Primer-template Junction Research Articles

The Rev1 translesion synthesis polymerase has multiple distinct DNA binding modes

Rev1 is a eukaryotic DNA polymerase of the Y family involved in translesion synthesis (TLS), a major damage tolerance pathway that allows DNA replication at damaged templates. Uniquely amongst the Y family polymerases, the N-terminal part of Rev1, dubbed the BRCA1 C- terminal homology (BRCT) region, includes a BRCT domain. While most BRCT domains mediate protein–protein interactions, Rev1 contains a predicted α-helix N-terminal to the BRCT domain and in human Replication Factor C (RFC) such a BRCT region endows the protein with DNA binding capacity. Here, we studied the DNA binding properties of yeast and mouse Rev1. Our results show that the BRCT region of Rev1 specifically binds to a 5′ phosphorylated, recessed, primer–template junction. This DNA binding depends on the extra α-helix, N-terminal to the BRCT domain. Surprisingly, a stretch of 20 amino acids N-terminal to the predicted α-helix is also critical for high-affinity DNA binding. In addition to 5′ primer–template junction binding, Rev1 efficiently binds to a recessed 3′ primer–template junction. These dual DNA binding characteristics are discussed in view of the proposed recruitment of Rev1 by 5′ primer–template junctions, downstream of stalled replication forks.

Time Dependence of Switching Between Polymerization and Editing Mode DNA Binding by Klenow Polymerase

Klenow polymerase is the large fragment of DNA polymerase I from E. coli. Klenow possesses 5’-3’ polymerase and intrinsic 3’-5’ exonuclease activities with two distinct active sites that are located ∼30 angstroms apart. During DNA replication, proofreading activity enhances replication fidelity by excising misincorporated nucleotides from the 3’ end of primer strand. The first step of the proofreading process is sending the 3’-primer terminus to the proofreading site, which requires separation of the primer terminus from the template strand. Here we examined the binding of Klenow to matched and mismatched primed-template DNA (pt-DNA) by monitoring the steady state fluorescence intensity change of a single 2-aminopurine base site-specifically substituted in the template strand within the duplex part of pt-DNA. The changes in fluorescence intensity allow us to follow shuttling of the primer terminus between the polymerization and proofreading sites. We have found that partitioning of the primer between the two active sites depends on: 1) the number of mismatched bases at the primer-template junction, 2) the presence or absence of divalent ions, 3) the type of divalent ions, and 4) the time in the presence of the divalent ions. A significant time dependence of the partitioning between the two sites was observed with the matched and single mismatched pt-DNA in the presence of Mg2+. The slow kinetic dependence of the primer partitioning between the polymerization and proofreading sites in Klenow polymerase helps explain previous conflicting reports of the equilibrium partitioning between sites.

DNA conformational changes at the primer-template junction regulate the fidelity of replication by DNA polymerase

Local conformational changes in primer-template (P/T) DNA are involved in the selective incorporation of dNTP by DNA polymerases (DNAP). Here we use near UV CD and fluorescence spectra of pairs of base analogue probes, substituted either at the primer terminus or in the coding region of the template strand, to monitor and interpret conformational changes at and near the coding base of the template in P/T DNA complexes with Klenow fragment (KF) DNAP as the polymerase moves through the nucleotide addition cycle. Incoming dNTPs and rNTPs encounter binary complexes in which the 3'-end of the primer shuttles between the polymerization (pol) and exonuclease (exo) sites of DNAPs, even for perfectly complementary P/T DNA sequences. We have used spectral changes of probes inserted in both strands to monitor this two-state distribution and determine how it depends on the formation of ternary complexes with both complementary ("correct") and noncomplementary ("incorrect") NTPs and on the local sequence of the P/T DNA. The results show that the relative occupancy of the exo and pol sites is coupled to conformational changes in the P/T DNA of the complex that are partially regulated by the incoming NTP. We find that the coding base on the template strand is unperturbed by the binding of incorrect dNTPs, while binding of complementary rNTPs induces a novel template conformation. We conclude that, in addition to its editing function, primer strand occupancy of the 3'-exo site may also serve as a regulatory checkpoint for accurate dNTP selection in DNA synthesis.

