DNA elongation by the human DNA polymerase lambda polymerase and terminal transferase activities are differentially coordinated by proliferating cell nuclear antigen and replication protein A

DNA polymerase lambda contains template-dependent (DNA polymerase) and template-independent (terminal transferase) activities. In this study we enzymologically characterized the terminal transferase activity of polymerase lambda (pol lambda-tdt). Pol lambda-tdt activity was strongly influenced by the nature of the 3'-terminal sequence of the DNA substrate, and it required a single-stranded (ss) DNA 3'-overhang of about 9-12 nucleotides for optimal activity. The strong preference observed for pyrimidine versus purine nucleotide incorporation was found to be due, at least partially, to a steric block imposed by the residue Tyr-505 in the active site of pol lambda. Pol lambda-tdt was found to be able to elongate a 3'-ssDNA end by two alternative mechanisms: first, a template-independent one resulting in addition of 1 or 2 nucleotides, and second, a template-dependent one where a homopolymeric tract as short as 3 nucleotides at the 3'-end could be used as a template to direct DNA polymerization by a looping back mechanism. Furthermore repetitive cycles of DNA synthesis resulted in the expansion of such a short homopolymeric terminal sequence. Most importantly we found that the proliferating cell nuclear antigen was able to selectively block the looping back mechanism while stimulating the single terminal nucleotide addition. Finally replication protein A completely suppressed the transferase activity of pol lambda while stimulating the polymerase activity, suggesting that proliferating cell nuclear antigen and replication protein A can coordinate the polymerase and the terminal transferase activities of pol lambda.     

DNA polymerase stabilization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1

To ensure proper replication and segregation of the genome, eukaryotic cells have evolved surveillance systems that monitor and react to impaired replication fork progression. In budding yeast, the intra-S phase checkpoint responds to stalled replication forks by downregulating late-firing origins, preventing spindle elongation and allowing efficient resumption of DNA synthesis after recovery from stress. Mutations in this pathway lead to high levels of genomic instability, particularly in the presence of DNA damage. Here we demonstrate by chromatin immunoprecipitation that when yeast replication forks stall due to hydroxyurea (HU) treatment, DNA polymerases alpha and epsilon are stabilized for 40-60 min. This requires the activities of Sgs1, a member of the RecQ family of DNA helicases, and the ATM-related kinase Mec1, but not Rad53 activation. A model is proposed whereby Sgs1 helicase resolves aberrantly paired structures at stalled forks to maintain single-stranded DNA that allows RP-A and Mec1 to promote DNA polymerase association.     

DNA polymerase D temporarily connects primase to the CMG-like helicase before interacting with proliferating cell nuclear antigen

The eukaryotic replisome is comprised of three family-B DNA polymerases (Polα, δ and ϵ). Polα forms a stable complex with primase to synthesize short RNA-DNA primers, which are subsequently elongated by Polδ and Polϵ in concert with proliferating cell nuclear antigen (PCNA). In some species of archaea, family-D DNA polymerase (PolD) is the only DNA polymerase essential for cell viability, raising the question of how it alone conducts the bulk of DNA synthesis. We used a hyperthermophilic archaeon, Thermococcus kodakarensis, to demonstrate that PolD connects primase to the archaeal replisome before interacting with PCNA. Whereas PolD stably connects primase to GINS, a component of CMG helicase, cryo-EM analysis indicated a highly flexible PolD–primase complex. A conserved hydrophobic motif at the C-terminus of the DP2 subunit of PolD, a PIP (PCNA-Interacting Peptide) motif, was critical for the interaction with primase. The dissociation of primase was induced by DNA-dependent binding of PCNA to PolD. Point mutations in the alternative PIP-motif of DP2 abrogated the molecular switching that converts the archaeal replicase from de novo to processive synthesis mode.     

