Stimulation of DNA Synthesis Activity of Human DNA Polymerase {kappa} by PCNA

Humans have three DNA polymerases, Pol ${eta}$ , Pol ${kappa}$ , and Pol ${iota}$ , whichare able to promote replication through DNA lesions. However,the mechanism by which these DNA polymerases are targeted tothe replication machinery stalled at a lesion site has remainedunknown. Here, we provide evidence for the physical interactionof human Pol ${kappa}$ (hPol ${kappa}$ ) with proliferating cell nuclear antigen(PCNA) and show that PCNA, replication factor C (RFC), and replicationprotein A (RPA) act cooperatively to stimulate the DNA synthesisactivity of hPol ${kappa}$ . The processivity of hPol ${kappa}$ , however, is notsignificantly increased in the presence of these protein factors.The efficiency (V_max/K_m) of correct nucleotide incorporationby hPol ${kappa}$ is enhanced $~$ 50- to 200-fold in the presence of PCNA,RFC, and RPA, and this increase in efficiency is achieved bya reduction in the apparent K_m for the nucleotide. Althoughin the presence of these protein factors, the efficiency ofthe insertion of an A nucleotide opposite an abasic site isincreased $~$ 40-fold, this reaction still remains quite inefficient;thus, it is unlikely that hPol ${kappa}$ would bypass an abasic site byinserting a nucleotide opposite the site.

INTRODUCTION

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Introduction

In both prokaryotes and eukaryotes, DNA polymerases belongingto the UmuC/Rad30/DinB family promote replication through DNAlesions (6, 16). In eukaryotes, DNA polymerase ${eta}$ (Pol ${eta}$ ) functionsin the error-free replication of UV-damaged DNA, and both yeastPol ${eta}$ and human Pol ${eta}$ (hPol ${eta}$ ) replicate through a cis-syn thymine-thymine(T-T) dimer with the same efficiency and accuracy with whichthey replicate through undamaged T’s (13, 17, 31). Geneticstudies of yeast have also implicated Pol ${eta}$ in the error-freebypass of cyclobutane dimers formed at 5'-TC-3' and 5'-CC-3'sites (32), and Pol ${eta}$ efficiently replicates through many otherDNA lesions as well (9, 11). Mutations in Pol ${eta}$ in humans causea cancer-prone syndrome, the variant form of xeroderma pigmentosum(12, 22).

In addition to Pol ${eta}$ , humans contain two other related polymerases,Pol ${iota}$ and Pol ${kappa}$ (14, 15, 25, 30). Unlike Pol ${eta}$ , Pol ${iota}$ does not bypassa cis-syn T-T dimer and it does not even insert a nucleotideopposite the 3' T of the dimer (15). A role of Pol ${iota}$ in lesionbypass, however, is indicated by its ability to incorporatenucleotides opposite the 3' T of the (6-4) T-T photoproductand opposite abasic sites (15). Subsequently, Pol ${zeta}$ extends fromthe nucleotide inserted by Pol ${iota}$ , thus completing the bypass process(15).

In contrast to Pol ${eta}$ and Pol ${iota}$ , which exist only in eukaryotes,the DinB protein and its homologs are found in both prokaryotesand eukaryotes (6, 16). In Escherichia coli, dinB-encoded PolIV shows little ability to bypass a cis-syn T-T dimer, a (6-4)T-T photoproduct, or an abasic site (29). However, genetic studieshave implicated Pol IV in the mutagenic replication of undamagedDNA, as deletion of the dinB gene in E. coli reduces the ratesof frameshift and base substitution mutations. This effect ofdinB in generating spontaneous mutations becomes more evidentwhen the replicative polymerase has been partially disabledand the mismatch repair system has been inactivated (27). Also,overexpression of dinB in E. coli increases the rate of singlebase deletions in a run of guanines (20).

The human DINB1-encoded Pol ${kappa}$ (previously called Pol ${theta}$ by us) isunable to bypass a cis-syn T-T dimer, a (6-4) T-T photoproduct,or an abasic site (14). However, Pol ${kappa}$ can replicate through aguanine-AAF (N-2-acetyl-aminofluorine) adduct, and it may contributeto the bypass of other, as yet unidentified, DNA lesions aswell (25). Transient expression of the mouse DINB1 gene in culturedmouse cells leads to an increase in the frequency of base substitutionand frameshift mutations (23), which suggests a role for Pol ${kappa}$ in the mutagenic replication of undamaged DNA. Thus, in bothprokaryotes and eukaryotes, DinB contributes to spontaneousmutagenesis.