Probing the Interaction of Archaeal DNA Polymerases with Deaminated Bases Using X-ray Crystallography and Non-Hydrogen Bonding Isosteric Base Analogues

Archaeal family-B DNA polymerases stall replication on encountering the pro-mutagenic bases uracil and hypoxanthine. This publication describes an X-ray crystal structure of Thermococcus gorgonarius polymerase in complex with a DNA containing hypoxanthine in the single-stranded region of the template, two bases ahead of the primer-template junction. Full details of the specific recognition of hypoxanthine are revealed, allowing a comparison with published data that describe uracil binding. The two bases are recognized by the same pocket, in the N-terminal domain, and make very similar protein-DNA interactions. Specificity for hypoxanthine (and uracil) arises from a combination of polymerase-base hydrogen bonds and shape fit between the deaminated bases and the pocket. The structure with hypoxanthine at position 2 explains the stimulation of the polymerase 3'-5' proofreading exonuclease, observed with deaminated bases at this location. A beta-hairpin element, involved in partitioning the primer strand between the polymerase and exonuclease active sites, inserts between the two template bases at the extreme end of the double-stranded DNA. This denatures the two complementary primer bases and directs the resulting 3' single-stranded extension toward the exonuclease active site. Finally, the relative importance of hydrogen bonding and shape fit in determining selectivity for deaminated bases has been examined using nonpolar isosteres. Affinity for both 2,4-difluorobenzene and fluorobenzimidazole, non-hydrogen bonding shape mimics of uracil and hypoxanthine, respectively, is strongly diminished, suggesting polar protein-base contacts are important. However, residual interaction with 2,4-difluorobenzene is seen, confirming a role for shape recognition.

Continued primer synthesis at stalled replication forks contributes to checkpoint activation.

Stalled replication forks activate and are stabilized by the ATR (ataxia-telangiectasia mutated and Rad3 related)-mediated checkpoint, but ultimately, they must also recover from the arrest. Although primed single-stranded DNA (ssDNA) is sufficient for checkpoint activation, it is still unknown how this signal is generated at a stalled replication fork. Furthermore, it is not clear how recovery and fork restart occur in higher eukaryotes. Using Xenopus laevis egg extracts, we show that DNA replication continues at a stalled fork through the synthesis and elongation of new primers independent of the checkpoint. This synthesis is dependent on the activity of proliferating cell nuclear antigen, Pol-delta, and Pol-epsilon, and it contributes to the phosphorylation of Chk1. We also used defined DNA structures to show that for a fixed amount of ssDNA, increasing the number of primer-template junctions strongly enhances Chk1 phosphorylation. These results suggest that new primers are synthesized at stalled replication forks by the leading and lagging strand polymerases and that accumulation of these primers may contribute to checkpoint activation.

RPA physically interacts with the human DNA glycosylase NEIL1 to regulate excision of oxidative DNA base damage in primer-template structures

The human DNA glycosylase NEIL1, activated during the S-phase, has been shown to excise oxidized base lesions in single-strand DNA substrates. Furthermore, our previous work demonstrating functional interaction of NEIL1 with PCNA and flap endonuclease 1 (FEN1) suggested its involvement in replication-associated repair. Here we show interaction of NEIL1 with replication protein A (RPA), the heterotrimeric single-strand DNA binding protein that is essential for replication and other DNA transactions. The NEIL1 immunocomplex isolated from human cells contains RPA, and its abundance in the complex increases after exposure to oxidative stress. NEIL1 directly interacts with the large subunit of RPA ( K d ∼20 nM) via the common interacting interface (residues 312–349) in NEIL1's disordered C-terminal region. RPA inhibits the base excision activity of both wild-type NEIL1 (389 residues) and its C-terminal deletion CΔ78 mutant (lacking the interaction domain) for repairing 5-hydroxyuracil (5-OHU) in a primer-template structure mimicking the DNA replication fork. This inhibition is reduced when the damage is located near the primer-template junction. Contrarily, RPA moderately stimulates wild-type NEIL1 but not the CΔ78 mutant when 5-OHU is located within the duplex region. While NEIL1 is inhibited by both RPA and Escherichia coli single-strand DNA binding protein, only inhibition by RPA is relieved by PCNA. These results showing modulation of NEIL1's activity on single-stranded DNA substrate by RPA and PCNA support NEIL1's involvement in repairing the replicating genome.

