distinct structural alterations in proliferating cell nuclear antigen block dna mismatch repair

Distinct Structural Alterations in Proliferating Cell Nuclear AntigenBlock DNA Mismatch RepairLynne M. Dieckman,†,§ Elizabeth M. Boehm,†,§ Manju M. Hingorani,‡ and M. Todd Washington*,†

†Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, United States‡Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459, United States

*S Supporting Information

ABSTRACT: During DNA replication, mismatches and smallloops in the DNA resulting from insertions or deletions arerepaired by the mismatch repair (MMR) machinery.Proliferating cell nuclear antigen (PCNA) plays an importantrole in both mismatch-recognition and resynthesis stages ofMMR. Previously, two mutant forms of PCNA were identifiedthat cause defects in MMR with little, if any, other defects. TheC22Y mutant PCNA protein completely blocks MutSα-dependent MMR, and the C81R mutant PCNA proteinpartially blocks both MutSα-dependent and MutSβ-dependentMMR. In order to understand the structural and mechanisticbasis by which these two amino acid substitutions in PCNA proteins block MMR, we solved the X-ray crystal structures of bothmutant proteins and carried out further biochemical studies. We found that these amino acid substitutions lead to subtle, distinctstructural changes in PCNA. The C22Y substitution alters the positions of the α-helices lining the central hole of the PCNA ring,whereas the C81R substitution creates a distortion in an extended loop near the PCNA subunit interface. We conclude that thestructural integrity of the α-helices lining the central hole and this loop are both necessary to form productive complexes withMutSα and mismatch-containing DNA.

I naccurate DNA replication can result in base−basemismatches and small loops arising from insertions or

deletions. These mismatches and loops are recognized andrepaired by the mismatch repair (MMR) machinery. Themechanisms of MMR in Escherichia coli have been studiedextensively and are relatively well understood.1−6 The MutSprotein is a homodimer that recognizes base−base mismatchesand small nucleotide insertions and deletions. The MutLprotein is a homodimer that interacts with MutS in an ATP-dependent manner to initiate MMR.7−11 The MutHendonuclease is then activated and generates a nick in thenewly synthesized, unmethylated DNA strand of a hemi-methylated duplex.12 Subsequent steps include unwinding anddegradation of the newly synthesized DNA strand and fillingthe resulting gap by DNA polymerase III.1−6

The mechanisms of MMR in eukaryotes are morecomplicated and are not as well understood. In yeast, thereare six MutS homologues designated Msh1 to Msh6; inmammals, there are five, Msh2 to Msh6. These proteinsfunction as heterodimers, such as MutSα (comprised of Msh2and Msh6) and MutSβ (comprised of Msh2 and Msh3), thathave partially overlapping mismatch recognition functions.13−15

In addition to the MutS homologues, there are several MutLhomologues, including Mlh1, Mlh2, Mlh3, and Pms1, whichalso function as heterodimers. The best characterized of these isMutLα (Mlh1/Pms1 in yeast and Mlh1/Pms2 in humans),which functions with both MutSα and MutSβ.16−20 Mutations

in both MutS and MutL homologues that disrupt mismatchrepair cause sporadic and hereditary human cancers, includinghereditary nonpolyposis colorectal cancer (HNPCC).2,21−25

Other key proteins involved in the subsequent excision andresynthesis steps include exonuclease I (ExoI),26−30 DNApolymerase delta (pol δ),31 replication protein A (RPA),32,33

replication factor C (RFC),34 and proliferating cell nuclearantigen (PCNA).35−37

The PCNA clamp is an essential replication accessoryprotein that forms a ring-shaped homotrimer and encirclesduplex DNA.38 In addition to serving as a processivity factor forDNA polymerases, it interacts with a wide variety of proteinsand plays important roles in DNA replication, repair,recombination, translesion synthesis, chromatin remodeling,sister chromatid cohesion, and cell cycle regulation.39−44

During MMR, PCNA functions in the initiation and mismatchrecognition stage as well as the excision and resynthesis stage.The role of PCNA in the initial stage of MMR is not wellunderstood. PCNA interacts with both MutSα and MutSβ andis thought to facilitate their recruitment to mismatches.37,45−49

Moreover, it has been suggested that PCNA plays a role instrand discrimination, that is, the recognition of the newlysynthesized daughter strand.35,50,51

Received: March 23, 2013Revised: July 18, 2013Published: July 19, 2013

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© 2013 American Chemical Society 5611 dx.doi.org/10.1021/bi400378e | Biochemistry 2013, 52, 5611−5619

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Various PCNA mutant alleles have been identified that leadto elevated mutation rates.36,52−54 Genetic studies have shownthat two of these mutant alleles, pol30-201 and pol30-204,specifically disrupt the MMR pathway with little, if any, effecton other DNA metabolic processes.54 The pol30-201 allele,which encodes the C22Y mutant PCNA protein, completelyblocks MutSα-dependent MMR, and the pol30-204 allele,which encodes the C81R mutant PCNA protein, partiallyblocks both MutSα-dependent and MutSβ-dependent MMR.54

In order to understand the structural and mechanistic basis bywhich these mutant PCNA proteins disrupt MMR, we solvedthe X-ray crystal structures of both mutant proteins and carriedout related biochemical studies. We found that these two aminoacid substitutions lead to distinct structural changes in PCNA.The C22Y substitution alters the positions of the α-heliceslining the inside of the PCNA ring, whereas the C81Rsubstitution disrupts an extended loop located near the PCNAsubunit interface. We conclude that the structural integrity ofthe α-helices lining the central hole and the structural integrityof this loop are both necessary to form productive complexeswith MutSα and mismatch-containing DNA.