Although Pol ${kappa}$ would need to be recruited to the stalled replicationfork to perform its function in translesion synthesis, or topromote replication through certain sites in undamaged DNA,the manner by which this polymerase gains access to the replicationmachinery is not known. Here, we examine if interaction withproliferating cell nuclear antigen (PCNA) targets hPol ${kappa}$ to thereplication machinery. PCNA, a ring-shaped homotrimeric protein,is loaded onto the primer-template junction by a multiproteinclamp loader, replication factor C (RFC), which couples thehydrolysis of ATP with the opening and closing of the PCNA ringaround the DNA. After the loading of PCNA, RFC stays on theDNA via interaction with replication protein A (RPA) bound tosingle-stranded DNA (ssDNA) (1, 18, 33). The replicative DNApolymerase, Pol ${delta}$ , then assembles with PCNA and carries out processivesynthesis of both the leading and lagging DNA strands (26, 28).Here, we provide evidence for the physical interaction of hPol ${kappa}$ with PCNA and show that PCNA, together with RFC and RPA, stimulatesthe DNA polymerizing activity of hPol ${kappa}$ .

MATERIALS AND METHODS

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Materials and Methods

Proteins. To obtain glutathione S-transferase (GST)-hPol ${kappa}$ and untaggedhPol ${kappa}$ , the hDinB1 cDNA from pBJ733 (14) was cloned in frame withthe GST gene (containing a PreScission Protease cleavage recognitionsequence) in plasmid pBJ842 (7). hPol ${kappa}$ fused with GST was purifiedas described previously (14) and was used for the interactionstudies described below (see Fig. 1). Untagged hPol ${kappa}$ was purifiedby treating GST-hPol ${kappa}$ (50 µg) bound to glutathione-Sepharose(Pharmacia) with 2 U of PreScission Protease (Amersham Pharmacia)overnight at 4°C and was used for DNA polymerase assays.Human PCNA, RFC, and RPA were purified as described previously(2, 4, 21). Six-His-tagged human PCNA (Six-His-hPCNA) used forthe interaction studies shown in Fig. 1 was overexpressed inE. coli and purified as described previously (19).

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FIG. 1. Physical interaction of hPol ${kappa}$ with PCNA. Human six-His-hPCNA (2 µg) was mixed with GST-hPol ${kappa}$ (2 µg) (lanes 1 through 6). As controls, the same amounts of GST-hPol ${kappa}$ (lanes 7 through 9) or six-His-hPCNA (lanes 10 through 12) were used alone. After incubation, samples were bound to Ni-NTA (lanes 4 through 9) or glutathione-Sepharose (lanes 1 through 3 and 10 through 12) beads, followed by the washing and elution of the bound proteins by imidazole- or glutathione-containing buffer, respectively. Aliquots of each sample before additions of the beads (lanes L), the flowthrough-plus-washing fractions (lanes F), and the eluted proteins (lanes E) were precipitated by trichloroacetic acid and analyzed on a sodium dodecyl sulfate-12% polyacrylamide gel stained with Coomassie blue. The positions of GST-hPol ${kappa}$ and six-His-hPCNA are shown on the left.

Physical interaction of hPol ${kappa}$ with PCNA. To constitute complexes, GST-hPol ${kappa}$ (2 µg) was mixed withSix-His-hPCNA (2 µg) in buffer I, which contained 50 mMTris-HCl (pH 7.5), 150 mM NaCl, 1 mM dithiothreitol (DTT), 0.01%NP-40, and 10% glycerol. The 60-µl samples were incubatedfor 30 min at 4°C, followed by 10 min at 25°C. To 20µl of these samples, 10 µl of glutathione-Sepharoseor 10 µl of Ni-nitrilotriacetic acid (Ni-NTA) (Qiagen)beads was added to bind GST-hPol ${kappa}$ or six-His-hPCNA and theircomplexes, respectively, and the samples were further incubatedwith constant rocking for 30 min at 4°C. The glutathione-Sepharoseand Ni-NTA beads were washed 5 times with buffer I, and thenbound proteins were eluted with 40 mM glutathione or 500 mMimidazole containing buffer I, respectively. The samples containingthe protein mixture before the additions of affinity beads,the flowthrough-plus-washing fractions, and the eluted proteinswere precipitated with 5% trichloroacetic acid and separatedon a sodium dodecyl sulfate-12% polyacrylamide gel, followedby Coomassie blue R-250 staining.

