A Single Domain in Human DNA Polymerase {iota} Mediates Interaction with PCNA: Implications for Translesion DNA Synthesis

DNA polymerases (Pols) of the Y family rescue stalled replicationforks by promoting replication through DNA lesions. Humans havefour Y family Pols, ${eta}$ , ${iota}$ , ${kappa}$ , and Rev1, of which Pols ${eta}$ , ${iota}$ , and ${kappa}$ havebeen shown to physically interact with proliferating cell nuclearantigen (PCNA) and be functionally stimulated by it. However,in sharp contrast to the large increase in processivity thatPCNA binding imparts to the replicative Pol, Pol ${delta}$ , the processivityof Y family Pols is not enhanced upon PCNA binding. Instead,PCNA binding improves the efficiency of nucleotide incorporationvia a reduction in the apparent K_m for the nucleotide. Herewe show that Pol ${iota}$ interacts with PCNA via only one of its conservedPCNA binding motifs, regardless of whether PCNA is bound toDNA or not. The mode of PCNA binding by Pol ${iota}$ is quite unlikethat in Pol ${delta}$ , where multisite interactions with PCNA providefor a very tight binding of the replicating Pol with PCNA. Wediscuss the implications of these observations for the accuracyof DNA synthesis during translesion synthesis and for the processof Pol exchange at the lesion site.

INTRODUCTION

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Introduction

Proliferating cell nuclear antigen (PCNA), a highly conserved,ring-shaped homotrimeric eukaryotic protein, forms a slidingclamp at the template-primer junction. PCNA is loaded onto theprimer-template junction in an ATP-dependent manner by a multiproteinclamp loader, replication factor C (RFC). After the loadingof PCNA, RFC stays on the DNA via interaction with replicationprotein A (RPA) bound to single-stranded DNA (1, 18, 37). Thebinding of the replicative DNA polymerase (Pol), Pol ${delta}$ , to PCNAendows it with a very high processivity (25, 30), and that presumablyis the essential function of PCNA in DNA replication.

In Saccharomyces cerevisiae, Pol ${delta}$ is comprised of three subunitsof 125, 55, and 40 kDa, encoded by the POL3, POL31, and POL32genes, respectively (6). While the Pol3 catalytic subunit andthe Pol31 subunit are highly conserved among eukaryotes, thePol32 subunit shows a high degree of divergence. The S. cerevisiaePol3, Pol31, and Pol32 subunits are the respective homologsof Schizosaccharomyces pombe Pol ${delta}$ subunits Pol3, Cdc1, and Cdc27.Whereas the Pol3 and Pol31 subunits and their counterparts areessential in both S. cerevisiae and S. pombe, the third subunit,Cdc27, is essential for completion of the S phase in S. pombe(22, 27), but its counterpart in S. cerevisiae, Pol32, is not.pol32 ${Delta}$ cells, however, grow poorly and exhibit DNA replicationdefects (6).

A series of genetic and biochemical observations with S. cerevisiaePol ${delta}$ have indicated that at least two separate domains on Pol ${delta}$ interact with at least two separate domains on PCNA, and furthermore,it has been suggested that, during replication, Pol ${delta}$ binds toat least two PCNA monomers (15). Overall, the various studieswith Pol ${delta}$ from S. cerevisiae, S. pombe, and humans have stronglyindicated that several distinct domains on Pol ${delta}$ interact withdifferent regions of PCNA, and these multiple interactions providethe high degree of processivity that PCNA binding imparts toPol ${delta}$ . Briefly, we review this evidence below.

A consensus PCNA binding motif, QXX(L/I)XXFF, is present atthe extreme C terminus of Pol32 in S. cerevisiae and also inits S. pombe and human counterparts Cdc27 and p66, respectively,and mutational inactivation of this domain in PCNA from S. cerevisiaeand S. pombe affects the processivity of Pol ${delta}$ (2, 15). In addition,biochemical studies with two mutant PCNAs from S. cerevisiae,pcna-79 and pcna-90, have shown that they both affect the processivityof Pol ${delta}$ (5, 15). In the pcna-79 mutant (I126A/L128A), the hydrophobicpocket in the interdomain connector loop (IDCL) of PCNA is impaired(5), and this mutant PCNA fails to interact with proteins viatheir consensus QXX(L/I)XXFF PCNA binding motif (8, 15, 32).The pcna-90 (P252A/K253A) mutant has mutational changes in thecarboxy-terminal tail of PCNA (5). Since the carboxy-terminaltail of PCNA does not interact with the consensus PCNA bindingmotif present in Pol32, the adverse effects of mutations inthis PCNA region on Pol ${delta}$ processivity (15) must derive from interactionsof PCNA with Pol ${delta}$ at a site different from the IDCL interactingdomain of Pol32. In keeping with this idea, at least two PCNAbinding sites have been identified in the p125 catalytic subunitof human Pol ${delta}$ : one of these is contained in the N2 region towardthe amino terminus (38), and the other is in the succeedingN4 region (36). The latter sequence is characterized by thepresence of a highly conserved KA motif. The association ofPol ${delta}$ with PCNA thus would be considerably strengthened by thesemultisite interactions.