The 3'-5' proofreading exonuclease of archaeal family-B DNA polymerase hinders the copying of template strand deaminated bases

Archaeal family B polymerases bind tightly to the deaminated bases uracil and hypoxanthine in single-stranded DNA, stalling replication on encountering these pro-mutagenic deoxynucleosides four steps ahead of the primer–template junction. When uracil is specifically bound, the polymerase–DNA complex exists in the editing rather than the polymerization conformation, despite the duplex region of the primer-template being perfectly base-paired. In this article, the interplay between the 3′–5′ proofreading exonuclease activity and binding of uracil/hypoxanthine is addressed, using the family-B DNA polymerase from Pyrococcus furiosus. When uracil/hypoxanthine is bound four bases ahead of the primer–template junction (+4 position), both the polymerase and the exonuclease are inhibited, profoundly for the polymerase activity. However, if the polymerase approaches closer to the deaminated bases, locating it at +3, +2, +1 or even 0 (paired with the extreme 3′ base in the primer), the exonuclease activity is strongly stimulated. In these situations, the exonuclease activity is actually stronger than that seen with mismatched primer-templates, even though the deaminated base-containing primer-templates are correctly base-paired. The resulting exonucleolytic degradation of the primer serves to move the uracil/hypoxanthine away from the primer–template junction, restoring the stalling position to +4. Thus the 3′–5′ proofreading exonuclease contributes to the inability of the polymerase to replicate beyond deaminated bases.

Local Conformations and Competitive Binding Affinities of Single- and Double-stranded Primer-Template DNA at the Polymerization and Editing Active Sites of DNA Polymerases

In addition to their capacity for template-directed 5' --> 3' DNA synthesis at the polymerase (pol) site, DNA polymerases have a separate 3' --> 5' exonuclease (exo) editing activity that is involved in assuring the fidelity of DNA replication. Upon misincorporation of an incorrect nucleotide residue, the 3' terminus of the primer strand at the primer-template (P/T) junction is preferentially transferred to the exo site, where the faulty residue is excised, allowing the shortened primer to rebind to the template strand at the pol site and incorporate the correct dNTP. Here we describe the conformational changes that occur in the primer strand as it shuttles between the pol and exo sites of replication-competent Klenow and Klentaq DNA polymerase complexes in solution and use these conformational changes to measure the equilibrium distribution of the primer between these sites for P/T DNA constructs carrying both matched and mismatched primer termini. To this end, we have measured the fluorescence and circular dichroism spectra at wavelengths of >300 nm for conformational probes comprising pairs of 2-aminopurine bases site-specifically replacing adenine bases at various positions in the primer strand of P/T DNA constructs bound to DNA polymerases. Control experiments that compare primer conformations with available x-ray structures confirm the validity of this approach. These distributions and the conformational changes in the P/T DNA that occur during template-directed DNA synthesis in solution illuminate some of the mechanisms used by DNA polymerases to assure the fidelity of DNA synthesis.

