Analysis of the solution structure of the UBZ domain of human Pol (6) has shown that it adopts a classical C₂H₂ zinc finger structure characterized by a ßß fold which is comprised of two short antiparallel ß-strands and a carboxy-terminal -helix (Fig. 1). The two cysteines are located on the fingertip made by the two ß-strands, and the two histidines are located on the -helix. A zinc ion is coordinated by these two cysteines and two histidines. The binding of the UBZ domain to ubiquitin is mediated through the -helix, and the residues present on the outward face of the -helix, particularly the residues D652, F655, A656, and L657, are important for this interaction (see Fig. 1A in reference 6). The residues in the C₂H₂ motif involved in zinc binding, and those in the -helix that are involved in ubiquitin binding, are strongly conserved in Pol from yeast to humans. The H654A mutation in the conserved histidine residue of the C₂H₂ motif of human Pol that would disrupt zinc binding abolishes the interaction of Pol with ubiquitin (24), indicating that zinc binding is an important prerequisite for ubiquitin binding by the UBZ domain. The D652A mutation of human Pol also prevents the interaction of Pol with ubiquitin, and in UV-damaged human cells, this mutant protein does not localize to the replication foci (5). From the inability of this mutant protein to localize to the replication foci in UV-damaged cells, the inference has been drawn that this lack of localization results from the inability of the mutant protein to bind the Ub moiety on PCNA (5). However, for the H654A protein, which also is defective in ubiquitin binding, localization to replication foci in UV-damaged cells is reduced only somewhat (approximately twofold) (24).

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FIG. 1. Sequence alignment of Pol UBZ domains. The UBZ domains of Homo sapiens (Hs), Mus musculus (Mm), Drosophila melanogaster (Dm), Schizosaccharomyces pombe (Sp), and Saccharomyces cerevisiae (Sc) Pol have been aligned. The first and last amino acids of each sequence are numbered. Highly conserved residues are depicted on a dark background. The asterisks indicate the residues that were changed to alanine in yeast Pol. The positions of the two short ß sheets and of the -helix, as determined from the solution structure of human Pol UBZ, are indicated above the sequences. See reference 6 for details of the UBZ structure.

To provide for a more comprehensive analysis of the biological significance of ubiquitin binding by the UBZ domain, we have examined the effects of mutations in the cysteine and histidine residues of the C₂H₂ motif and of a mutation in the conserved aspartate residue present in the -helix on Pol function in TLS in vivo and we have examined these mutant Pol proteins for their ability to functionally interact with PCNA and Ub-PCNA in vitro.

Mutations in the C₂H₂ zinc binding motif of the UBZ domain do not affect Pol function in TLS during replication. To test the functional significance of ubiquitin binding by the UBZ domain, we examined the ability of the C552A, CC552,553AA, and HH568,572AA mutant rad30 genes carried on a low-copy-number CEN/ARS plasmid to complement the UV sensitivity of the rad30 strain. As shown in Fig. 2A and C, all the mutant genes restored wild-type levels of UV sensitivity to the rad30 strain. Since Pol promotes the efficient and error-free replication through cyclobutane pyrimidine dimers, the frequency of UV-induced mutations rises to a much greater level in the rad30 strain than in the wild-type strain. Introduction of the plasmids carrying the C552A, CC552,553AA, or HH568,572AA mutant rad30 genes into the rad30 strain restored UV mutagenesis to a level similar to that seen in the wild-type strain. Thus, the mutational inactivation of the zinc binding and ubiquitin binding ability of the UBZ domain has no perceptible effect on UV sensitivity or on UV mutagenesis, which suggests that the function of Pol in promoting synthesis through UV-induced DNA lesions during replication is not impaired by mutations in the C₂H₂ motif of the UBZ domain.

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FIG. 2. Effects of mutations in the UBZ of Pol (Rad30) on UV sensitivity and UV-induced mutagenesis. (A) Survival after UV irradiation of wild-type strain EMY747, its isogenic rad30 derivative YR30.35 carrying the CEN ARS LEU2 vector YCplac111, and the rad30 strain carrying the rad30 HH568,572AA, the C552A, the CC552, 553AA, or the D570A mutant Rad30 encoding gene on the CEN ARS LEU2 plasmid. (B) UV-induced can1r mutations in the same strains as shown in panel A. For both panel A and panel B, each point on the curve represents the average results of at least three experiments. (C) Serial dilutions for determining the UV sensitivity of UBZ mutants. SC-leu plates containing equal volumes of serial 10-fold dilutions of exponentially growing yeast cells were UV irradiated and incubated in the dark at 30°C. For this experiment, the RAD30+ strain EMY747 was also transformed with the vector YCplac111 so that it would grow on SC-leu medium. All the other strains were the same as in panel A.