Direct interaction between mammalian DNA polymerase beta and proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the DNA replication and DNA repair machinery. As such, PCNA interacts with many proteins that have a sequence motif termed the PCNA interacting motif (PIM) and also with proteins lacking a PIM. Three regions in human and rat DNA polymerases beta (beta-pol) that resemble the consensus PIM were identified, and we show here that beta-polymerase and PCNA can form a complex both in vitro and in vivo. Immunoprecipitation experiments, yeast two-hybrid analysis, and overlay binding assays were used to examine the interaction between the two proteins. Competition experiments with synthetic PIM-containing peptides suggested the importance of a PIM in the interaction, and studies of a beta-polymerase PIM mutant, H222A/F223A, demonstrated that this alteration blocked the interaction with PCNA. The results indicate that at least one of the PIM-like sequences in beta-polymerase appears to be a functional PIM and was required in the interaction between beta-polymerase and PCNA.     

Ubiquitylation of proliferating cell nuclear antigen and recruitment of human DNA polymerase eta

This study investigated the requirement for ubiquitylation of PCNA at lysine 164 during polymerase eta-dependent translesion synthesis (TLS) of site-specific cis-syn cyclobutane thymine dimers (T (wedge)T). The in vitro assay recapitulated origin-dependent initiation, fork assembly, and semiconservative, bidirectional replication of double-stranded circular DNA substrates. A phosphocellulose column was used to fractionate HeLa cell extracts into two fractions; flow-through column fraction I (CFI) contained endogenous PCNA, RPA, ubiquitin-activating enzyme E1, and ubiquitin conjugase Rad6, and eluted column fraction II (CFII) included pol delta, pol eta, and RFC. CFII supplemented with purified recombinant RPA and PCNA (wild type or K164R, in which lysine was replaced with arginine) was competent for DNA replication and TLS. K164R-PCNA complemented CFII for these activities to the same extent and efficiency as wild-type PCNA. CFII mixed with CFI (endogenous PCNA, E1, Rad6) exhibited enhanced DNA replication activity, but the same TLS efficiency determined with the purified proteins. These results demonstrate that PCNA ubiquitylation at K164 of PCNA is not required in vitro for pol eta to gain access to replication complexes at forks stalled by T (wedge)T and to catalyze TLS across this dimer.     

The replication factor C clamp loader requires arginine finger sensors to drive DNA binding and proliferating cell nuclear antigen loading

Replication factor C (RFC) is an AAA+ heteropentamer that couples the energy of ATP binding and hydrolysis to the loading of the DNA polymerase processivity clamp, proliferating cell nuclear antigen (PCNA), onto DNA. RFC consists of five subunits in a spiral arrangement (RFC-A, -B, -C, -D, and -E, corresponding to subunits RFC1, RFC4, RFC3, RFC2, and RFC5, respectively). The RFC subunits are AAA+ family proteins and the complex contains four ATP sites (sites A, B, C, and D) located at subunit interfaces. In each ATP site, an arginine residue from one subunit is located near the gamma-phosphate of ATP bound in the adjacent subunit. These arginines act as "arginine fingers" that can potentially perform two functions: sensing that ATP is bound and catalyzing ATP hydrolysis. In this study, the arginine fingers in RFC were mutated to examine the steps in the PCNA loading mechanism that occur after RFC binds ATP. This report finds that the ATP sites of RFC function in distinct steps during loading of PCNA onto DNA. ATP binding to RFC powers recruitment and opening of PCNA and activates a gamma-phosphate sensor in ATP site C that promotes DNA association. ATP hydrolysis in site D is uniquely stimulated by PCNA, and we propose that this event is coupled to PCNA closure around DNA, which starts an ordered hydrolysis around the ring. PCNA closure severs contact to RFC subunits D and E (RFC2 and RFC5), and the gamma-phosphate sensor of ATP site C is switched off, resulting in low affinity of RFC for DNA and ejection of RFC from the site of PCNA loading.     

Mammalian DNA polymerase auxiliary proteins: analysis of replication factor C-catalyzed proliferating cell nuclear antigen loading onto circular double-stranded DNA.