The Y family DNA Pols, such as Pol ${eta}$ , Pol ${iota}$ , and Pol ${kappa}$ , promote replicationthrough distorting DNA lesions, but they replicate DNA witha low fidelity and low processivity (24). Although all thesePols interact with PCNA, physically as well as functionally,binding to PCNA does not improve their processivity (9-11, 13).For example, PCNA, when loaded onto DNA by RFC in the presenceof RPA, stimulates the DNA synthetic activity of both yeastand human Pol ${eta}$ approximately 10- to 15-fold, but the processivityin the presence of these protein factors remains the same asin their absence, at about three or four nucleotides per DNAbinding event (9, 11). Instead, the increase in the efficiencyof nucleotide incorporation is achieved primarily by a reductionin the apparent K_m for the nucleotide (9, 11). PCNA, in thepresence of RFC and RPA, also greatly stimulates the DNA syntheticactivity of Pol ${iota}$ and Pol ${kappa}$ , and this again is achieved by a decreasein the apparent K_m for the nucleotide, whereas the processivityremains unaffected (10, 13).

Since PCNA binding does not improve the processivity of Y familyDNA Pols, these Pols must differ in their mode of PCNA bindingfrom Pol ${delta}$ . As multisite interactions would provide for the strongassociation of Pol ${delta}$ with PCNA and the ensuing large increasein Pol ${delta}$ processivity, we have examined the possibility that thelack of PCNA stimulation of the processivity of Y family Polsderives from their binding to PCNA rather weakly. The presenceof multiple putative PCNA binding motifs in Pol ${iota}$ prompted usto determine whether this Pol made multisite contacts with PCNAor whether only one of the sites was involved. Here we showthat Pol ${iota}$ interacts with PCNA via only one of these sites anddiscuss the implications of this observation for translesionDNA synthesis (TLS) by Pol ${iota}$ as well as other Y family Pols.

MATERIALS AND METHODS

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Materials and Methods

Proteins. Human PCNA, RFC, and RPA were purified as described previously(3, 7, 20). Six-His-tagged human PCNA used for the interactionstudies was overexpressed in Escherichia coli and purified asdescribed previously (19). Wild-type and mutant human Pol ${iota}$ proteinsin fusion with glutathione S-transferase (GST) were expressedin the yeast strain BJ5464 and bound to a glutathione-Sepharose4B column as described previously (17). To purify Pol ${iota}$ , the GST-Pol ${iota}$ -containingbeads were incubated overnight at 4°C with PreScission protease,which cleaves the GST-Pol ${iota}$ fusion protein at 7 amino acids amino-terminalfrom the first methionine of Pol ${iota}$ , in buffer E containing 50mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM dithiothreitol, 0.01%NP-40, and 10% glycerol. The purified Pol ${iota}$ was concentrated witha Microcon 30 (Amicon) concentrator, aliquoted, and frozen at–70°C.

Pull-down assay of Pol ${iota}$ and PCNA complexes. To constitute complexes, Pol ${iota}$ (2 µg) was mixed with His₆-hPCNA(4 µg) in buffer E in 30-µl samples and incubatedfor 30 min at 4°C followed by 10 min at 25°C. To 20µl of these samples, 10 µl of nickel-nitrilotriaceticacid (Ni-NTA; Qiagen) beads was added to bind His₆-PCNA andHis₆-PCNA-Pol ${iota}$ complex, and the samples were further incubatedwith constant rocking for 30 min at 4°C followed by theelution of bound proteins with 500 mM imidazole-containing bufferE. The samples containing the protein mixture before the additionof affinity beads, the flowthrough plus washing fractions, andthe eluted proteins were precipitated with 5% trichloroaceticacid and separated on a sodium dodecyl sulfate-12% polyacrylamidegel followed by Coomassie blue R-250 staining.