■ EXPERIMENTAL PROCEDURESProtein Expression and Purification. The wild-type and

the C22Y and C81R mutant PCNA proteins from Saccha-romyces cerevisiae were overexpressed as N-terminally His6-tagged proteins in E. coli and were purified as describedpreviously.55 Replication factor C (RFC) from S. cerevisiae wasoverexpressed in E. coli and purified as previously described.56

DNA polymerase δ (pol δ) from S. cerevisiae was overexpressedin S. cerevisiae and purified as previously described.57 MutSαfrom S. cerevisiae was overexpressed in E. coli and purified asdescribed previously.58

DNA and Nucleotide Substrates. The template strandused to measure pol δ activity was a 68-mer oligodeoxynucleo-tide with the sequence 5′-GAC GGC ATT GGA TCG ACCTCX AGT TGG TTG GAC GGG TGC GAG GCT GGCTAC CTG CGA TGA GGA CTA GC with biotins on bothends. The X represents the position of a nondamaged G or anabasic site. The primer strand was a 31-mer oligodeoxynucleo-tide with the sequence 5′-TCG CAG GTA GCC AGC CTCGCA CCC GTC CAA C. The primer strand was 5′-32P-end-labeled with T4 polynucleotide kinase and (γ-32P)ATP andannealed to the template strand at 1 μM in 25 mM TrisCl, pH7.5, and 100 mM NaCl at 90 °C for 2 min and slowly cooled to30 °C. A mixture of all four dNTPs (10 mM each) waspurchased from New England Biolabs.Two 37-mer duplex DNA substrates were used in the

sedimentation analysis, one with a G/C pair at position 19 (thehomoduplex) and one with a G/T mispair at position 19 (theheteroduplex). The DNA substrates were formed by annealinga top strand to a bottom strand. The top strand, which had aCy3 fluorescent tag on the 3′ end, had the sequence 5′-ATTTCC TTC AGC AGA TAG GAA CCA TAC TGA TTC ACAT. The bottom strand had the sequence 5′-ATG TGA ATCAGT ATG GTT XCT ATC TGC TGA AGG AAA T, wherethe X represents the position of either a C in the case of thehomoduplex or a T in the case of the heteroduplex. Theannealing reactions were carried out as described above.Crystallization of the C22Y and C81R Mutant PCNA

Proteins. The C22Y mutant PCNA protein and the C81Rmutant PCNA protein were crystallized using the hanging dropmethod with 400 nL drops prepared using a Mosquito

crystallization robot (TTP Labtech). The best diffractingC22Y mutant PCNA protein crystals were obtained bycombining an equal volume of protein (30 mg/mL) with areservoir containing 1.6 M ammonium sulfate and 0.1 M citricacid. Crystals formed after 3 days at 18 °C. The best diffractingC81R mutant PCNA protein crystals were obtained bycombining an equal volume of protein (20 mg/mL) with areservoir containing 20% PEG1000, 0.2 M MgCl2 hexahydrate,and 0.1 M sodium cacodylate trihydrate, pH 6.5. Crystalsformed after 3 days at 18 °C.

Data Collection and Structural Determination. TheC22Y and C81R mutant PCNA protein crystals were soaked ina mother-liquor solution containing 10% (v/v) glycerol prior toflash-cooling in liquid nitrogen. In the case of the C22Y mutantPCNA protein, data were collected using a Rigaku rotatinganode X-ray source. In the case of the C81R mutant PCNAprotein, data were collected at the 4.2.2 synchrotron beamlineat the Advanced Light Source in the Ernest Orlando LawrenceBerkeley National Laboratory. The data were processed andscaled using d*TREK.59 For the C22Y mutant protein crystals,the space group was determined to be P212121, and there wasone PCNA trimer in the asymmetric unit. For the C81Rmutant protein crystals, the space group was determined to beP213, and there was one PCNA subunit in the asymmetric unit.Molecular replacement was performed using the knownstructure of wild-type PCNA [PDB 1PLQ] with PHASER.60

Refinement was done using REFMAC5 from CCP4 61 andPHENIX.62 All model building was carried out using Coot.63

The 2Fo − Fc omit maps were generated in PHENIX using thefully refined structure by setting the occupancies of the relevantresidues to zero and introducing random shifts in the positionsof the atoms by up to 0.5 Å and running a few rounds ofrefinement.

PCNA Trimer Stability Assays. For nondenaturingpolyacrylamide gel electrophoresis (PAGE), the wild-type,C22Y mutant, and C81R mutant PCNA proteins (0.1−1.0 mg/mL) were incubated in 25 mM TrisCl, pH 7.4, 150 mM NaCl,0.01% bromophenol blue, and 10% glycerol for 5 min and thenrun on a TrisCl precast 4−20% gradient nondenaturingpolyacrylamide gel (Bio-Rad) at 4 °C at a current of 25 mAusing 25 mM Tris, pH 8.3, and 0.2 M glycine running buffer.Protein bands were visualized using Coomassie blue staining.For size exclusion chromatography, wild-type and mutantPCNA proteins were diluted to various concentrations (0.01−10 mg/mL) in 25 mM Tris, pH 7.4, 150 mM NaCl, 5% glyceroland run on a 120 mL HiLoad 16/60 Superdex 200 PG column(GE Healthcare). The size exclusion chromatography columnwas calibrated using thyroglobin (670 kDa), γ-globin (158kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitaminB12 (1.35 kDa), which eluted at 51.9, 69.5, 85.5, 98.6, and 118.0mL, respectively.

Polymerase δ Activity Assays. Running start assays wereperformed as described previously.64 Reactions were carried outin the absence of PCNA and in the presence of 90 nM wild-type or mutant PCNA proteins (trimer concentration) andcontained 20 nM pol δ, 25 nM DNA, and 100 μM of each ofthe four dNTPs. Reactions were stopped after 30 min, and theextension products were analyzed on a 15% polyacrylamidesequencing gel containing 8 M urea.

Enzyme-Linked Immunosorbent Assays. The wells of a96 well EIA/RIA plate (Corning) were coated with 0.75 μg ofMutSα in PBS (4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4,137 mM NaCl, 2.7 mM KCl) for 2 h. The wells were then

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washed four times with PBS, 0.2% Tween-20, blocked for 1 hwith PBS with 5% milk, and washed again. Various amounts ofthe wild-type, the C22Y mutant, or the C81R mutant PCNAproteins (1−20 μg) in 100 μL of PBS with 5% milk were thenadded to the wells and incubated for 1 h. After washing, a1:1000 dilution of rabbit polyclonal anti-PCNA antibody inPBS with 5% milk was added to the wells and incubated for 30min. The wells were washed again, and a 1:10000 dilution ofgoat anti-rabbit antibody conjugated with horseradish perox-idase (Jackson ImmunoResearch) in PBS with 5% milk wasadded and incubated for 30 min. The plate was then washed,and 0.8 mg/mL of O-phenylenediamine (Aldrich) in 0.05 Mphosphate−citrate buffer with 0.03% sodium perborate (Sigma)was added. Absorbance was measured at 450 nm after variousamounts of time (5−35 min) using an iMark microplate reader(BioRad). Parallel control reactions using bovine serumalbumin instead of MutSα were carried out, and thesebackground absorbance values were subtracted from theabsorbance of each sample at the corresponding PCNA proteinconcentration. All steps were performed at 25 °C.Sedimentation Assays. Samples (100 μL) were prepared