To understand the mechanism of action of the two eukaryotic replication auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C), we constructed a plasmid for producing PCNA which could be 32P labelled in vitro. This allowed us to analyze the assembly of the auxiliary proteins directly on DNA and to examine this process in the absence of DNA synthesis. By using closed circular double-stranded DNA or gapped circular DNA for protein-DNA complex formation, the following results were obtained, (i) RF-C can load PCNA in an ATP-dependent manner directly on double-stranded DNA, and no 3'-OH ends are required for this reaction; (ii) the RF-C-PCNA complex assembled on closed circular DNA differs from those assembled on gapped or nicked circular DNA; (iii) the stable RF-C-PCNA complex can be assembled on circular but not on linear DNA; and (iv) only gapped DNA can partially retain the assembled RF-C-PCNA complex upon the linearization of the template. We propose that RF-C first binds unspecifically to double-stranded DNA in the presence of ATP and then loads PCNA onto DNA to yield a protein complex able to track along DNA. The RF-C-PCNA complex could slide along the template until it encounters a 3'-OH primer-template junction, where it is likely transformed into a competent clamp. The latter complex, finally, might still be able to slide along double-stranded DNA.     

Mechanism of proliferating cell nuclear antigen clamp opening by replication factor C

The eukaryotic replication factor C (RFC) clamp loader is an AAA+ spiral-shaped heteropentamer that opens and closes the circular proliferating cell nuclear antigen (PCNA) clamp processivity factor on DNA. In this study, we examined the roles of individual RFC subunits in opening the PCNA clamp. Interestingly, Rfc1, which occupies the position analogous to the delta clamp-opening subunit in the Escherichia coli clamp loader, is not required to open PCNA. The Rfc5 subunit is required to open PCNA. Consistent with this result, Rfc2.3.4.5 and Rfc2.5 subassemblies are capable of opening and unloading PCNA from circular DNA. Rfc5 is positioned opposite the PCNA interface from Rfc1, and therefore, its action with Rfc2 in opening PCNA indicates that PCNA is opened from the opposite side of the interface that the E. coli delta wrench acts upon. This marks a significant departure in the mechanism of eukaryotic and prokaryotic clamp loaders. Interestingly, the Rad.RFC DNA damage checkpoint clamp loader unloads PCNA clamps from DNA. We propose that Rad.RFC may clear PCNA from DNA to facilitate shutdown of replication in the face of DNA damage.     

Calf thymus DNA polymerase delta independent of proliferating cell nuclear antigen (PCNA).

DNA polymerase delta from calf thymus was purified under conditions that minimized proteolysis to a specific activity of 27,000 units/mg. The four step isolation procedure included phosphocellulose, hydroxyapatite, heparin-Sepharose and FPLC-MonoS. This enzyme consists of four polypeptides with Mr of 140, 125, 48 and 40 kilodaltons. Velocity gradient sedimentation in glycerol removed the 48 kDa polypeptide while the other three sedimented with the DNA polymerase activity. The biochemical properties of the three subunit enzyme and the copurification of 3'----5' exonuclease activity were typical for a bona fide DNA polymerase delta. Tryptic peptide analysis showed that the 140 kDa polypeptide was different from the catalytic 180 kDa polypeptide of calf thymus DNA polymerase alpha. Both high Mr polypeptides (140 and 125 kDa) were catalytically active as analysed in an activity gel. Four templates were used by DNA polymerase delta with different preferences, namely poly(dA)/oligo(dT)12-18 much much greater than activated DNA greater than poly(dA-dT) greater than primed single-stranded M13DNA. Calf thymus proliferating cell nuclear antigen (PCNA) could not stimulated this DNA polymerase delta in any step of the isolation procedure. If tested on poly(dA)/oligo(dT)12-18 (base ratio 10:1), PCNA had no stimulatory effect on DNA polymerase delta when tested with low enzyme DNA ratio nor did it change the kinetic behaviour of the enzyme. DNA polymerase delta itself did not contain PCNA. The enzyme had an intrinsic processivity of several thousand bases, when tested either on the homopolymer poly(dA)/oligo(dT)12-18 (base ratio 64:1) or on primed single-stranded M13DNA. Contrary to DNA polymerase alpha, no pausing sites were seen with DNA polymerase delta. Under optimal in vitro replication conditions the enzyme could convert primed single-stranded circular M13 DNA of 7,200 bases to its double-stranded form in less than 10 min. This supports that a PCNA independent DNA polymerase delta exists in calf thymus in addition to a PCNA dependent enzyme (Lee, M.Y.W.T. et al. (1984) Biochemistry 23, 1906-1913).     