Gel filtration analysis of Pol ${iota}$ -PCNA interaction. Gel filtration of Pol ${iota}$ , PCNA, and their complexes was performedat 4°C with a Superdex 200 PC 3.2/30 column (Amersham PharmaciaBiotech, Piscataway, N.J.) equilibrated with buffer I, containing20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol,0.01% NP-40, and 10% glycerol. To constitute complexes, Pol ${iota}$ (4 µg), His₆-PCNA (5 µg), or the mixture of theseproteins was incubated in 25 µl of buffer I for 60 minat 4°C followed by incubation for 10 min at 25°C. Theprotein mixture was then gel filtered at a 20-µl/min flowrate at 4°C. Fractions were collected and analyzed on 10%sodium dodecyl sulfate-polyacrylamide gels stained with Coomassieblue R-250.

DNA Pol assays. DNA substrate was generated by annealing a 75-nucleotide oligomertemplate, 5'-biotin-AGCAAGTCACCAATGTCTAAGAGTTCGTATTATGCCTACACTGGAGTACCGGAGCATCGTCGTGACTGGGAAAAC-biotin-3',which contained one biotin molecule attached at each end, tothe 5' ³²P-labeled oligonucleotide primer N4577, 5'-GTTTTCCCAGTCACGACGATGCTCCGGTA-3'.To bind streptavidin to biotin present at the ends of the linearDNA substrates, these primer-templates (2.5 pmol) were preincubatedwith streptavidin (5 µg) in 25 µl of DNA Pol bufferwhich contained no MgCl₂, for 10 min at 30°C, before theiraddition to the DNA Pol reaction mixtures. The standard DNAPol 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 of bovine serum albumin/ml; 500 µMATP; and 100 µM (each) dGTP, dATP, dTTP, and dCTP. Asindicated in the figure legends, mutant and wild-type Pol ${iota}$ proteins(1 nM) were mixed with PCNA (50 ng), RFC (5 ng), and RPA (50ng) and the linear primer-template DNA (10 nM). Assay mixtureswere assembled on ice and incubated at 37°C for 10 min,and assays were stopped by the addition of loading buffer (40µl) containing 20 mM EDTA, 95% formamide, 0.3% bromphenolblue, and 0.3% cyanol blue. The reaction products were resolvedon 10% polyacrylamide gels containing 8 M urea. Visualizationof the results was done using a Molecular Dynamics Storm PhosphoImagerand ImageQuant software.

Two-hybrid analyses. The HF7c yeast strain was transformed with the Pol ${iota}$ and PCNAfusion constructs by the lithium acetate method. Transformantsharboring both the GAL4 DNA binding domain (BD) and the GAL4activation domain (AD) fusion constructs were grown on syntheticcomplete medium, lacking leucine and tryptophan. ß-Galactosidaseactivity was examined to determine the interaction between Pol ${iota}$ and PCNA, as described in the Clontech yeast protocols handbook(PT3024-1, chapter VI). ß-Galactosidase activitieswere quantitated using O-nitrophenyl-ß-D-galactopyranosideas substrate. Experiments were performed at least three timesin triplicate samples.

RESULTS

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Results

Putative PCNA binding motifs in Pol ${iota}$ . A consensus PCNA binding motif QXX(I, L, or M)XXF(F or Y), alsoreferred to as the PCNA interaction protein box (PIP box), isfound in many proteins involved in DNA metabolic processes,such as DNA replication and repair, DNA methylation, and chromatinassembly (4, 21, 28). Structural and mutational studies haveindicated the involvement of the conserved hydrophobic residueswithin this motif in interaction with PCNA (23, 26, 33). Pol ${iota}$ has two putative PIP boxes, KKGLIDYY and SRGVLSFF, which arelocated toward the C terminus after the five conserved Pol domainsand the polymerase-associated domain (PAD), and they encompassresidues 420 to 427 (PIP1 box) and 540 to 547 (PIP2 box), respectively,of the 715-amino-acid protein (Fig. 1A).

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FIG. 1. Putative PCNA binding motifs of human DNA Pol ${iota}$ . (A) Amino acids 418 to 430 and 538 to 550 of human Pol ${iota}$ were aligned with the PIP motif identified in various PCNA binding proteins and shown to bind the IDCL of PCNA. The highly conserved residues are indicated in boldface. Hs, Homo sapiens; Sc, S. cerevisiae. (B) Residues 412 to 424 of human Pol ${iota}$ were aligned with the PCNA binding KA box of Pol ${delta}$ , which is present also in many other PCNA binding proteins.

Recently, another conserved PCNA binding sequence characterizedby a KA motif has been identified in the catalytic subunit ofhuman Pol ${delta}$ (36), and a KA motif is present also in Pol ${iota}$ betweenresidues 412 and 424 (Fig. 1B). The presence of three potentialPCNA binding motifs in Pol ${iota}$ raised the possibility that Pol ${iota}$ bindsto PCNA at more than one site.