with 300 nM MutSα, 300 nM of either the wild-type or mutantPCNA proteins (trimer concentration), and 300 nM of eitherthe homoduplex or heteroduplex DNA in 1× TBS buffer. Thesamples were incubated on ice for 30 min prior to loading on a5 mL glycerol gradient (15−30%) and were then spun for 20 hat 45 000 rpm at 4 °C in a Thermo Sorvall WX ultracentrifugeusing an AH-651 swing bucket rotor. Sixteen 300 μL aliquotswere collected from the bottom of each gradient, wereconcentrated using Millipore Amicon Ultra 10K centrifugalfilters, and were analyzed by SDS−PAGE using 4−15% precastgradient gels (BioRad). The Cy3-labeled DNA in each fractionwas visualized using a BioRad ChemiDoc-MP Imaging Systemafter which the gels were silver stained according to the BioRadpolyacrylamide gel staining procedure.

■ RESULTSWe have examined two mutant forms of PCNA that are knownto cause defects in MMR with little if any defects in other DNAmetabolic processes.54 The C22Y mutant PCNA proteincompletely blocks MutSα-dependent MMR, and the C81Rmutant PCNA protein partially blocks both MutSα-dependentand MutSβ-dependent MMR. In order to understand thestructural and mechanistic basis by which they disrupt MMR,we solved the X-ray crystal structures of both mutant proteinsand analyzed their biochemical properties.Structure of the C22Y Mutant PCNA Protein. We first

determined the X-ray crystal structure of the C22Y mutantPCNA protein to a resolution of 2.7 Å (Table 1). Overall, itsstructure resembles that of the wild-type PCNA protein witheach subunit comprised of two domains, an N-terminal domain(residues 1−117) and a C-terminal domain (residues 135−258) linked by a long, interdomain connector loop (residues118−134) (Figure 1A). The subunits are arranged in a head-to-tail fashion to form a ring-shaped trimer. Residue 22 is locatedin the N-terminal domain on loop B (residues 21−25). Thisloop follows α-helix A1 (residues 9−20), which along with α-helices B1, A2, and B2 forms the inside ring of the PCNA trimer(Figure 1B). Because of its larger size, the substituted tyrosineside chain is unable to occupy the same position as the wild-type cysteine side chain. Consequently, the tyrosine side chainpoints toward the front of the PCNA ring rather than toward α-helix B2 (Figure 1C,D). This rearrangement induces a set of

other structural changes that ultimately alter the positions ofboth α-helix A2 and α-helix B2. First, the α-carbon of residue 22

Table 1. Data Collection and Refinement Statistics

C22Y mutant protein C81R mutant protein

(A) Data Collection Statisticsresolution (Å)a 20.9−2.7 (2.8−2.7) 21.5−3.0 (3.1−3.0)wavelength (Å) 1.54 1.00space group P212121 P213cell (Å) a = 85.9, b = 90.6,

c = 140.6a = b = c = 121.8

completeness (%)a 100 (100) 100 (100)redundancya 7.1 (7.2) 19.9 (20.2)⟨I/σ(I)⟩a 7.0 (2.0) 9.1 (2.0)Rmerge (%)

a 13.3 (65.9) 14.2 (77.3)(B) Refinement StatisticsResolution Range (Å) 19.9−2.7 20.5−3.0R (%) 20.5 19.8Rfree (%) 28.7 25.4rms bonds (Å) 0.017 0.013rms angles (deg) 1.78 1.57no. of protein atoms 6026 (761 residues) 1975 (254 residues)no. of water molecules 0 0ramachandran analysis(%)

most favored 92.5 92.0allowed 6.3 8.0

PDB ID code 4L6P 4L60aValues in parentheses are for the highest resolution shell.

Figure 1. Structure of the C22Y mutant PCNA protein. (A) Frontview of the C22Y mutant PCNA protein trimer with one of thesubunits shown in red. (B) Side view of one subunit of the C22Ymutant PCNA protein with the α-helices A2, B2, and A1 shown in red.The wild-type PCNA structure is overlaid with the same α-helicesshown in purple. (C) Close up of the region near the C22Ysubstitution. The mutant PCNA protein is shown in red and the wild-type PCNA protein is shown in purple. (D) 2Fo − Fc omit mapcontoured at 1σ and carved at 1.7 Å around the substituted tyrosine atresidue 22, Asp-21, and Lys-217.



is shifted 1.5 Å from its position in the wild-type proteinstructure. This in turn causes the α-carbon of Asp-21 to move1.1 Å and the δ-oxygen of Asp-21 to move 1.6 Å from theirpositions in the wild-type protein structure. This causes the ε-nitrogen of Lys-217, which is located in α-helix B2 and forms ahydrogen bond with the δ-oxygen of Asp-21, to move 3.4 Å,and this causes the α-carbons in α-helix B2 (residues 209−221)to move up to 2.1 Å from their positions in the structure of thewild-type protein and the α-carbons in α-helix A2 (residues141−153) to move up to 1.8 Å from their positions in the wild-type protein structure. The changes in the positions of the twoα-helices in the C-terminal domain are the most notablestructural alterations in the C22Y mutant PCNA protein.Structure of the C81R Mutant PCNA Protein. We next

determined the X-ray crystal structure of the C81R mutantPCNA protein to a resolution of 3.0 Å (Table 1 and Figure2A). Residue 81 is located in the N-terminal domain on loop H

(residues 80−86). This loop follows α-helix B1 (residues 72−79) and is near the subunit interface (Figure 2B). Thesubstituted arginine side chain points toward β-strand I1(residues 109−117), which comprises the subunit interface,and forms a new hydrogen bond with the η-oxygen of Tyr-114(Figure 2C,D). This interaction causes the α-carbon of Tyr-114, located in β-strand I1, to move 0.5 Å from its position inthe wild-type protein structure. This is a minor change anddoes not alter the β strands comprising the subunit interface inany substantive way. By contrast, the interaction between thesubstituted arginine at residue 81 and Tyr-114 causes the α-

carbon of residue 81 to move 3.2 Å from its position in thewild-type protein structure thereby distorting the structure ofloop H. This distortion of loop H is the only notable structuralalteration in the C81R mutant PCNA protein.