DNA polymerase assays. The circular DNA substrate used for DNA synthesis studies (seeFig. 2A) was a 7.2-kb M13mp18 ssDNA primed with a nonlabeled36-nucleotide (nt)-long oligomer spanning nt 6330 to 6294. Forthe DNA polymerase processivity studies (see Fig. 2B), the M13derivative M13mp7L2 ssDNA was primed with the 35-nt-long, 5'³²P-labeled oligomer primer LP-272 (5'-GGGTTTTCCCAGTCACGACGTTGTAAAACGACGGC-3').Linear, running-start DNA substrates used for examining theDNA damage bypass ability of hPol ${kappa}$ (see Fig. 3) were generatedby annealing a 75-nt oligomer template (5'-biotin-AGCAAGTCACCAATGTCTAAGAGTTCGTAXXATGCCTACACTGGAGTACCGGAGCATCGTCGTGACTGGGAAAAC-biotin-3')or a 75-nt oligomer, 75AP-2biotin (5'-biotin-AAAAAAAAAAAAAAAAAAAAAAAAAAAGGG0ATGCCTACACTGGAGTACCGGAGCATCGTCGTGACTGGGAAAAC-biotin-3'),which contained one biotin molecule attached at each end andtwo undamaged T-residues, a cis-syn T-T dimer, or a (6-4) T-Tphotoproduct at positions 30 and 31 (XX) or an abasic site (atetrahydrofuran moiety; Midland Company) at position 31 (0),respectively, attached to the 5' ³²P-labeled 26-nt primer LP283(5'-CGACGATGCTCCGGTACTCCAGTGTA-3'). For steady-state kineticanalysis of nucleotide insertion opposite an undamaged C, G,T, or A or an abasic site template residue (see Fig. 4), the75AP-2biotin template oligomer was annealed to the 5' ³²P-labeledoligomer primers LP-158 (5'-CGACGATGCTCCGGTACTCCAGTGTAG-3'),N4577 (5'-GTTTTCCCAGTCACGACGATGCTCCGGTA-3'), N4578 (5'-GTTTTCCCAGTCACGACGATGCTCCGGTACTCCAGTGT-3'),N4267 (5'-GTTTTCCCAGTCACGACGATGCTCCGGTACTCCAGTGTAGGCA-3'), orLP-159 (5'-CGACGATGCTCCGGTACTCCAGTGTAGGCAT-3'), respectively.To bind streptavidin to the biotin present at the ends of thelinear DNA substrates, these primers-templates (2.5 pmol) werepreincubated for 10 min at 30°C with streptavidin (5 µg)in 25 µl of DNA polymerase buffer which contained no MgCl₂before their addition to the DNA polymerase reactions. The standardDNA polymerase reaction buffer (10 µl) contained 40 mMTris-HCl (pH 7.5), 8 mM MgCl₂, 150 mM NaCl, 1 mM DTT, 10% glycerol,100 µg of bovine serum albumin/ml, 500 µM ATP, and100 µM (each) dGTP, dATP, dTTP, and dCTP. For the reactionsin which circular DNA substrate was primed with nonlabeled oligonucleotide(Fig. 2A), [ ${alpha}$ -³²P]dATP ( $~$ 200 to 500 cpm/pmol, final [dATP]) wasadded. As indicated in the figure legends, hPol ${kappa}$ (1 to 10 ng),PCNA (100 ng), RFC (50 ng), and/or RPA (50 to 250 ng) was incubatedwith 25 ng of M13 DNA substrate or with linear primer-templateDNA (20 nM). Assay mixtures were assembled on ice, incubatedat 37°C for 10 min, and stopped by the addition of loadingbuffer (40 µl) containing EDTA (20 mM), 95% formamide,0.3% bromphenol blue, and 0.3% cyanole blue. The reaction productswere resolved on 10% polyacrylamide gels containing 8 M urea.Quantitation of the results was done with Molecular DynamicsSTORM PhosphorImager and ImageQuant software.