Generation of mutations in the putative PCNA binding motifs of Pol ${iota}$ . To test if the three PCNA binding motifs of Pol ${iota}$ have roles inthe interaction with PCNA, we generated several Pol ${iota}$ mutations(Fig. 2A). In the Pol ${iota}$ (1-420) deletion mutant, both the PIP1and PIP2 motifs are deleted, and only the KA motif is retained.In the Pol ${iota}$ (1-451) deletion mutant, the PIP2 motif is deleted,but the KA and PIP1 motifs are retained. In the Pol ${iota}$ (1-451) deletionmutant, we also changed the conserved lysine 414 residue ofthe KA motif to alanine, resulting in the Pol ${iota}$ (1-451)K414A mutant,and changed the two tyrosines at positions 426 and 427 in thePIP1 motif to alanine, resulting in Pol ${iota}$ (1-451)YY426,427,AA.Additionally, we mutated the YY residues of PIP1 and the FFresidues of PIP2 in full-length Pol ${iota}$ , generating Pol ${iota}$ YY426,427,AA,Pol ${iota}$ FF546,547,AA, and Pol ${iota}$ YY426,427,AA,FF546,547,AA.

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FIG. 2. Identification of a PCNA binding motif in Pol ${iota}$ (A) Mutations made in the putative PCNA binding motifs of human Pol ${iota}$ . In the schematic representation of Pol ${iota}$ , the boxes indicate the five conserved motifs characteristic of Y family DNA Pols. The location and sequences of the three putative PCNA binding motifs, KA, PIP1, and PIP2, present in Pol ${iota}$ are indicated. Arrows indicate the amino acid residues of PCNA binding motifs which were changed to alanine in the various mutant Pol ${iota}$ proteins. In the Pol ${iota}$ (1-420) deletion mutant protein, the two PIP motifs have been deleted, which leaves only the KA motif, while the Pol ${iota}$ (1-451) deletion mutant protein contains the KA box as well as the PIP1 motif. In the Pol ${iota}$ (1-451) deletion mutant, the K residue at 414 and the Y residues at 426 and 427 were changed to alanine, resulting in Pol ${iota}$ (1-451)K414A and Pol ${iota}$ (1-451)YY426,427,AA mutant proteins, respectively. (B) The PIP1 motif mediates complex formation of Pol ${iota}$ with PCNA. As indicated on the top, wild-type and mutant Pol ${iota}$ proteins (2 µg each) were mixed with His₆-PCNA (4 µg). After incubation, samples were bound to Ni-NTA beads followed by washing and elution of the bound proteins with imidazole-containing buffer. Aliquots of each sample before addition to the beads (L), the flowthrough plus wash (F), and the eluted proteins (E) were analyzed on a sodium dodecyl sulfate-12% polyacrylamide gel stained with Coomassie blue. The positions of Pol ${iota}$ and His₆-PCNA are shown on the left. (C) Pol ${iota}$ (1-451) mutant protein is able to form a stable complex with PCNA. The mixture of Pol ${iota}$ (1-451) (4 µg) and His₆-PCNA (5 µg) (I) and the mixture of Pol ${iota}$ (1-451)YY426,427,AA (4 µg) and His₆-PCNA (5 µg) (II) were gel filtered after incubation in a 30-µl volume of reaction mixture for 60 min at 4°C followed by 10 min at 25°C. The elution positions of Pol ${iota}$ (1-451) in complex with PCNA, PCNA homotrimer, and the free Pol ${iota}$ (1-451) are indicated on top. His₆-PCNA and Pol ${iota}$ mutant proteins are identified on the right.

PIP1 motif of Pol ${iota}$ mediates physical interaction with PCNA. To examine the physical interactions of mutant Pol ${iota}$ proteinswith PCNA, the Pol ${iota}$ proteins were incubated with His₆-PCNA anda pull-down assay was carried out using the Ni-NTA affinitybeads (Fig. 2B). Because Pol ${iota}$ alone cannot bind to Ni-NTA, Pol ${iota}$ could be pulled down only if it interacted with PCNA.