Stability of the Mutant PCNA Proteins. It was previouslysuggested that the C81R mutant PCNA protein was amonomer in vitro.54 This, however, seemed inconsistent withthe structure of the C81R mutant PCNA protein, whichshowed it to be a trimer with no significant structuralperturbations to the β strands comprising the subunit interface.Thus, we re-examined the stability of the mutant PCNAtrimers. First, we used native polyacrylamide gel electrophoresis(PAGE) to determine the oligomeric form of the wild-type andthe two mutant PCNA proteins at various concentrations(Figure 3A). Both the wild-type and the C22Y mutant PCNAproteins were trimers at all concentrations tested (0.1−1.0 mg/mL). By contrast, the C81R mutant PCNA protein did notform stable trimers as indicated by the higher mobility speciesat low concentrations (0.1 and 0.2 mg/mL) and the smearedgel bands at higher concentrations (0.5 and 1.0 mg/mL).In order to further assess PCNA trimer stability, we analyzed

the proteins by size exclusion chromatography (Figure 3B).When loaded onto the size exclusion column at highconcentration (10 mg/mL), the wild-type PCNA protein andthe C22Y mutant protein eluted in a narrow peak as expectedfor the 90-kDa trimer. When the C81R mutant protein wasloaded at high concentration (10 mg/mL), it eluted in a broadpeak corresponding to a mixture of mostly trimer with somedimer present. At lower protein concentration (0.01 mg/mL),the C81R mutant protein eluted as the 30-kDa monomer(Figure S1 in the Supporting Information). Taken together,both the native PAGE and size exclusion chromatographyresults show that the C81R mutant PCNA protein formstrimers in vitro at higher protein concentrations but that thesetrimers are less stable than in the cases of either the wild-typePCNA protein or the C22Y mutant PCNA protein. Thusdistortions to loop H, such as those occurring with the C81Rmutant PCNA protein, do impact the stability of the PCNAtrimer. It should be noted, however, that this mutant PCNAprotein supports normal cell growth and likely forms stabletrimers in vivo.

Impact of the Mutant PCNA Proteins on DNAPolymerase δ Activity. DNA polymerase δ (pol δ) isresponsible for the majority of lagging strand synthesis duringnormal DNA replication and is also involved in base excisionrepair, nucleotide excision repair, mismatch repair, and doublestrand break repair.65−68 To determine whether the two mutantPCNA proteins can stimulate DNA synthesis by pol δ, we usedrunning start experiments to measure pol δ activity in theabsence and presence of the wild-type and mutant PCNAproteins on nondamaged DNA (Figure 4A). The wild-typePCNA protein stimulates DNA synthesis by pol δ with ∼5-foldmore full-length runoff products formed in the presence of thewild-type PCNA protein than in its absence. The C22Y mutantPCNA protein and the C81R mutant PCNA protein stimulatedDNA synthesis by pol δ to differing extents. In the presence ofthe C22Y mutant protein, ∼3-fold more full-length runoffproducts were observed than in its absence. In the presence ofthe C81R mutant protein, ∼2-fold more full-length runoffproducts were observed.PCNA also facilitates the bypass of abasic sites by pol δ, and

we examined whether this activity was affected by the mutantPCNA proteins (Figure 4B). In the presence of the wild-type

Figure 2. Structure of the C81R mutant PCNA protein. (A) Frontview of the C81R mutant PCNA protein trimer with one of thesubunits shown in orange. (B) Side view of one subunit of the C81Rmutant PCNA protein with the α-helix B1 and the β-strands H1 and I1shown in orange. The wild-type PCNA structure is overlaid with thesame α-helix and β-strands shown in purple. (C) Close up of theregion near the C81R substitution. The mutant PCNA protein isshown in orange and the wild-type PCNA protein is shown in purple.(D) 2Fo − Fc omit map contoured at 1σ and carved at 1.7 Å aroundthe substituted arginine at residue 81 and Tyr-114.



PCNA protein, ∼5-fold more extension products resulting fromincorporation opposite the abasic site were observed than in theabsence of PCNA. Moreover, ∼4-fold more full-length runoffproducts were observed in the presence of the wild-type PCNAprotein. Similarly, in the presence of the C22Y mutant PCNAprotein, there were ∼6-fold more extension products resultingfrom incorporation opposite the abasic site and ∼3-fold morefull-length runoff products than in the absence of PCNA. In thepresence of the C81R mutant PCNA protein, there were ∼2-fold more extension products resulting from incorporationopposite the abasic site and no increase in the amount of full-length products than in the absence of PCNA.Taken together, these experiments show that, like the wild-

type PCNA protein, the C22Y mutant PCNA protein fullysupports pol δ function in both normal and translesion DNAsynthesis, whereas the C81R mutant PCNA protein onlypartially supports pol δ function. It should be noted that underthese assay conditions, the concentration of PCNA was low(0.01 mg/mL), and most of the C81R mutant PCNA proteinwas not expected to be trimeric. Thus, the reduced ability of theC81R mutant PCNA protein to stimulate the activity of pol δ isvery likely due to this mutant protein not forming stable trimersin these assays.

Figure 3. Trimer stability of the PCNA mutant proteins. (A) Analysis of the PCNA proteins by native gel electrophoresis. Solutions containing thewild-type or mutant PCNA proteins (0.1−1.0 mg/mL) were run on a nondenaturing polyacrylamide gradient gel (4−20%) and Coomassie stained.The position of the PCNA trimer is indicated. (B) Analysis of the PCNA proteins by size exclusion chromatography. The elution profile of a sizeexclusion chromatography column in which a solution of the wild-type PCNA protein, the C22Y mutant PCNA protein, and the C81R mutantPCNA protein (10 mg/mL) was run is shown.

Figure 4. DNA synthesis by pol δ in the presence of the PCNAproteins. (A) An autoradiogram of the products of pol δ-catalyzedDNA synthesis on a nondamaged DNA substrate in the presence of noPCNA, the wild-type PCNA protein, the C22Y mutant PCNA protein,and the C81R mutant PCNA protein. The position of the fullyextended, runoff product is indicated with an arrow. (B) Anautoradiogram of the products of pol δ-catalyzed DNA synthesis onan abasic site-containing DNA substrate in the presence of no PCNA,the wild-type PCNA protein, the C22Y mutant PCNA protein, and theC81R mutant PCNA protein. The position of the abasic site isindicated by an X, and the position of the fully extended, runoffproduct is indicated with an arrow.