Synthetic activity of Sso DNA polymerase Y1, an archaeal DinB-like DNA polymerase, is stimulated by processivity factors proliferating cell nuclear antigen and replication factor C

DNA replication efficiency is dictated by DNA polymerases (pol) and their associated proteins. The recent discovery of DNA polymerase Y family (DinB/UmuC/RAD30/REV1 superfamily) raises a question of whether the DNA polymerase activities are modified by accessory proteins such as proliferating cell nuclear antigen (PCNA). In fact, the activity of DNA pol IV (DinB) of Escherichia coli is enhanced upon interaction with the beta subunit, the processivity factor of DNA pol III. Here, we report the activity of Sso DNA pol Y1 encoded by the dbh gene of the archaeon Sulfolobus solfataricus is greatly enhanced by the presence of PCNA and replication factor C (RFC). Sso pol Y1 per se was a distributive enzyme but a substantial increase in the processivity was observed on poly(dA)-oligo(dT) in the presence of PCNA (039p or 048p) and RFC. The length of the synthesized DNA product reached at least 200 nucleotides. Sso pol Y1 displayed a higher affinity for DNA compared with pol IV of E. coli, suggesting that the two DNA polymerases have distinct reason(s) to require the processivity factors for efficient DNA synthesis. The abilities of pol Y1 and pol IV to bypass DNA lesions and their sensitive sites to protease are also discussed.     

Human DNA polymerase epsilon colocalizes with proliferating cell nuclear antigen and DNA replication late, but not early, in S phase.

DNA polymerase epsilon (pol epsilon) has been implicated in DNA replication, DNA repair, and cell cycle control, but its precise roles are unclear. When the subcellular localization of human pol epsilon was examined by indirect immunofluorescence, pol epsilon appeared in discrete nuclear foci that colocalized with proliferating cell nuclear antigen (PCNA) foci and sites of DNA synthesis only late in S phase. Early in S phase, pol epsilon foci were adjacent to PCNA foci. In contrast to PCNA foci that were only present in S phase, pol epsilon foci were present throughout mitosis and the G(1) phase of cycling cells. It is hypothesized from these observations that pol epsilon and PCNA have separate but associated functions early in S phase and that pol epsilon participates with PCNA in DNA replication late in S phase.     

Direct interaction of proliferating cell nuclear antigen with the small subunit of DNA polymerase delta

The interaction between proliferating cell nuclear antigen (PCNA) and DNA polymerase delta is essential for processive DNA synthesis during DNA replication/repair; however, the identity of the subunit of DNA polymerase delta that directly interacts with PCNA has not been resolved until now. In the present study we have used reciprocal co-immunoprecipitation experiments to determine which of the two subunits of core DNA polymerase delta, the 125-kDa catalytic subunit or the 50-kDa small subunit, directly interacts with PCNA. We found that PCNA co-immunoprecipitated with human p50, as well as calf thymus DNA polymerase delta heterodimer, but not with p125 alone, suggesting that PCNA directly interacts with p50 but not with p125. A PCNA-binding motif, similar to the sliding clamp-binding motif of bacteriophage RB69 DNA polymerase, was identified in the N terminus of p50. A 22-amino acid oligopeptide containing this sequence (MRPFL) was shown to bind PCNA by far Western analysis and to compete with p50 for binding to PCNA in co-immunoprecipitation experiments. The binding of p50 to PCNA was inhibited by p21, suggesting that the two proteins compete for the same binding site on PCNA. These results establish that the interaction of PCNA with DNA polymerase delta is mediated through the small subunit of the enzyme.     