When His₆-PCNA is bound to the Ni-NTA beads, a large proportionof the wild-type Pol ${iota}$ is retained on the beads via PCNA (Fig.2B, lanes 1 to 3). The Pol ${iota}$ (1-420) mutant protein, however, isimpaired in interaction with PCNA, indicating that the KA motifalone is not able to mediate the binding of Pol ${iota}$ to PCNA (Fig.2B, lanes 4 to 6). The Pol ${iota}$ (1-451) mutant protein, on the otherhand, interacts with PCNA as well as the wild-type Pol ${iota}$ , whichsuggests a role for the PIP1 motif in the binding of Pol ${iota}$ toPCNA. To provide evidence that the conserved PIP1 motif is,in fact, involved in PCNA binding, we tested the ability ofthe Pol ${iota}$ (1-451)YY426,427,AA mutant protein to bind PCNA. As shownin Fig. 2B, lanes 13 to 15, this mutational alteration of thePIP1 motif in Pol ${iota}$ (1-451) inactivated the interaction with PCNA.The Pol ${iota}$ (1-451) K414A mutation, however, did not affect PCNAbinding (Fig. 2B, lanes 10 to 12). These results indicate thatthe PIP1 PCNA binding domain in Pol ${iota}$ is sufficient to mediatethe physical interaction of Pol ${iota}$ with PCNA.

To examine further whether the PIP1 domain of Pol ${iota}$ alone is ableto mediate a stable interaction with PCNA, we analyzed the complexesof mutant Pol ${iota}$ proteins and PCNA by gel filtration. Pol ${iota}$ (1-451)or Pol ${iota}$ (1-451)YY426,427,AA proteins were mixed with PCNA in almosta 1:1 molar ratio, and after incubation, their interaction wasexamined by gel filtration, a nonequilibrium technique whereinonly rather stable protein complexes survive. While PCNA aloneeluted mainly in fractions 6 and 7 and Pol ${iota}$ (1-451) alone elutedaround fractions 9 and 10 (data not shown; also Fig. 2C, panelII), when PCNA was preincubated with Pol ${iota}$ (1-451), both of themtogether eluted earlier around fractions 5 and 6 (Fig. 2C, panelI). This shift in the elution position of both proteins indicatesthe formation of a complex between Pol ${iota}$ (1-451) and PCNA. By contrast,Pol ${iota}$ (1-451)YY426,427,AA and PCNA did not elute together afterpreincubation (Fig. 2C, panel II). These observations supportthe requirement of the PIP1 motif in Pol ${iota}$ (1-451) in PCNA binding.

PIP1 motif of Pol ${iota}$ mediates the functional interaction with PCNA. Many PCNA binding proteins interact with PCNA via at least twodifferent domains; one of these is more important when PCNAis in solution, and the other becomes essential when PCNA isloaded onto the DNA (8, 15, 32). To determine if the bindingof Pol ${iota}$ to PCNA on DNA requires the PIP1 motif or some othermotif, we compared the effects of PCNA on the DNA syntheticactivity of the wild-type and mutant Pol ${iota}$ proteins. First, weloaded PCNA by RFC onto an RPA-coated singly primed 75-nucleotide-longtemplate DNA containing biotin-streptavidin at both ends, whichprevented the PCNA from sliding off. DNA synthesis was theninitiated by adding the wild-type or mutant Pol ${iota}$ protein. TheDNA synthetic activity of wild-type Pol ${iota}$ is greatly enhancedupon the addition of PCNA, RFC, and RPA (Fig. 3, compare lanes1 and 2), and PCNA also stimulated the synthetic activity ofPol ${iota}$ (1-451) (Fig. 3, compare lanes 5 and 6) and Pol ${iota}$ (1-451)K414A(Fig. 3, compare lanes 7 and 8) mutant enzymes, indicating thatthe KA motif has no role in the functional interaction of Pol ${iota}$ with PCNA. By contrast, Pol ${iota}$ (1-420) (Fig. 3, compare lanes 3and 4) and Pol ${iota}$ (1-451)YY426,427,AA (Fig. 3, compare lanes 9 and10) mutant proteins lost their ability to be stimulated by PCNA.The PIP1 motif of Pol ${iota}$ thus is sufficient to mediate the physicaland functional interactions of Pol ${iota}$ with PCNA.

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FIG. 3. The PIP1 motif of Pol ${iota}$ is sufficient to mediate the stimulatory effect of PCNA on the DNA synthetic activity of Pol ${iota}$ . The reaction mixtures contained wild-type or mutant Pol ${iota}$ proteins (1 nM each), along with singly primed 75-nucleotide-long DNA substrate (10 nM) in which the 29-nucleotide primer was ³²P labeled at its 5' end, and the template contained biotin-streptavidin complex at both ends to prevent the PCNA sliding off the DNA and all four deoxynucleotides (100 µM each), in the presence or absence of PCNA (50 ng), RFC (5 ng), and RPA (50 ng). After incubation for 10 min at 37°C, samples were quenched and run on a 10% polyacrylamide gel containing 8 M urea followed by PhosphorImager analysis.