Interactions of the Mutant PCNA Proteins withMutSα. Since PCNA is known to interact with MutSα duringMMR and since the C22Y and C81R mutant PCNA proteinsare defective in MMR, we examined the ability of these mutantproteins to bind MutSα. We used an enzyme-linkedimmunosorbent assay (ELISA) to monitor this interaction(Figure 5). A fixed concentration of the MutSα was

immobilized in a microtiter plate and titrated with variousconcentrations of the wild-type PCNA protein, the C22Ymutant PCNA protein, or the C81R mutant PCNA protein.The absorbance signal is proportional to the amount of PCNAbound to MutSα. No significant difference was observedbetween the binding of the wild-type PCNA protein and theC22Y mutant PCNA protein to MutSα. The C81R mutantPCNA protein, by contrast, exhibited weaker binding than thewild-type and the C22Y mutant PCNA proteins, though stillsignificantly greater than the background. These results differsomewhat from previously published cosedimentation studiesthat showed that the C22Y mutant PCNA protein binds MutSαin vitro, but the C81R mutant PCNA protein does not.54 Itshould be pointed out again that the concentrations of PCNAused in both the ELISA assays reported here and thecosedimentation assay reported previously are less than 0.2mg/mL. Under these conditions, most of the C81R mutantprotein is not expected to be trimeric. Thus, the apparentweaker binding of the C81R mutant PCNA protein to MutSα isvery likely due to this mutant protein not forming stable trimersin these assays.Interactions of the Mutant PCNA Proteins with MutSα

and DNA. It has previously been shown that PCNA, MutSα,and mismatch-containing DNA form a stable ternarycomplex.37 We carried out sedimentation analysis to determinewhether these amino acid substitutions in PCNA affect itsability to form ternary complexes with MutSα and DNA. TheDNA contained a G:T mismatched base-pair flanked on bothsides by 18 matched base-pairs. In the absence of PCNA,MutSα was found mainly in fractions 5−9 along with DNA(Figure 6B), suggesting that a single MutSα protein binds tothe centrally positioned G:T mismatch. In the presence of thewild-type PCNA protein, MutSα was found mainly in fractions5−9 along with PCNA and DNA (Figure 6C), suggesting that a

single MutSα protein and a single PCNA protein bind to themismatch. Interestingly, in the presence of either the C22Ymutant PCNA protein or the C81R mutant PCNA protein,MutSα was found mainly in fractions 1−4 along with themutant PCNA proteins and DNA (Figure 6D,E). This meansthat the MutSα-containing complex is larger in the presence ofthe mutant PCNA proteins than in the presence of the wild-type PCNA protein. This may be because in the case of themutant PCNA proteins, MutSα no longer specifically binds thecentrally positioned G:T mismatch, and more than one MutSαprotein can bind the same DNA molecule. The precise natureof these larger complexes with the mutant PCNA proteinsremains unclear, and further structural analysis will be requiredto understand how and why they form. Nevertheless, theseresults convincingly demonstrate that the complexes of themutant PCNA proteins, MutSα, and mismatch-containingDNA are aberrant. Moreover, these aberrant complexes arestrictly dependent both on the presence of the mutant PCNAproteins and on the presence of a mismatch in the DNA,because they do not form in the absence of DNA (FigureS2D,E,F in the Supporting Information) or in the presence of

Figure 5. Interactions of the PCNA proteins with MutSα. The resultsof an ELISA assay showing the interactions of the wild-type PCNA(●), the C22Y mutant PCNA protein (○), and the C81R mutantPCNA protein (▼) with MutSα. Control experiments using BSAinstead of MutSα have been subtracted from each of the values.

Figure 6. Sedimentation analysis of the interactions of the PCNAproteins with MutSα and DNA. Fractions of a glycerol gradients (15−30%) containing the complex components were analyzed bydenaturing polyacrylamide gradient gel electrophoresis (4−15%).The fractions ranged from 1 (the bottom of the gradient) to 14 (thetop of the gradient). The DNA substrate was visualized by Cy3fluorescence. The proteins were visualized by silver staining. (A)Fractions containing the heteroduplex DNA in the absence of PCNAand MutSα (Msh2−Msh6). (B) Fractions containing MutSα andheteroduplex DNA in the absence of PCNA. (C) Fractions containingMutSα and heteroduplex DNA in the presence of the wild-type PCNAprotein. (D) Fractions containing MutSα and heteroduplex DNA inthe presence of the C22Y mutant PCNA protein. (E) Fractionscontaining MutSα and heteroduplex DNA in the presence of theC81R mutant PCNA protein.



fully matched G:C DNA (Figure S2H,I,J in the SupportingInformation).

■ DISCUSSIONPCNA plays a critical role in many aspects of DNA metabolismand the maintenance of genome stability. It interacts with awide variety of proteins, recruits them, and coordinates theiractivities at sites of DNA synthesis. It functions in DNAreplication, translesion synthesis, base excision repair, nucleo-tide excision repair, mismatch repair, homologous recombina-tion, chromatin remodeling, sister chromatid cohesion, and cellcycle regulation.39−44 Genetic studies, especially in yeast, haveidentified a number of mutations in PCNA that disrupt one ormore of these processes. For example, simple amino acidsubstitutions in PCNA have been identified that interfere withtranslesion synthesis,69,70 error-free postreplication repair,71

MMR,36,52−54 and chromatin remodeling.72,73 In this study, wefocused on two important substitutions that block MMR.A systematic study of amino acid substitutions in PCNA that

interfere with MMR has shown that many of the changesimpact cell growth at nonpermissive temperatures and aresensitive to DNA damaging agents such as methyl methanesul-fonate, ultraviolet radiation, and hydroxyurea.54 Most of thesesubstitutions in PCNA cause an increase in spontaneousmutations in assays measuring the frequency of reversionmutations of a one-nucleotide insertion in the hom3-10 allele,the frequency of reversion mutations of a four-nucleotideinsertion in the lys2-bgl allele, and the frequency of forwardmutations in the CAN1 gene. Two of these amino acidsubstitutions in PCNA, the C22Y substitution (encoded by thepol30-201 allele) and the C81R substitution (encoded by thepol30-204 allele), specifically impact MMR and do not causenotable defects in other DNA replication and repairprocesses.54 Neither substitution causes temperature-depend-ent growth defects or increased sensitivity to DNA damagingagents. Both substitutions lead to an increase in the frequencyof reversion mutations in the hom3-10 and lys2-bgl alleles and inthe frequency of forward mutations in the CAN1 gene. Geneticanalysis of these mutant forms of PCNA in combination withthe msh2Δ, msh3Δ, and msh6Δ mutations, which disruptMutSα or MutSβ, imply that the C22Y mutant PCNA proteincompletely blocks MutSα-dependent MMR and the C81Rmutant PCNA protein partially blocks both MutSα-dependentand MutSβ-dependent MMR.54