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FIG. 2. PCNA, RFC, and RPA stimulate the DNA polymerase activity of hPol ${kappa}$ . (A) Effect of PCNA, RFC, and RPA and various combinations of these proteins on DNA synthesis by hPol ${kappa}$ . The complete reaction mixture contained hPol ${kappa}$ (10 ng), along with singly primed M13 ssDNA (25 ng), PCNA (100 ng), RFC (50 ng), and RPA (250 ng), and all four deoxynucleotides (100 µM each) containing [ ${alpha}$ -³²P]dATP (lane 3). PCNA, RFC, and RPA and combinations of these proteins were omitted from the reaction mixture as indicated at the tops of the lanes. The amount of DNA synthesis is indicated at the bottom of each lane as the relative nucleotide incorporation. The HaeIII-digested ${phi}$ X174 DNA labeled by polynucleotide kinase is shown on the left as a size standard. (B) Processivity of hPol ${kappa}$ in the presence of PCNA, RFC, and RPA. As indicated at the top, different concentrations of hPol ${kappa}$ (1.5 to 80 nM), alone (lanes 1 through 3 and 7 through 9) or in the presence of PCNA (100 ng), RFC (50 ng), and RPA (250 ng) (lanes 4 through 6 and 10 through 12), were preincubated with circular M13 template ssDNA (5 nM) singly primed with 5' ³²P-labeled oligonucleotide for 5 min at 37°C. Primer extension reactions were initiated by adding all four deoxynucleotides (500 µM each) (lanes 1 through 6) or all four deoxynucleotides and excess sonicated herring sperm DNA (0.5 mg/ml) as a trap (lanes 7 through 12). After 10 min of incubation at 37°C, the samples were quenched and run on a 10% polyacrylamide gel. To demonstrate the effectiveness of the trap, hPol ${kappa}$ plus PCNA, RFC, and RPA were preincubated with excess herring sperm DNA together with the primer-template substrate for 5 min at 37°C before the addition of dNTPs (lane 13).

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FIG. 3. Effects of PCNA, RFC, and RPA on synthesis by hPol ${kappa}$ on a lesion containing DNA substrates. A portion of the DNA substrate containing undamaged T-T residues, a cis-syn T-T dimer, or a (6-4) T-T photoproduct is shown in schematic I. The positions of the two undamaged or damaged T’s are indicated by asterisks. In the abasic site containing DNA substrate shown in schematic II, the abasic site in the template DNA is indicated by a "0" and is marked by an asterisk. The primer was ³²P-labeled at its 5' end, and the template contained biotin-streptavidin complex at both ends. Pol ${kappa}$ (2 nM) was incubated with the DNA substrate (20 nM) in the presence of each of four dNTPs under standard reaction conditions. As indicated, the reactions were carried out in the presence or absence of PCNA (100 ng), RFC (50 ng), and RPA (50 ng).

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FIG. 4. Deoxynucleotide incorporation opposite undamaged C and T residues, and opposite an abasic site, by hPol ${kappa}$ in the presence or absence of PCNA, RFC, and RPA. hPol ${kappa}$ (1 nM) was incubated with primer-template DNA substrate (20 nM) and increasing concentrations of a single deoxynucleotide in the absence or presence of PCNA (100 ng), RFC (50 ng), and RPA (50 ng) for 10 min opposite C and T and for 30 min opposite an abasic site at 37°C. The DNA substrates contained biotin-streptavidin complexes at both ends. The nucleotide incorporation rate was plotted against dNTP concentration, and the data were fit to the Michaelis-Menten equation describing a hyperbola. Apparent K_m and V_max values were obtained from the fit and used to calculate the efficiency of deoxynucleotide incorporation (V_max/K_m). AP, abasic.

Processivity assays. Different concentrations of hPol ${kappa}$ (1.5 to 80 nM) in the presenceor absence of PCNA, RFC, and RPA were preincubated for 5 minat 37°C with circular M13 ssDNA substrate primed with a5' ³²P-labeled oligomer primer (5 nM) in the standard reactionbuffer but without the deoxynucleotides. Reactions were initiatedby the addition of all four deoxynucleotides (500 µM each)or all four deoxynucleotides plus excess sonicated herring spermDNA (0.5 mg/ml) used as a trap, followed by 10 min of incubationat 37°C. To demonstrate the effectiveness of the trap, hPol ${kappa}$ was preincubated with the DNA trap and the primer-template substratebefore the addition of deoxynucleotide triphosphates (dNTPs).