Direct rescue of stalled DNA replication forks via the combined action of PriA and RecG helicase activities

The PriA protein of Escherichia coli plays a key role in the rescue of replication forks stalled on the template DNA. One attractive model of rescue relies on homologous recombination to establish a new fork via PriA-mediated loading of the DnaB replicative helicase at D loop intermediates. We provide genetic and biochemical evidence that PriA helicase activity can also rescue a stalled fork by an alternative mechanism that requires manipulation of the fork before loading of DnaB on the lagging strand template. This direct rescue depends on RecG, which unwinds forks and Holliday junctions and interconverts these structures. The combined action of PriA and RecG helicase activities may thus avoid the potential dangers of rescue pathways involving fork breakage and recombination.     

Interplay between DNA polymerase and proliferating cell nuclear antigen switches off base excision repair of uracil and hypoxanthine during replication in archaea

Archaeal family-B DNA polymerases bind tightly to uracil and hypoxanthine (the deamination products of cytosine and adenine), resulting in profound inhibition of DNA replication. Investigation of the mechanism of inhibition, using single-turnover kinetics with polymerase in excess of DNA, indicated that deoxy-NTPs were efficiently bound to the polymerase-DNA complex but very poorly incorporated into the extending chain. Addition of the processivity factor proliferating cell nuclear antigen (PCNA) resulted in increased affinity of the polymerase for all primer-templates, producing extremely tight complexes when uracil (K_d = 16 pM) or hypoxanthine (K_d = 65 pM) was present. Analytical ultracentrifugation confirmed the stability of these complexes and revealed a polymerase/PCNA/DNA stoichiometry of 1:1:1. However, PCNA had no influence on the ability of the polymerase to read through uracil and hypoxanthine, the same kinetic parameters being observed with or without the processivity factor. The specificity constants determined using single-turnover kinetics showed that uracil and hypoxanthine slowed the polymerase by factors of ∼ 5000 and 3000, respectively. Uracil and hypoxanthine are removed from DNA by base excision repair, initiated by uracil-DNA glycosylase and endonuclease V, respectively. Both enzymes are profoundly inhibited by the simultaneous binding of both PCNA and polymerase to primer-templates, with polymerase alone being much less effective. Thus, when the PCNA-polymerase complex encounters uracil/hypoxanthine in DNA templates, base excision repair is switched off, protecting the complex from a repair pathway that is dangerous in the context of single-stranded DNA formed during replication.     

Cell cycle specific plasmid DNA replication in the nuclear extract of Saccharomyces cerevisiae: modulation by replication protein A and proliferating cell nuclear antigen

Plasmid DNA replication in nuclear extracts of Saccharomyces cerevisiae in vitro has been shown to be S-phase specific, similar to that observed in vivo. We report here a reconstituted in vitro system with partially purified replication proteins, purified replication protein A (RPA), and recombinant proliferating cell nuclear antigen (PCNA). Nuclear extracts from S-phase, G(1)-phase, and unsynchronized yeast cells were fractionated by phosphocellulose chromatography. Protein fraction (polymerase fraction) enriched with replication proteins, including DNA polymerases (alpha, delta, etc.), was isolated, which was not capable of in vitro replication of supercoiled plasmid DNA. However, when purified yeast RPA and recombinant PCNA together were added to the polymerase fraction obtained from S-phase synchronized cells, in vitro plasmid DNA replication was restored. In vitro plasmid DNA replication with polymerase fractions from unsynchronized and G(1)-phase cells could not be reconstituted upon addition of purified RPA and PCNA. RPA and PCNA isolated from various phases of the cell cycle complemented the S-phase polymerase pool to the same extent. Reconstituted systems with the S-phase polymerase pool, complemented with either the RPA- and PCNA-containing fraction or purified RPA and recombinant PCNA together, were able to produce replication intermediates (ranging in size from 50 to 1500 bp) similar to that observed with the S-phase nuclear extract. Results presented here demonstrate that both RPA and PCNA are cell cycle-independent in their ability to stimulate in vitro plasmid DNA replication, whereas replication factors in the polymerase fractions are strictly S-phase dependent.     