In the present study, we examined the structural changes inPCNA induced by these two amino acid substitutions tounderstand the basis for their specific defects in MMR.Interestingly, these two substitutions caused two distinctstructural alterations in PCNA. The C22Y mutant PCNAprotein has shifts in the α-helices that line the central hole ofthe PCNA ring that encircles DNA. This mutant protein formsstable trimers and stimulates DNA synthesis by pol δ. TheC81R mutant PCNA protein has a localized distortion in loopH, which follows α-helix B1 near the PCNA subunit interface.This mutant protein forms less stable trimers compared withthe wild-type and the C22Y mutant PCNA proteins.Consequently, this mutant protein exhibits only a moderatestimulation of DNA synthesis by pol δ, which is still higher thanin the absence of PCNA.Since both PCNA mutant proteins are known to block

MutSα-dependent MMR, we examined their ability to interactwith MutSα. The C22Y mutant PCNA protein interacts withMutSα with the same affinity as does the wild-type PCNA. The

C81R mutant PCNA protein also interacts with MutSα butdoes so with lower affinity compared with the wild-type andC22Y mutant proteins. Again, this apparent weaker binding islikely due to the instability of the C81R mutant PCNA proteintrimers, and we believe that the C81R mutant PCNA proteintrimers that do form under these experimental conditions stillbind MutSα. This is in contrast to the previous suggestion thatthis mutant PCNA protein does not interact with MutSα.54 Itshould be noted that the conformational changes induced bythese amino acid substitutions do not perturb the hydrophobicbinding pocket on PCNA, which binds the canonical PCNA-interacting protein (PIP) motif in the N-terminal region of theMsh6 subunit of MutSα. Overall, these findings suggest that thedisruption of MMR by these PCNA mutant proteins does notarise from substantial defects in the interaction with MutSα,especially in the case of the C22Y mutant PCNA protein.We then examined the ability of the PCNA mutant proteins

to form ternary complexes with MutSα and mismatch-containing DNA. Surprisingly, we found that despite relativelysubtle changes in the structures of the C22Y and C81R mutantPCNA proteins, they both formed aberrant complexes withMutSα and DNA. In the presence of the mutant PCNAproteins and a mismatch, the MutSα-containing complexeswere larger than in the presence of the wild-type PCNAprotein. A possible explanation for this is that impropermismatch recognition leads to multiple MutSα proteins on theDNA substrate. Understanding why these higher-orderedcomplexes form awaits further structural studies. However, weconclude that these complexes are aberrant and that theformation of proper, productive complexes between MutSα andmismatch-containing DNA depends on both the structuralintegrity of the α-helices lining the central hole of the PCNAring and the structural integrity of loop H near the PCNAsubunit interface.

■ ASSOCIATED CONTENT

*S Supporting InformationSize exclusion chromatography experiments and sedimentationanalysis experiments. This material is available free of charge viathe Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*Mailing address: Department of Biochemistry, 4-403 BowenScience Building, University of Iowa, Iowa City, IA 52242-1109.Phone: 319-335-7518. Fax: 319-335-9570. E-mail: [email protected].

Author Contributions§L.M.D. and E.M.B. contributed equally to this work.

FundingThe project described was supported by Award NumberGM081433 from the National Institute of General MedicalSciences to M.T.W.

NotesThe content of this work is solely the responsibility of theauthors and does not necessarily represent the official views ofthe National Institute of General Medical Sciences or theNational Institutes of Health.The authors declare no competing financial interest.



http://pubs.acs.org

mailto:[email protected]

mailto:[email protected]

■ ACKNOWLEDGMENTS

We thank Dr. Christine Kondratick, Dr. John Pryor, Dr. MariaSpies, Dr. Marc Wold, and Kyle Powers for valuablediscussions. We also thank Lokesh Gakhar and Nicole Sipfleboth for technical assistance in data collection and refinementand for discussions.

■ ABBREVIATIONS

BSA, bovine serum albumin; ELISA, enzyme-linked immuno-sorbent assay; HNPCC, hereditary nonpolyposis colorectalcancer; MMR, mismatch repair; PAGE, polyacrylamide gelelectrophoresis; PCNA, proliferating cell nuclear antigen; PIP,PCNA-interacting protein; Pol, polymerase; RFC, replicationfactor C; RPA, replication protein A; TLS, translesion synthesis