Kinetic Analysis of Arginine-Finger Motif Function in S. cerevisiae Clamp Loader

Replication factor C (RFC) loads a circular clamp, proliferating cell nuclear antigen (PCNA), onto a primer-template junction at the replication fork during DNA replication. RFC consists of five subunits, A-E, each of which belongs to the AAA+ family member of ATPases. The subunits are arranged in an open ring form with four ATPase sites located at the interfaces of subunits A-B, B-C, C-D and D-E. In each ATPase site, an Arginine-finger motif (SRC), which is provided by the adjacent subunit, is implicated in sensing ATP binding and regulating ATP hydrolysis. According to a recent study of SRC→SAC point mutations, it appears that RFC subunits play distinct roles during clamp assembly (Johnson, et al. 2006). We analyzed these mutants by measuring their pre-steady state functions of DNA binding and ATPase activities. We found that CSAC (Binactive) and DSAC (Cinactive) mutants have severe defects in DNA binding, while BSAC (Ainactive) and ESAC (Dinactive) mutants have severe defects in DNA dissociation. These disruptions in RFC interactions with DNA, and corresponding effects of RFC ATPase activities, elucidate further the distinct functions of individual RFC subunits in the clamp assembly reaction.Reference: Johnson A. et al., (2006) J. Biol. Chem. 281, 35531-35543.

Spectroscopic Studies of Position-specific DNA ‘Breathing’ Fluctuations at Replication Forks and Primer-Template Junctions

The properties of single- (ss) and double-stranded (ds) DNA sequences at replication forks and primer-template DNA junctions are central to the function of the protein complexes that drive DNA replication, transcription, recombination and repair. Significant base-pair ‘breathing’ (or ‘fraying’) occurs at these ss-dsDNA junctions and the physiological consequences of this ‘breathing’ is not well understood, in part because it has been difficult to measure in a sequence-specific manner in biological systems. We have used single and dimer pairs of 2-aminopurine (2-AP) residues, site-specifically placed at various positions on both sides of the ss-dsDNA junctions of DNA constructs, as spectroscopic probes of this breathing. Replacing adenosine with 2-AP in DNA has minimal biological or physical consequences, and these moieties absorb, fluoresce and display CD spectra at wavelengths >300 nm where other nucleic acid and protein components are transparent. The optical properties of these constructs have been used to measure the position-specific extent and nature of the fluctuations of forked and P/T DNA junctions. We find that spectroscopically measurable melting penetrates ∼2 bps into the interior of the duplex region of these junctions under physiological conditions. In addition 2-AP bases in ssDNA loci directly adjacent to these junctions display significantly more unstacked character than do 2-AP probes located within long ssDNA sequences. Quenching of 2-AP fluorescence with acrylamide has independently confirmed these results. These local and transient DNA conformations have possible biological significance as interaction targets for DNA-manipulating enzymes, and we are using these breathing properties of DNA in isolation as a platform to study helicase activity on duplex DNA and ultimately the coupling of these helicases to the other components of the ‘macromolecular machines of gene expression’.

DNA models of trinucleotide frameshift deletions: the formation of loops and bulges at the primer–template junction

Although mechanisms of single-nucleotide residue deletion have been investigated, processes involved in the loss of longer nucleotide sequences during DNA replication are poorly understood. Previous reports have shown that in vitro replication of a 3′-TGC TGC template sequence can result in the deletion of one 3′-TGC. We have used low-energy circular dichroism (CD) and fluorescence spectroscopy to investigate the conformations and stabilities of DNA models of the replication intermediates that may be implicated in this frameshift. Pyrrolocytosine or 2-aminopurine residues, site-specifically substituted for cytosine or adenine in the vicinity of extruded base sequences, were used as spectroscopic probes to examine local DNA conformations. An equilibrium mixture of four hybridization conformations was observed when template bases looped-out as a bulge, i.e. a structure flanked on both sides by duplex DNA. In contrast, a single-loop structure with an unusual unstacked DNA conformation at its downstream edge was observed when the extruded bases were positioned at the primer–template junction, showing that misalignments can be modified by neighboring DNA secondary structure. These results must be taken into account in considering the genetic and biochemical mechanisms of frameshift mutagenesis in polymerase-driven DNA replication.