Recruitment to stalled replication forks of the PriA DNA helicase and replisome-loading activities is essential for survival

PriA, a 3′ → 5′ superfamily 2 DNA helicase, acts to remodel stalled replication forks and as a specificity factor for origin-independent assembly of a new replisome at the stalled fork. The ability of PriA to initiate replication at stalled forked structures ensures complete genome replication and helps to protect the cell from illegitimate recombination events. This review focuses on the activities of PriA and its role in replication fork assembly and maintaining genomic integrity.     

DNA polymerase delta is highly processive with proliferating cell nuclear antigen and undergoes collision release upon completing DNA

In most cells, 100-1000 Okazaki fragments are produced for each replicative DNA polymerase present in the cell. For fast-growing cells, this necessitates rapid recycling of DNA polymerase on the lagging strand. Bacteria produce long Okazaki fragments (1-2 kb) and utilize a highly processive DNA polymerase III (pol III), which is held to DNA by a circular sliding clamp. In contrast, Okazaki fragments in eukaryotes are quite short, 100-250 bp, and thus the eukaryotic lagging strand polymerase does not require a high degree of processivity. The lagging strand polymerase in eukaryotes, polymerase delta (pol delta), functions with the proliferating cell nuclear antigen (PCNA) sliding clamp. In this report, Saccharomyces cerevisiae pol delta is examined on model substrates to gain insight into the mechanism of lagging strand replication in eukaryotes. Surprisingly, we find pol delta is highly processive with PCNA, over at least 5 kb, on Replication Protein A (RPA)-coated primed single strand DNA. The high processivity of pol delta observed in this report contrasts with its role in synthesis of short lagging strand fragments, which require it to rapidly dissociate from DNA at the end of each Okazaki fragment. We find that this dilemma is solved by a "collision release" process in which pol delta ejects from PCNA upon extending a DNA template to completion and running into the downstream duplex. The released pol delta transfers to a new primed site, provided the new site contains a PCNA clamp. Additional results indicate that the collision release mechanism is intrinsic to the pol3/pol31 subunits of the pol delta heterotrimer.     

Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase delta and DNA polymerase epsilon.

The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenesis. Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro, the mutant PCNAs showed defects in DNA synthesis. Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Poldelta, whereas pcna-90 was defective in replication with Pol epsilon. Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol delta and Pol epsilon, respectively. In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Poldelta, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) x oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks.     

Distinct pools of proliferating cell nuclear antigen associated to DNA replication sites interact with the p125 subunit of DNA polymerase delta or DNA ligase I

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and cell cycle control. PCNA is a homotrimeric ring that, when encircling DNA, is not easily extractable. Consequently, the dynamics of protein-protein interactions established by PCNA at DNA replication sites is not well understood. We have used DNase I to release DNA-bound PCNA together with replication proteins including the p125-catalytic subunit of DNA polymerase delta (p125-pol delta), DNA ligase I, cyclin A, and cyclin-dependent kinase 2 (CDK2). Interaction with these proteins was investigated by immunoprecipitation with antibodies binding near the interdomain connector loop or to the C-terminal domain of PCNA, respectively, or with antibodies to p125-pol delta or DNA ligase I. PCNA interaction with p125-pol delta or DNA ligase I was detected only by the latter antibodies, and found to be mutually exclusive. In contrast, antibodies to PCNA co-immunoprecipitated only CDK2. A GST-p21(waf1/cip1) C-terminal peptide displaced p125-pol delta and DNA ligase I, but not CDK2, from PCNA. These results suggest that PCNA trimers bound to DNA during the S phase are organized as distinct pools able to bind selectively different partners. Among them, p125-pol delta and DNA ligase I interact with PCNA in a mutually exclusive manner.