PIP2 motif of Pol ${iota}$ has no role in physical or functional interactions with PCNA. Our observations with Pol ${iota}$ (1-451) protein indicating that thePIP1 motif of Pol ${iota}$ is required for the physical and functionalinteractions of Pol ${iota}$ with PCNA do not exclude the possibilitythat the PIP2 motif also influences the PCNA binding of Pol ${iota}$ .For this reason, we examined the interactions of full-lengthPol ${iota}$ proteins carrying mutations in the PIP1 or PIP2 motifs withPCNA. From pull-down analysis with His₆-PCNA on Ni-NTA beads,we found that, whereas mutational inactivation of the PIP2 motifof Pol ${iota}$ has no effect on the physical interaction of Pol ${iota}$ withPCNA (Fig. 4A, lanes 4 to 6), mutational inactivation of thePIP1 motif in full-length Pol ${iota}$ impairs the interaction of Pol ${iota}$ with PCNA (Fig. 4A, lanes 7 to 9).

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FIG. 4. Only the PIP1 PCNA binding domain of Pol ${iota}$ mediates interactions with PCNA. (A) Pull-down analysis of complex formation between Pol ${iota}$ proteins and PCNA. Wild-type or mutant Pol ${iota}$ (2 µg each) was incubated with His₆-PCNA (4 µg) and pulled down on Ni-NTA beads. Aliquots of each sample before addition to the beads (L), the flowthrough plus wash (F) and the eluted proteins (E) were analyzed on a polyacrylamide gel. (B) Wild-type or mutant Pol ${iota}$ (1 nM each) was incubated with the DNA substrate (10 nM) in the presence of each of four deoxynucleoside triphosphates under standard reaction conditions. As indicated, the reactions were carried out in the presence or absence of PCNA (50 ng), RFC (5 ng), and RPA (50 ng).

Next, we examined the effect of addition of PCNA, RFC, and RPAon the DNA synthetic activity of full-length Pol ${iota}$ proteins carryingmutations in the PIP1 or PIP2 motif (Fig. 4B). While the additionof PCNA, RFC, or RPA alone did not enhance the DNA syntheticactivity of the wild-type or mutant Pol ${iota}$ proteins (Fig. 4B, comparelanes 1, 9 and 17 to lanes 6 to 8, 14 to 16, and 22 to 24, respectively),robust stimulation of DNA synthesis was observed with the Pol ${iota}$ (1-715)FF546,547,AAprotein upon the addition of PCNA, RFC, and RPA or only PCNAand RFC (Fig. 4B, compare lanes 9, 11, and 12), and the degreeof stimulation was the same as for the wild-type Pol ${iota}$ (1-715)protein (Fig. 4B, lanes 1, 3, and 4). DNA synthesis by the Pol ${iota}$ (1-715)YY426,427,AAmutant, however, was not stimulated upon the addition of PCNA,RFC, and RPA (Fig. 4B, compare lanes 17, 19, and 20). Thus,the presence of the C-terminal PCNA binding motif, PIP2, inthe complete Pol ${iota}$ protein with the YY426,427,AA mutation doesnot enable Pol ${iota}$ to bind PCNA or be stimulated by it, and conversely,the mutational inactivation of the PIP2 domain has no adverseeffect on the physical or functional interactions of Pol ${iota}$ withPCNA. From these results, we conclude that the PIP2 domain hasno role in the binding of Pol ${iota}$ with PCNA, whereas the PIP1 domainis essential for the physical as well as functional interactionsof Pol ${iota}$ with PCNA.