Determination of steady-state kinetic parameters. Steady-state kinetic analyses for deoxynucleotide incorporationopposite a nondamaged G, A, T, or C residue or an abasic sitewere performed as described previously (3, 5). Briefly, hPol ${kappa}$ alone or in the presence of PCNA, RFC, and RPA was incubatedwith increasing concentrations of a single deoxynucleotide for10 to 30 min under standard reaction conditions. Gel band intensitiesof the substrates and products were quantitated with a PhosphorImager,and the percentage of primer extension was plotted as a functionof dNTP concentration. The data were fit by nonlinear regressionby using SigmaPlot 5.0 to the Michaelis-Menten equation describinga hyperbola, v = (V_max x [dNTP])/(K_m + [dNTP]). Apparent K_mand V_max steady-state parameters were obtained from the fitand used to calculate the efficiency of deoxynucleotide incorporation(V_max/K_m).

RESULTS

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Results

Complex formation between hPol ${kappa}$ and PCNA. To examine whether hPol ${kappa}$ physically interacts with PCNA, GST-hPol ${kappa}$ was incubated with six-His-hPCNA and a pull-down assay was performedusing Ni-NTA and glutathione-Sepharose affinity beads (Fig.1). The Ni-NTA beads have a high specificity for six-His-taggedproteins and bind six-His-hPCNA, whereas the glutathione-Sepharosebeads bind GST-hPol ${kappa}$ . Our control experiments demonstrated thatNi-NTA beads do not bind GST-hPol ${kappa}$ (Fig. 1, lanes 7 through 9)and that glutathione-Sepharose beads do not bind six-His-hPCNA(Fig. 1, lanes 10 through 12). Hence, GST-hPol ${kappa}$ and six-His-hPCNAcould have been pulled down together only if they had interactedwith one another.

We preincubated GST-hPol ${kappa}$ and the six-His-hPCNA homotrimer ata 1:1 molar ratio. When GST-hPol ${kappa}$ was bound to glutathione-Sepharosebeads, almost all of the input PCNA was retained on the beads(Fig. 1, lanes 1 through 3), and similarly, when six-His-hPCNAwas bound to the Ni-NTA beads, nearly all of the input hPol ${kappa}$ remained bound to PCNA (Fig. 1, lanes 4 through 6). These observationsprovide evidence for the physical interaction of hPol ${kappa}$ with PCNA,and they indicate that the two proteins are able to form a complexin vitro.

PCNA, RFC, and RPA cooperatively stimulate DNA synthesis by hPol ${kappa}$ . Next we examined the effect of replication accessory proteinsPCNA, RFC, and RPA on the DNA synthesis activity of hPol ${kappa}$ . Anunlabeled oligonucleotide primer annealed to a unique positionof M13 circular ssDNA was elongated by hPol ${kappa}$ alone or in thepresence of either PCNA, RFC, or RPA or different combinationsof these proteins (Fig. 2A), and DNA synthesis was monitoredby determining the incorporation of radioactive deoxynucleotides.Addition of either PCNA, RFC, or RPA did not significantly enhancethe DNA synthesis activity of hPol ${kappa}$ (Fig. 2A, compare lane 1to lanes 6 through 8). However, a robust ( $~$ 23-fold) stimulationof DNA synthesis by hPol ${kappa}$ was observed upon the addition of allthree proteins, PCNA, RFC, and RPA (Fig. 2A, lane 3). This enhancementof the DNA synthesis activity of hPol ${kappa}$ requires all three accessoryproteins, since in the absence of either PCNA, RFC, or RPA,only a weak stimulation was observed (Fig. 2A, compare lanes1 through 5). From these results, we concluded that PCNA, RFC,and RPA cooperate to enhance the DNA synthesis activity of hPol ${kappa}$ .