A mutation of the conserved aspartate in the -helix of the UBZ domain impairs Pol function. We also examined the effects of the D570A mutation on Pol function in promoting replication through UV-induced DNA lesions. This residue corresponds to D652 in human Pol, and nuclear magnetic resonance studies have indicated that this residue, along with many other conserved residues present in the -helix, mediates the binding of the UBZ domain to ubiquitin (6). Moreover, the D652A mutant protein does not localize to replication foci in UV-damaged human cells (5). As shown in Fig. 2, introduction of the D570A mutant rad30 gene carried on a CEN/ARS plasmid into the yeast rad30 strain did not restore wild-type levels of UV sensitivity or UV mutagenesis. The rad30 strain harboring the D570A mutant plasmid exhibited levels of UV sensitivity (Fig. 2A and C) and UV mutagenesis (Fig. 2B) that were intermediate between those seen with the wild-type strain and those seen with the rad30 strain. Although this observation supports the previously reported results obtained with the D570A mutation in that this mutation adversely affects Pol function, we found that the effect on UV sensitivity and UV mutagenesis is not as severe as was reported in the previous study (23). Regardless of these quantitative differences seen in the two studies, all the available evidence from both yeast and human Pol implies an important role for this conserved aspartate residue in Pol function.

Mutations in the UBZ domain do not affect functional interaction of Pol with Ub-PCNA. Based upon the observation that the TLS Pols such as human Pol or Pol are coimmunoprecipitated to a greater extent with monoubiquitinated PCNA than with nonubiquitinated PCNA (5, 21) and that the Ub binding domains present in the TLS Pols promote their interaction with ubiquitin, the inference has been drawn that the ubiquitin moiety attached to lysine 164 of PCNA via Rad6-Rad18 action in DNA-damaged cells serves as a receptor for the binding of TLS Pols to PCNA through their UBDs. This model, that the access to PCNA of TLS Pols is mediated via their binding to the Ub moiety on PCNA, makes several predictions. First, it suggests that Ub-PCNA would be stimulatory to synthesis by TLS Pols. Second, it suggests that mutations in the ubiquitin binding domain of TLS Pols would inactivate this stimulation. Third, and importantly, it suggests that mutations which impair the ubiquitin binding ability of the ubiquitin binding domain will have a biological effect on polymerase function. We have already shown that the third and most important prediction of the model is not supported by our observation that mutations in the C₂H₂ motif of the UBZ domain do not affect the biological function of Pol in TLS. We have previously shown that PCNA monoubiquitinated at its lysine 164 residue by Rad6-Rad18 is not any more effective in stimulating the activity of yeast Pol than unmodified PCNA (15), that mutations in the PIP domain of yeast Pol inactivate the physical and functional interaction of Pol with PCNA, and that they abrogate the biological role of Pol in promoting replication through UV-induced DNA lesions (11).

Even though Ub-PCNA stimulates synthesis by Pol to the same extent as unmodified PCNA, we have now examined whether mutations in the UBZ domain impair the binding of Pol to Ub-PCNA, purified as described previously (15), to a greater extent than that to PCNA. As shown in Fig. 3, we found that PCNA and Ub-PCNA are equally stimulatory to synthesis by Pol and that the C552A, CC552,553AA, HH568,572AA, and D570A mutant Pol proteins are all stimulated by PCNA or Ub-PCNA to the same degree as wild-type Pol. Mutations in the UBZ domain thus have no adverse effect on the DNA synthesis proficiency of Pol with PCNA or Ub-PCNA.