Interaction of Pol ${iota}$ with PCNA—two-hybrid analysis. We used the yeast two-hybrid system to examine the interactionof Pol ${iota}$ with PCNA in vivo. In one of the plasmids, the GAL4 DNABD was fused with either the complete wild-type RAD30B Pol ${iota}$ -encodinggene or the mutant rad30B A⁵⁴⁶-A⁵⁴⁷ and rad30B A⁴²⁶-A⁴²⁷-A⁵⁴⁶-A⁵⁴⁷genes, and in the other plasmid, the GAL4 AD was fused withhuman PCNA. The HF7c yeast reporter strain harboring the GAL4-ADplasmid was transformed with one of the GAL4-BD plasmids. Interactionof the wild-type and mutant Pol ${iota}$ proteins with PCNA in thesetransformants was analyzed by a ß-galactosidase liquidassay, and the results are summarized in Table 1. Compared tothe low level of ß-galactosidase activity measuredwith GAL4-BD and the GAL4-AD-PCNA plasmids, the wild-type GAL4-BDPol ${iota}$ protein showed strong interaction with PCNA bound to GAL4-AD,resulting in 38-fold-higher ß-galactosidase activity.The A⁵⁴⁶-A⁵⁴⁷ point mutations in the PIP2 motif of Pol ${iota}$ did notaffect the interaction of Pol ${iota}$ and PCNA. The additional inactivationof the PIP1 motif, however, as in Pol ${iota}$ A⁴²⁶-A⁴²⁷-A⁵⁴⁶-A⁵⁴⁷, completelyabolished the interaction of Pol ${iota}$ with PCNA, yielding ß-galactosidaseactivity similar to that in the control. These results providefurther evidence that the interaction of Pol ${iota}$ with PCNA occursvia the PIP1 motif of Pol ${iota}$ .

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TABLE 1. Interaction of Pol ${iota}$ with PCNA in the yeast two-hybrid system

DISCUSSION

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Discussion

Pol ${iota}$ contains three potential PCNA binding motifs, the KA motif,and the PIP1 and PIP2 motifs; however, only one of these, PIP1,mediates the physical and functional interactions of Pol ${iota}$ withPCNA. We consider it very unlikely that Pol ${iota}$ could also interactwith PCNA via some other domain, because the PIP1 domain followsright after the PAD. The region of Pol ${iota}$ N-terminal to PAD containsthe highly conserved motifs I to V, characteristic of Y familyPols, and these are all essential for DNA Pol activity togetherwith the PAD. The PIP1 domain of Pol ${iota}$ , KKGLIDYY, resembles theconserved PCNA binding motif that has been identified in a numberof proteins and shown to interact with the IDCL region of PCNA.

The observation that the PIP1 domain of Pol ${iota}$ is both necessaryand sufficient for the interaction of Pol ${iota}$ with PCNA in the absenceof DNA as well as when PCNA is bound to the DNA substrate atthe template-primer junction makes a clear distinction betweenthe mode of PCNA binding by Pol ${iota}$ , a TLS Pol, and Pol ${delta}$ , a replicativePol. Thus, in contrast to Pol ${delta}$ , where distinct domains in differentsubunits mediate its interactions with PCNA (2, 15, 36, 38),contributing to the tight binding and the concomitant increasein processivity of Pol ${delta}$ , Pol ${iota}$ binds PCNA at only one site—theIDCL region. In contrast, and as indicated from biochemicalanalysis of PCNA mutants of S. cerevisiae, Pol ${delta}$ binds DNA-boundPCNA in at least two regions, the IDCL region and the C-terminalregion (15).

The mode of PCNA binding by Pol ${iota}$ differs also from that in Fen1,a 5' $->$ 3' nuclease that functions in the removal of RNA primersduring Okazaki fragment maturation, and Apn2, a nuclease whichfunctions in base excision repair (8, 32). Whereas in the absenceof DNA both these nucleases bind PCNA in the IDCL region, whenPCNA is bound to the DNA substrate, they additionally requireits C-terminal region for enforcing productive binding. Suchan inference is supported from the effects that mutations inthe IDCL and C-terminal regions of PCNA have upon the Fen1 andApn2 binding of PCNA (8, 32): while in the absence of DNA mutationsin the IDCL region impair Fen1 and Apn2 binding to PCNA andmutations in the C-terminal region have little effect, in thepresence of DNA mutations in both the IDCL and C-terminal regionsaffect their binding to PCNA, but mutations in the C-terminalregion have a more profound effect than do mutations in theIDCL region.

The ability of Y family Pols to replicate through DNA lesionsimplies that they are not as sensitive to geometric distortionsof DNA as are the replicative Pols, which are unable to replicatethrough DNA lesions. As a consequence, the Y family DNA Polssynthesize DNA with a low fidelity, the fidelity of Pol ${iota}$ beingparticularly poor (24). In contrast to almost all other DNAPols, Pol ${iota}$ incorporates nucleotides opposite the four templatebases with very different efficiencies (k_cat/K_m) and fidelities,and opposite template T, it even misincorporates a G approximately10-fold better than an A (10, 16, 31, 34, 39). Also, Pol ${iota}$ synthesizesDNA with a very low processivity, regardless of whether it isbound to PCNA. Under conditions of DNA synthesis, where Pol ${iota}$ was allowed to bind the DNA substrate only once, even with PCNApresent Pol ${iota}$ incorporated only one nucleotide before dissociationfrom the DNA (10). Since Pol ${iota}$ functions primarily at the nucleotideincorporation step of lesion bypass, wherein it incorporatesnucleotides opposite from highly distorting lesions, with thesubsequent extension step being performed by another TLS Pol(16, 35), Pol ${iota}$ 's role in TLS would then mostly be limited tothe incorporation of a single nucleotide. Therefore, the verylow processivity of PCNA-bound Pol ${iota}$ is in accord with its rolein lesion bypass; moreover, this attribute would contributeto keeping the incidence of inadvertent nucleotide misincorporationsat undamaged sites low.