Effect of PCNA on the processivity of hPol ${kappa}$ . In a search for the mechanism by which PCNA, RFC, and RPA stimulatethe DNA synthesis activity of hPol ${kappa}$ , we examined the effect ofthese accessory proteins on the processivity of hPol ${kappa}$ . Althougha low processivity is desirable for a low-fidelity polymerasesuch as hPol ${kappa}$ (14, 24), as that would limit its activity to synthesizingonly short stretches of DNA, thereby preventing high mutationrates, the possibility existed that an increase in processivitywas in fact responsible for the stimulation of the DNA synthesisactivity of hPol ${kappa}$ . To test whether PCNA, together with RFC andRPA, increased the processivity of hPol ${kappa}$ , we used a circularM13 template ssDNA primed with a 5' ³²P-labeled oligonucleotideprimer (Fig. 2B) and we ensured in two different ways that wewere observing deoxynucleotide incorporation resulting froma single DNA binding event of hPol ${kappa}$ . First, we decreased hPol ${kappa}$ concentration to a point where only a small fraction of theprimer was extended, and second, we monitored DNA synthesisin the presence of an excess of nonradiolabeled, sonicated herringsperm DNA used as a trap. At a low enzyme concentration, hPol ${kappa}$ alone incorporated only 1 to 4 nt (Fig. 2B, lane 3). Althoughthe addition of PCNA, RFC, and RPA greatly stimulated DNA synthesisby hPol ${kappa}$ (Fig. 2B, compare lanes 3 and 6), even in the presenceof these accessory proteins, hPol ${kappa}$ incorporated only a few, $~$ 1to 18 nt (Fig. 2B, lane 6). In the presence of excess nonlabeledherring sperm DNA, any hPol ${kappa}$ molecules that dissociated fromthe labeled DNA substrate would be trapped by the excess nonlabeledDNA, thereby preventing the rebinding of the enzyme to the primer-templatejunction. These reactions were performed by first preincubatinghPol ${kappa}$ either alone (Fig. 2B, lanes 7 through 9) or in the presenceof PCNA, RFC, and RPA (Fig. 2B, lanes 10 through 12) with thelabeled DNA substrate. A mixture of excess herring sperm DNAand all four deoxynucleotides was then added to initiate thereaction. The effectiveness of the trap was verified by preincubatinghPol ${kappa}$ with the DNA substrate and the excess herring sperm DNAbefore the addition of the nucleotides (Fig. 2B, lane 13). Thelack of any DNA synthesis in this sample showed that excessherring sperm DNA ( $~$ 200-fold) was sufficient to trap all hPol ${kappa}$ molecules. In these reactions, wherein single-hit conditionswere provided by excess herring sperm DNA, PCNA, RFC, and RPAdid not significantly increase the processivity, since the overallpattern of nucleotide incorporation remained nearly the samein the presence and in the absence of these protein factors(Fig. 2B, compare lanes 7 through 9 and lanes 10 through 12).

Ability of hPol ${kappa}$ to bypass DNA lesions in the presence of PCNA, RFC, and RPA. Although hPol ${kappa}$ belongs to the family of translesion synthesisDNA polymerases, it is unable to bypass a cis-syn T-T dimer,a (6-4) T-T photoproduct, or an abasic site (14). We next examinedwhether PCNA, RFC, and RPA promote translesion DNA synthesisby hPol ${kappa}$ through these DNA lesions. For this purpose, we wereunable to use short, linear, damage-containing primer-templateDNA substrates, probably because PCNA does not remain stablybound on such DNAs and may slide off at the ends. Therefore,we constructed DNA substrates wherein biotin was attached toboth ends of the 75-nt oligonucleotide template, and followingits annealing to the 5' ³²P-labeled oligonucleotide primer,both of these biotins were coupled to streptavidin, an $~$ 50-kDaprotein (Fig. 3). The presence of streptavidin on both endsof the DNA substrate would inhibit PCNA from falling off afterhaving been loaded onto the template-primer junction by RFC.On such undamaged linear DNA substrate, PCNA, RFC, and RPA stimulatedthe DNA synthesis activity of hPol ${kappa}$ (Fig. 3, compare lanes 1and 2). However, even in the presence of these protein factors,hPol ${kappa}$ remained ineffective in bypassing a cis-syn T-T dimer,a (6-4) T-T photoproduct, or an abasic site (Fig. 3). In thepresence of PCNA, RFC, and RPA, hPol ${kappa}$ exhibited some nucleotideinsertion ability opposite an abasic site (Fig. 3, lane 8),but the presence of a strong stall site just before the lesionindicated an inhibition of insertion across from the abasicsite, and the stall site opposite the lesion indicated an inhibitionof extension of the 3' primer end opposite the abasic site (Fig.3, lane 8). The strong stall sites present 1 or 2 nt beforethe 3' T of the cis-syn T-T dimer or the (6-4) T-T photoproduct(Fig. 3, lanes 4 and 6) indicated that PCNA, RFC, and RPA areunable to stimulate hPol ${kappa}$ ’s ability to insert nucleotidesopposite these DNA lesions.