■ REFERENCES(1) Iyer, R. R., et al. (2006) DNA mismatch repair: Functions andmechanisms. Chem. Rev. 106 (2), 302−323.(2) Modrich, P., and Lahue, R. (1996) Mismatch repair in replicationfidelity, genetic recombination, and cancer biology. Annu. Rev. Biochem.65, 101−133.(3) Jiricny, J. (2006) The multifaceted mismatch-repair system. Nat.Rev. Mol. Cell Biol. 7 (5), 335−346.(4) Li, G.-M. (2008) Mechanisms and functions of DNA mismatchrepair. Cell Res. 18 (1), 85−98.(5) Kunz, C., Saito, Y., and Schaer, P. (2009) DNA Repair inMammalian Cells. Cell. Mol. Life Sci. 66 (6), 1021−1038.(6) Kunkel, T. A., and Erie, D. A. (2005) DNA mismatch repair.Annu. Rev. Biochem., 681−710.(7) Grilley, M., et al. (1989) Isolation and Characterization of theEscherichia-coli MutL Gene-Product. J. Biol. Chem. 264 (2), 1000−1004.(8) Galio, L., Bouquet, C., and Brooks, P. (1999) ATP hydrolysis-dependent formation of a dynamic ternary nucleoprotein complexwith MutS and MutL. Nucleic Acids Res. 27 (11), 2325−2331.(9) Spampinato, C., and Modrich, P. (2000) The MutL ATPase isrequired for mismatch repair. J. Biol. Chem. 275 (13), 9863−9869.(10) Schofield, M. J., et al. (2001) Interaction of Escherichia coliMutSand MutL at a DNA mismatch. J. Biol. Chem. 276 (30), 28291−28299.(11) Acharya, S., et al. (2003) The coordinated functions of the E-coliMutS and MutL proteins in mismatch repair. Mol. Cell 12 (1), 233−246.(12) Au, K. G., Welsh, K., and Modrich, P. (1992) Initiation ofMethyl-Directed Mismatch Repair. J. Biol. Chem. 267 (17), 12142−12148.(13) Marsischky, G. T., et al. (1996) Redundancy of Saccharomycescerevisiae MSH3 and MSH6 in MSH2-dependent mismatch repair.Genes Dev. 10 (4), 407−420.(14) Sia, E. A., et al. (1997) Microsatellite instability in yeast:Dependence on repeat unit size and DNA mismatch repair genes. Mol.Cell. Biol. 17 (5), 2851−2858.(15) Genschel, J., et al. (1998) Isolation of MutS beta from humancells and comparison of the mismatch repair specificities of MutS betaand MutS alpha. J. Biol. Chem. 273 (31), 19895−19901.(16) Li, G. M., and Modrich, P. (1995) Restoration of MismatchRepair to Nuclear Extracts of H6 Colorectal Tumor-Cells by aHeterodimer of Human Mutl Homologs. Proc. Natl. Acad. Sci. U. S. A.92 (6), 1950−1954.(17) Habraken, Y., et al. (1997) Enhancement of MSH2-MSH3-mediated mismatch recognition by the yeast MLH1-PMS1 complex.Curr. Biol. 7 (10), 790−793.(18) Habraken, Y., et al. (1998) ATP-dependent assembly of aternary complex consisting of a DNA mismatch and the yeast MSH2-MSH6 and MLH1-PMS1 protein complexes. J. Biol. Chem. 273 (16),9837−9841.

(19) Bowers, J., et al. (2001) MSH-MLH complexes formed at aDNA mismatch are disrupted by the PCNA sliding clamp. J. Mol. Biol.306 (5), 957−968.(20) Raschle, M., et al. (2002) Mutations within the hMLH1 andhPMS2 subunits of the human MutL alpha mismatch repair factoraffect its ATPase activity, but not its ability to interact with hMutSalpha. J. Biol. Chem. 277 (24), 21810−21820.(21) Kolodner, R. D., and Marsischky, G. T. (1999) Eukaryotic DNAmismatch repair. Curr. Opin. Genet. Dev. 9 (1), 89−96.(22) Lynch, H. T., and de la Chapelle, A. (1999) Geneticsusceptibility to non-polyposis colorectal cancer. J. Med. Genet. 36(11), 801−818.(23) Kolodner, R. D. (1995) Mismatch Repair - Mechanisms andRelationship to Cancer Susceptibility. Trends Biochem. Sci. 20 (10),397−401.(24) de la Chapelle, A. (2004) Genetic predisposition to colorectalcancer. Nat. Rev. Cancer 4 (10), 769−780.(25) Rowley, P. T. (2005) Inherited susceptibility to colorectalcancer. Annu. Rev. Med., 539−554.(26) Tishkoff, D. X., et al. (1998) Identification of a human geneencoding a homologue of Saccharomyces cerevisiae EXO1, anexonuclease implicated in mismatch repair and recombination. CancerRes. 58 (22), 5027−5031.(27) Tishkoff, D. X., et al. (1997) Identification and characterizationof Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease thatinteracts with MSH2. Proc. Natl. Acad. Sci. U. S. A. 94 (14), 7487−7492.(28) Bertrand, P., et al. (1998) Physical interaction betweencomponents of DNA mismatch repair and nucleotide excision repair.Proc. Natl. Acad. Sci. U. S. A. 95 (24), 14278−14283.(29) Schmutte, C., et al. (1998) Human exonuclease I interacts withthe mismatch repair protein hMSH21. Cancer Res. 58 (20), 4537−4542.(30) Genschel, J., Bazemore, L. R., and Modrich, P. (2002) Humanexonuclease I is required for 5′ and 3′ mismatch repair. J. Biol. Chem.277 (15), 13302−13311.(31) Longley, M. J., Pierce, A. J., and Modrich, P. (1997) DNApolymerase delta is required for human mismatch repair in vitro. J.Biol. Chem. 272 (16), 10917−10921.(32) Lin, Y. L., et al. (1998) The evolutionarily conserved zinc fingermotif in the largest subunit of human replication protein a is requiredfor DNA replication and mismatch repair but not for nucleotideexcision repair. J. Biol. Chem. 273 (3), 1453−1461.(33) Ramilo, C., et al. (2002) Partial reconstitution of human DNAmismatch repair in vitro: Characterization of the role of humanreplication protein A. Mol. Cell. Biol. 22 (7), 2037−2046.(34) Dzantiev, L., et al. (2004) Defined human system that supportsbidirectional mismatch-provoked excision. Mol. Cell 15 (1), 31−41.(35) Umar, A., et al. (1996) Requirement for PCNA in DNAmismatch repair at a step preceding DNA resynthesis. Cell 87 (1), 65−73.(36) Johnson, R. E., et al. (1996) Evidence for involvement of yeastproliferating cell nuclear antigen in DNA mismatch repair. J. Biol.Chem. 271 (45), 27987−27990.(37) Flores-Rozas, H., Clark, D., and Kolodner, R. D. (2000)Proliferating cell nuclear antigen and Msh2p-Msh6p interact to forman active mispair recognition complex. Nat. Genet. 26 (3), 375−378.(38) Krishna, T. S. R., et al. (1994) Crystal structure of the eukaryoticDNA polymerase processivity factor PCNA. Cell 79 (7), 1233−1243.(39) Maga, G., and Hubscher, U. (2003) Proliferating cell nuclearantigen (PCNA): A dancer with many partners. J. Cell Sci. 116 (15),3051−3060.(40) Moldovan, G. L., Pfander, B., and Jentsch, S. (2007) PCNA, themaestro of the replication fork. Cell 129, 665−679.(41) Naryzhny, S. N. (2008) Proliferating cell nuclear antigen: Aproteomics view. Cell. Mol. Life Sci. 65 (23), 3789−3808.(42) Tsurimoto, T. (1999) PCNA binding proteins. Frontiers inBioscience 4, d849−858.