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FIG. 3. DNA polymerase activity of wild-type and mutant Pol proteins with PCNA or Ub-PCNA. The complete standard reaction mixture contained a 0.5 nM concentration of Pol, PCNA (50 ng), RFC (25 ng), RPA (200 ng), a mix of all four deoxynucleoside triphosphates (each at 100 µM), 100 µM ATP, and DNA substrate (10 nM). The level of Ub-PCNA added was the same as that of PCNA. Various combinations of PCNA or Ub-PCNA, RFC, and RPA were added to the reaction mixture as indicated. Reaction mixtures were incubated at 30°C for 10 min. The DNA substrate was generated by annealing the 44-nucleotide (nt) 5' 32P-labeled oligonucleotide primer to the 75-nucleotide-long template containing biotin at the ends, to which streptavidin was bound right before the polymerase reaction. Lane 1, DNA substrate.

Secondary structure determination for D570A mutant Pol by CD. Since, by contrast to mutations in the C₂H₂ motif, which have no effect on Pol biological function, the D570A mutation has an adverse effect on Pol function, we wanted to rule out the possibility that this effect resulted from a significant change in Pol conformation. For this reason, we determined the CD spectra of the wild-type and D570A mutant Pol proteins (Fig. 4). The CD spectra determined in the "far-UV" region (180 to 260 nM) indicate that the mutant protein retains about the same level of -helical structure (37%) as the wild-type protein (40%), which would suggest that the D570A mutation does not cause a major perturbation of Pol structure.

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FIG. 4. Far UV-CD spectra of wild-type and D570A mutant Pol. CD spectra for yeast Pol at pH 7.5 for the wild type and the D570A mutant between 180 nm and 260 nm are shown. Data represent values determined after solvent correction and after averaging each set (n = 5). []MRE values at 222 nm (where MRE represents molar residue ellipticity) for the wild type and the mutant are –12,966.71 and –10,792.29, respectively.

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Here we have examined the plausibility of the recently proposed model for the role of the Ub moiety on PCNA and of the UBD on the TLS Pol (5). This model posits that the access of TLS Pols to PCNA at the stalled replication fork is modulated by the binding of UBD to Ub on PCNA and that this PCNA binding mode is indispensable for the ability of TLS Pols to access PCNA. However, since the evidence that has been garnered in support of this model is based upon the phenotypic effects of a limited number of mutations in the UB binding domain of Pol or of other Pols (5, 24), here we have carried out a more thorough genetic and biochemical analysis of a number of mutations in the UBZ domain of yeast Pol.

Our observation that none of the mutations in the C₂H₂ motif have a significant effect on Pol function in promoting TLS though UV-induced DNA lesions, as judged by the lack of any effect on UV sensitivity or on UV mutagenesis, implies that the lack of zinc binding, which is a prerequisite for Ub binding by the -helix in the UBZ domain (6, 24), confers no significant defect upon the ability of Pol to carry out TLS. These findings obtained with yeast Pol are additionally supported by the observation that the H654A mutation in the C₂H₂ motif of the UBZ of human Pol, which has been shown to be defective in interactions with ubiquitin, confers only a small reduction in the localization of Pol to the replication foci in UV-damaged cells (24). Since the mutational inactivation of the zinc binding ability and of the consequent ubiquitin binding ability has no significant effect on the function of Pol in TLS, we are led to conclude that the binding of the Ub moiety on PCNA is not a necessary prerequisite for the ability of Pol to gain access to PCNA at the site of stalled replication.

By contrast to the lack of any significant effect of mutations in the C₂H₂ motif in yeast Pol, a mutation of the conserved D652 residue in the -helix portion of the UBZ in human Pol confers a defect in its localization to replication foci (5), and the corresponding D570A mutation in yeast Pol confers a considerable increase in UV sensitivity and UV mutability (reference 23 and our results). Since mutations in the C₂H₂ motif have no significant effect on Pol function, but a mutation in this highly conserved aspartate residue adversely affects Pol function in both yeast and humans, we presume that the conserved aspartate, and perhaps the -helix which contains this residue, contributes to Pol function in some other way that is not related to the Ub binding role of the UBZ. It is plausible that an -helical structure is still retained by the UBZ domain in the absence of the zinc binding that would occur in the C₂H₂ mutants and that this -helical form can contribute to the ability of Pol to physically interact with the proteins at the replication fork. That would suggest two separate roles for the UBZ domain: one role in ubiquitin binding, requiring the zinc binding C₂H₂ finger and the -helix, and the other role in mediating protein-protein interactions, requiring the -helical form adopted by the UBZ domain in the absence of zinc binding. Such a possibility of dual roles for the UBZ domain is supported by the observation that the UBA domains of proteins, required for ubiquitin binding, are also capable of mediating protein-protein interactions (3, 4).