Kinetic analyses of the efficiency of nucleotide incorporation by hPol ${kappa}$ in the presence of PCNA, RFC, and RPA. To understand further the mechanism by which PCNA, RFC, andRPA stimulate hPol ${kappa}$ , we examined their effects on the steady-statekinetic parameters K_m and V_max for nucleotide insertion by hPol ${kappa}$ .DNA substrates bearing biotin-streptavidin complex on both endswere generated by annealing the 5' ³²P-labeled oligonucleotideprimers to different positions of a linear 75-nt template oligonucleotide.The kinetics of insertion of a single deoxynucleotide oppositean undamaged G, A, T, or C or an abasic site, in a standingstart reaction, were determined as a function of the deoxynucleotideconcentration under steady-state conditions (Fig. 4). From thekinetics of deoxynucleotide incorporation, the steady-stateapparent K_m and V_max values for each deoxynucleotide were obtainedfrom the curve fit to the Michaelis-Menten equation. These K_mand V_max values and the efficiencies of single nucleotide incorporation(V_max/K_m) for hPol ${kappa}$ in the presence or absence of PCNA, RFC,and RPA are summarized in Table 1.

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TABLE 1. Kinetic parameters of nucleotide insertion reactions catalyzed by hPol ${kappa}$ opposite undamaged template residues and opposite an abasic site

As indicated by the V_max/K_m values, PCNA, together with RFCand RPA, increased the efficiency of nucleotide incorporationby hPol ${kappa}$ opposite undamaged residues as well as opposite theabasic site. Opposite undamaged template residues and in thepresence of PCNA, RFC, and RPA, hPol ${kappa}$ incorporates the correctnucleotide $~$ 50- to 200-fold more efficiently than it does inthe absence of these proteins (Table 1). These increased efficienciesfor nucleotide incorporation arise primarily from a marked reductionin the K_m for the dNTP substrate, whereas only minor changeswere observed in V_max values. For example, in the presence ofPCNA, RFC, and RPA, the $~$ 60-fold increase in the efficiency ofG insertion opposite the C template nucleotide was accompaniedby an $~$ 70-fold decrease in the K_m for G, whereas the V_max stayedabout the same (Table 1). Opposite the abasic site, PCNA, RFC,and RPA increased the efficiency of a nucleotide A insertion $~$ 40-fold; nucleotide A is the nucleotide inserted most oftenby hPol ${kappa}$ , but the efficiency of this reaction still remainedvery low.

The incorporation of incorrect nucleotides is also enhancedby PCNA, RFC, and RPA; however, because of the low V_max valuesfor the insertion of incorrect nucleotides in the absence ofthese proteins, we did not quantitate this enhancement. Basedon the comparison of the V_max/K_m values for the incorporationof correct and incorrect nucleotides, the fidelity of nucleotideinsertion of hPol ${kappa}$ in the presence of PCNA, RFC, and RPA is low,ranging from 6.6 x 10^-3 to 3.8 x 10^-4.

DISCUSSION

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Discussion

Here, we show that hPol ${kappa}$ physically interacts with PCNA and thatPCNA, together with RFC and RPA, stimulates the DNA synthesisactivity of hPol ${kappa}$ . Similar evidence for the physical and functionalinteraction of yeast Pol ${eta}$ and hPol ${eta}$ with PCNA that has been reportedrecently has shown that this interaction is mediated via theconserved PCNA binding motif located at the extreme C terminusof these proteins (7, 8). A similar motif is also present atthe C terminus of the human DinB1 protein (Fig. 5), and it maycontribute to PCNA binding.

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FIG. 5. Putative PCNA binding motif of human DINB1-encoded Pol ${kappa}$ . C-terminal amino acids 859 to 870 of hPol ${kappa}$ are aligned with the PCNA binding motifs in various PCNA binding proteins. The highly conserved residues are shown in bold. Hs, human; Sc, Saccharomyces cerevisiae.