(43) Zhuang, Z. H., and Ai, Y. X. (2010) Processivity factor of DNApolymerase and its expanding role in normal and translesion DNAsynthesis. Biochim. Biophys. Acta, Proteins Proteomics 1804 (5), 1081−1093.(44) Dieckman, L. M., Freudenthal, B. D., and Washington, M. T.(2012) PCNA Structure and Function: Insights from Structures ofPCNA Complexes and Post-translationally Modified PCNA. Subcell.Biochem. 62, 281−299.(45) Clark, A. B., et al. (2000) Functional interaction of proliferatingcell nuclear antigen with MSH2-MSH6 and MSH2-MSH3 complexes.J. Biol. Chem. 275 (47), 36498−36501.(46) Kleczkowska, H. E., et al. (2001) hMSH3 and hMSH6 interactwith PCNA and colocalize with it to replication foci. Genes Dev. 15 (6),724−736.(47) Lau, P. J., and Kolodner, R. D. (2003) Transfer of theMSH2·MSH6 complex from proliferating cell nuclear antigen tomispaired bases in DNA. J. Biol. Chem. 278 (1), 14−17.(48) Shell, S. S., Putnam, C. D., and Kolodner, R. D. (2007) The Nterminus of Saccharomyces cerevisiae Msh6 is an unstructured tether toPCNA. Mol. Cell 26 (4), 565−578.(49) Hombauer, H., et al. (2011) Visualization of Eukaryotic DNAMismatch Repair Reveals Distinct Recognition and Repair Inter-mediates. Cell 147 (5), 1040−1053.(50) Pluciennik, A., et al. (2010) PCNA function in the activationand strand direction of MutL alpha endonuclease in mismatch repair.Proc. Natl. Acad. Sci. U. S. A. 107 (37), 16066−16071.(51) Gu, L. Y., et al. (1998) ATP-dependent interaction of humanmismatch repair proteins and dual role of PCNA in mismatch repair.Nucleic Acids Res. 26 (5), 1173−1178.(52) Chen, C., et al. (1999) Saccharomyces carevisiae pol30(proliferating cell nuclear antigen) mutations impair replication fidelityand mismatch repair. Mol. Cell. Biol. 19 (11), 7801−7815.(53) Kokoska, R. J., et al. (1999) A mutation of the yeast geneencoding PCNA destabilizes both microsatellite and minisatellite DNAsequences. Genetics 151 (2), 511−519.(54) Lau, P. J., Flores-Rozas, H., and Kolodner, R. D. (2002)Isolation and characterization of new proliferating cell nuclear antigen(POL30) mutator mutants that are defective in DNA mismatch repair.Mol. Cell. Biol. 22 (19), 6669−6680.(55) Freudenthal, B. D., et al. (2008) Structure of a Mutant Form ofProliferating Cell Nuclear Antigen That Blocks Translesion DNASynthesis. Biochemistry 47 (50), 13354−13361.(56) Finkelstein, J., et al. (2003) Overproduction and analysis ofeukaryotic multiprotein complexes in Escherichia coli using a dual-vector strategy. Anal. Biochem. 319 (1), 78−87.(57) Dieckman, L. M., et al. (2010) Pre-Steady State Kinetic Studiesof the Fidelity of Nucleotide Incorporation by Yeast DNA Polymerasedelta. Biochemistry 49 (34), 7344−7350.(58) Antony, E., and Hingorani, M. M. (2003) Mismatchrecognition-coupled stabilization of Msh2-Msh6 in an ATP-boundstate at the initiation of DNA repair. Biochemistry 42 (25), 7682−7693.(59) Pflugrath, J. W. (1999) The finer things in X-ray diffraction datacollection. Acta Crystallogr., Sect. D: Biol. Crystallogr. 55, 1718−1725.(60) Read, R. J. (2001) Pushing the boundaries of molecularreplacement with maximum likelihood. Acta Crystallogr., Sect. D: Biol.Crystallogr. 57, 1373−1382.(61) Bailey, S. (1994) The Ccp4 Suite - Programs for ProteinCrystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 50, 760−763.(62) Adams, P. D., et al. (2002) PHENIX: Building new software forautomated crystallographic structure determination. Acta Crystallogr.,Sect. D: Biol. Crystallogr. 58, 1948−1954.(63) Emsley, P., and Cowtan, K. (2004) Coot: Model-building toolsfor molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60,2126−2132.(64) Freudenthal, B. D., et al. (2011) Crystal structure of SUMO-modified proliferating cell nuclear antigen. J. Mol. Biol. 406 (1), 9−17.(65) Burgers, P. M. J. (2009) Polymerase Dynamics at the EukaryoticDNA Replication Fork. J. Biol. Chem. 284 (7), 4041−4045.

(66) Kunkel, T. A., and Burgers, P. M. (2008) Dividing the workloadat a eukaryotic replication fork. Trends Cell Biol. 18 (11), 521−527.(67) McElhinny, S. A. N., et al. (2008) Division of labor at theeukaryotic replication fork. Mol. Cell 30 (2), 137−144.(68) Shcherbakova, P. V., Bebenek, K., and Kunkel, T. A. (2003)Functions of eukaryotic DNA polymerases. Sci. Aging Knowl. Environ.2003 (8), RE3.(69) Zhang, H. S., Gibbs, P. E. M., and Lawrence, C. W. (2006) TheSaccharomyces cerevisiae rev6-1 mutation, which inhibits both the lesionbypass and the recombination mode of DNA damage tolerance, is anallele of POL30, encoding proliferating cell nuclear antigen. Genetics173 (4), 1983−1989.(70) Northam, M. R., et al. (2006) A novel function of DNApolymerase zeta regulated by PCNA. EMBO J. 25 (18), 4316−4325.(71) TorresRamos, C. A., et al. (1996) Requirement of proliferatingcell nuclear antigen in RAD6-dependent postreplicational DNA repair.Proc. Natl. Acad. Sci. U. S. A. 93 (18), 9676−9681.(72) Zhang, Z. G., Shibahara, K., and Stillman, B. (2000) PCNAconnects DNA replication to epigenetic inheritance in yeast. Nature408 (6809), 221−225.(73) Sharp, J. A., et al. (2001) Yeast histone deposition protein Asf1prequires Hir proteins and PCNA for heterochromatic silencing. Curr.Biol. 11 (7), 463−473.



distinct structural alterations in proliferating cell nuclear antigen block dna mismatch repair

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