Our previous studies indicating that the binding of yeast andhuman Pol ${eta}$ to DNA-bound PCNA or to PCNA in the absence of DNAis also mediated by a consensus IDCL binding motif present atthe C terminus of these proteins (9, 11) support the idea thatall Y family Pols resemble one another in their manner of PCNAbinding. Although the mutational studies for the identificationof PCNA binding domains in Pol ${eta}$ were not as exhaustive as thosethat we have done for Pol ${iota}$ , the fact that mutations in the IDCLbinding motif in both yeast and human Pol ${eta}$ inactivated theirinteractions with PCNA in both the absence and presence of DNAimplies that this PCNA binding region of Pol ${eta}$ makes a paramountcontribution to interactions with PCNA in both situations. Insummary, then, we suggest that Y family Pols differ strikinglyfrom Pol ${delta}$ and from other PCNA binding proteins, such as Fen1and Apn2, as they limit their interaction with DNA-bound PCNAonly to the IDCL region, and that contributes to their low processivityeven with PCNA.

The ability of Pol ${iota}$ and other Y family Pols to contact PCNA atonly the IDCL site could have important consequences for Polexchange at the lesion site. By virtue of its ability to contactPCNA at many sites, Pol ${delta}$ would remain stably bound to PCNA whenreplicating undamaged DNA (Fig. 5A). However, upon encounteringa DNA lesion, Pol ${delta}$ would stall, and that presumably activatesthe Rad6-Rad18-dependent ubiquitylation of PCNA (14), resultingin the displacement of Pol ${delta}$ from the template-primer junction(Fig. 5B). Importantly, however, because of its multisite bindingto PCNA, we expect Pol ${delta}$ to still remain bound to PCNA. Furthermore,the ubiquitylation of PCNA at the lysine 164 residue could beimportant for exposing the IDCL region to allow access of Pol ${iota}$ and other Y family Pols to PCNA (12, 14, 29) (Fig. 5B). However,because Pol ${iota}$ and other Y family Pols would be loosely anchoredto PCNA at only one site, they would dissociate from DNA soonafter their role in lesion bypass has been accomplished, whereuponPol ${delta}$ would regain access to the template-primer junction.

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FIG. 5. Model for the binding of PCNA by Pol ${delta}$ versus Pol ${iota}$ and for Pol exchange at the lesion site. (A) Binding of Pol ${delta}$ and Pol ${iota}$ on or off DNA. In the absence of DNA, both Pols could bind to PCNA via contacts with the IDCL region. On DNA, Pol ${delta}$ , via its multiple subunits, interacts with PCNA at multiple sites, whereas Pol ${iota}$ contacts only the IDCL region of PCNA. Although Pol ${delta}$ could also be bound to different PCNA monomers, Pol ${delta}$ binding to only one monomer is shown. K164 in PCNA is the site of ubiquitin (Ub) attachment by the Rad6-Rad18 enzyme complex. (B) Pol exchange at the lesion site. Rad6-Rad18-dependent PCNA ubiquitylation destabilizes the interactions of Pol ${delta}$ with PCNA, resulting in the displacement of Pol ${delta}$ from the primer end and allowing for the access of Pol ${iota}$ to the IDCL region of PCNA. Pol ${delta}$ presumably still remains bound to PCNA through multisite contacts (data not shown) and would regain access to the primer junction soon after the completion of lesion bypass and the concomitant exit of Pol ${iota}$ from the replication ensemble.

ACKNOWLEDGMENTS

This work was supported by National Institute of EnvironmentalHealth Sciences grant ES012411, the Wellcome Trust InternationalSenior Research Fellowship, the Hungarian Science Foundationgrant T043354, and the EMBO Restart Fellowship.

FOOTNOTES

* Corresponding author. Mailing address: Sealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Medical Research Building, 11th and Mechanic Streets, Galveston, TX 77555-1061. Phone: (409) 747-8602. Fax: (409) 747-8608. E-mail: s.prakash{at}utmb.edu.

REFERENCES

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