PCNA, along with RFC and RPA, greatly stimulates the processivityof Pol ${delta}$ (26, 28). However, these protein factors do not significantlyenhance the processivity of hPol ${kappa}$ . This result is similar toprevious observations with yeast Pol ${eta}$ and hPol ${eta}$ , where processivitywas also not altered in the presence of these accessory proteins(7, 8). The absence of a significant increase in processivityis desirable for low-fidelity enzymes such as Pol ${eta}$ and Pol ${kappa}$ , forotherwise, there would be a large increase in mutation rates.

As judged from the V_max/K_m values, PCNA, RFC, and RPA stimulatethe efficiency of correct nucleotide incorporation by $~$ 50- to200-fold, and this increase in efficiency is achieved primarilyby a reduction in the apparent K_m for the nucleotide. Althoughthe processivity of hPol ${kappa}$ is not increased in the presence ofPCNA, RFC, and RPA, it remains possible that the binding ofthe enzyme to the 3' primer end is stabilized in the presenceof PCNA and that this accounts for the apparent increase inthe affinity of the enzyme for the nucleotide. Alternatively,PCNA may contribute to the conformation of the active site,affording a higher affinity of hPol ${kappa}$ for the nucleotide.

Even with PCNA, RFC, and RPA, hPol ${kappa}$ was unable to insert a nucleotideopposite a cis-syn T-T dimer or a (6-4) T-T photoproduct andhence did not bypass these DNA lesions. Also, in the presenceof these accessory factors, an abasic site remained a strongblock to hPol ${kappa}$ and only a low level of nucleotide insertion occurredacross from the lesion. Furthermore, although in the presenceof PCNA, RFC, and RPA, steady-state kinetic analyses indicatean $~$ 40-fold increase in the efficiency of a nucleotide A insertionopposite the abasic site, this reaction still remained veryinefficient. Compared to the insertion of an A opposite thetemplate T, hPol ${kappa}$ inserted an A opposite an abasic site morethan 250-fold less efficiently. Previously, hPol ${kappa}$ has been reportedto bypass an abasic site via a looping-out mechanism, in whichit first inserts an A opposite the lesion, and this was followedby the looping out of the lesion in the template strand andthe pairing of the A with the T residue present 5' to the abasicsite in the template strand (25). However, even in the presenceof PCNA, RFC, and RPA, the highly inefficient incorporationof an A, the nucleotide incorporated most frequently by hPol ${kappa}$ opposite the abasic site, makes this possibility untenable.Other considerations also provide no support for such a roleof hPol ${kappa}$ . Genetic studies of yeast have indicated that althoughA is incorporated most frequently opposite an abasic site, theother nucleotides, C, G, and T, are also incorporated (10).Further, it has been shown previously that the bypass of anabasic site in eukaryotes requires the sequential action oftwo DNA polymerases, wherein the extension step depends solelyupon Pol ${zeta}$ but many different polymerases could contribute tothe insertion step (10). Because of its proximity to the lesionsite and its ability to insert an A opposite an abasic site,Pol ${delta}$ would play a major role in the bypass of this lesion. Rev1and Pol ${eta}$ , however, also could contribute to lesion bypass bypromoting the insertion of a C or a G nucleotide, respectively(10). Although on its own, Pol ${eta}$ is highly inefficient at insertinga nucleotide opposite an abasic site, the efficiency for nucleotideinsertion rises dramatically in the presence of PCNA, RFC, andRPA (8). Thus, whereas yeast Pol ${eta}$ incorporates a G opposite anabasic site more than 500-fold less efficiently than it insertsa G opposite an undamaged C template residue, in the presenceof PCNA, RFC, and RPA, G is incorporated opposite an abasicsite only 20-fold less efficiently than its incorporation oppositea C residue (8).

The interaction of hPol ${kappa}$ with PCNA implies that this polymerasegains access to the stalled replication fork via its bindingto PCNA. This targeting mechanism is also used by another eukaryotictranslesion synthesis DNA polymerase, Pol ${eta}$ (7, 8). However, themechanism by which Pol ${delta}$ dissociates and a translesion synthesisDNA polymerase gains access to the 3' primer end remains tobe identified.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grantsGM19261 and GM38559.

FOOTNOTES

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

REFERENCES

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