UBDs can promote the monoubiquitination of proteins that contain them, and interestingly, UBDs of different types, including UBZ, UBA, UIM, UBM, and NFZ, can directly cooperate with the Ub-charged E2 enzymes to promote the monoubiquitination of the host protein in an E3-independent manner (17). Human Pol and Pol and yeast Pol have been shown to be monoubiquitinated in vivo, and mutations in the UBZ domain of Pol and the UBM domain of Pol inactivate this modification (5). Since monoubiquitinated Pol or Pol is no longer able to bind ubiquitin, the proposal has been made that monoubiquitination of these Pols inhibits their binding to Ub-PCNA and thus promotes their exit from PCNA (5). Such a regulatory role for polymerase ubiquitination would require that this modification be damage dependent and Rad6-Rad18 dependent and also that the UBD be indispensable for the TLS role of the polymerase. However, genetic studies with yeast Pol have indicated that Pol monoubiquitination occurs independently of DNA damage and does not require Rad6-Rad18 (23). This observation, coupled with our results showing that the C₂H₂ mutations confer no defect in Pol function, would suggest that the monoubiquitinated form of Pol or of the other TLS Pols does not provide for such a regulatory mechanism.

In summary, the absence of a significant effect of C₂H₂ mutations on Pol function and the observations that Ub-PCNA is no more stimulatory to DNA synthesis by Pol than unmodified PCNA and that mutations in the UBZ domain have no debilitating effect on the stimulation of Pol by PCNA regardless of whether it is ubiquitinated or not, whereas mutations in the PIP domain inactivate Pol function in vivo as well as the PCNA-dependent stimulation in vitro, all run counter to the proposal that the binding of ubiquitin on PCNA via the UBZ domain is indispensable for the ability of Pol to access PCNA at the stalled replication fork. Rather, in the context of the replication fork, the ubiquitin on PCNA could serve some other function. The observation that a linear fusion of ubiquitin with PCNA, which apparently can substitute for lysine 164-linked PCNA ubiquitination in promoting TLS, does not rescue the lethality of a pol30 mutation (23) might suggest that Ub-PCNA is inhibitory for normal replication because it destabilizes the PCNA binding ability of the replicative polymerase and/or of other PCNA-associated proteins. Whether the ubiquitin on PCNA alters the binding of the replicative polymerase or of some protein factor(s) which prevents the binding of Pol and of other TLS Pols to PCNA via their PIP motifs remains to be seen.

Although in the context of the stalled replication fork, the potential ability of Pol and of other TLS Pols to directly bind the Ub moiety on PCNA via their UBDs may be dispensable for their ability to access PCNA, we cannot exclude the possibility that the TLS Pols bind PCNA not only via their PIP motif (which is essential for their function in TLS) but also through their UBD to the Ub on PCNA (this PCNA binding mode, however, not being essential). Also, we can conceive of situations in which the binding of the Ub moiety on PCNA by the TLS Pols takes on an added significance. For example, TLS that occurs during a repair synthesis reaction, such as that on gapped plasmids or following the removal of a DNA lesion, is unlikely to involve the myriad of proteins that modulate and coordinate the function of the TLS polymerase with that of the replicative polymerases and other protein components at the fork. In the repair synthesis reaction, the targeting of Pol and of other TLS Pols to PCNA may well be affected by the Ub moiety on PCNA, as that could serve as the initial recognition signal in promoting the binding of the TLS Pol to PCNA. Thus, in a repair synthesis reaction opposite from a lesion, there may well be a bona fide polymerase exchange in which the Ub moiety on PCNA promotes the exit of the repair synthesis polymerase from PCNA and then facilitates the binding of the TLS polymerase to PCNA. However, during replication, mechanisms may exist that coordinate the functions of the replicative and TLS polymerases such that the direct binding of the Ub moiety on PCNA by the TLS polymerase becomes dispensable.

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

 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, 6.104 Blocker Medical Research Building, 301 University Blvd., Galveston, TX 77555-1061. Phone: (409) 747-8602. Fax: (409) 747-8608. E-mail: s.prakash{at}utmb.edu

Published ahead of print on 20 August 2007.

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Molecular and Cellular Biology, October 2007, p. 7266-7272, Vol. 27, No. 20
0270-7306/07/$08.00+0     doi:10.1128/MCB.01196-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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