Catalysis of Strand Annealing by Replication Protein A Derives from Its Strand Melting Properties

Eukaryotic DNA-binding protein replication protein A (RPA) has a strand melting property that assists polymerases and helicases in resolving DNA secondary structures. Curiously, previous results suggested that human RPA (hRPA) promotes undesirable recombination by facilitating annealing of flaps produced transiently during DNA replication; however, the mechanism was not understood. We designed a series of substrates, representing displaced DNA flaps generated during maturation of Okazaki fragments, to investigate the strand annealing properties of RPA. Until cleaved by FEN1 (flap endonuclease 1), such flaps can initiate homologous recombination. hRPA inhibited annealing of strands lacking secondary structure but promoted annealing of structured strands. Apparently, both processes primarily derive from the strand melting properties of hRPA. These properties slowed the spontaneous annealing of unstructured single strands, which occurred efficiently without hRPA. However, structured strands without hRPA displayed very slow spontaneous annealing because of stable intramolecular hydrogen bonding. hRPA appeared to transiently melt the single strands so that they could bind to form double strands. In this way, melting ironically promoted annealing. Time course measurements in the presence of hRPA suggest that structured single strands achieve an equilibrium with double strands, a consequence of RPA driving both annealing and melting. Promotion of annealing reached a maximum at a specific hRPA concentration, presumably when all structured single-stranded DNA was melted. Results suggest that displaced flaps with secondary structure formed during Okazaki fragment maturation can be melted by hRPA and subsequently annealed to a complementary ectopic DNA site, forming recombination intermediates that can lead to genomic instability.

Motion of a DNA Sliding Clamp Observed by Single Molecule Fluorescence Spectroscopy

DNA sliding clamps attach to polymerases and slide along DNA to allow rapid, processive replication of DNA. These clamps contain many positively charged residues that could curtail the sliding due to attractive interactions with the negatively charged DNA. By single-molecule spectroscopy we have observed a fluorescently labeled sliding clamp (polymerase III beta subunit or beta clamp) loaded onto freely diffusing, single-stranded M13 circular DNA annealed with fluorescently labeled DNA oligomers of up to 90 bases. We find that the diffusion constant for the beta clamp diffusing along DNA is on the order of 10(-14) m(2)/s, at least 3 orders of magnitude less than that for diffusion through water alone. We also find evidence that the beta clamp remains at the 3' end in the presence of Escherichia coli single-stranded-binding protein. These results may imply that the clamp not only acts to hold the polymerase on the DNA but also prevents excessive drifting along the DNA.

Conserved residues in the δ subunit help the E. coli clamp loader, γ complex, target primer-template DNA for clamp assembly

The Escherichia coli clamp loader, γ complex (γ3δδ′λψ), catalyzes ATP-driven assembly of β clamps onto primer-template DNA (p/tDNA), enabling processive replication. The mechanism by which γ complex targets p/tDNA for clamp assembly is not resolved. According to previous studies, charged/polar amino acids inside the clamp loader chamber interact with the double-stranded (ds) portion of p/tDNA. We find that dsDNA, not ssDNA, can trigger a burst of ATP hydrolysis by γ complex and clamp assembly, but only at far higher concentrations than p/tDNA. Thus, contact between γ complex and dsDNA is necessary and sufficient, but not optimal, for the reaction, and additional contacts with p/tDNA likely facilitate its selection as the optimal substrate for clamp assembly. We investigated whether a conserved sequence—HRVW279QNRR—in δ subunit contributes to such interactions, since Tryptophan-279 specifically cross-links to the primer-template junction. Mutation of δ-W279 weakens γ complex binding to p/tDNA, hampering its ability to load clamps and promote proccessive DNA replication, and additional mutations in the sequence (δ-R277, δ-R283) worsen the interaction. These data reveal a novel location in the C-terminal domain of the E. coli clamp loader that contributes to DNA binding and helps define p/tDNA as the preferred substrate for the reaction.

DNA Damage-induced Ubiquitylation of RFC2 Subunit of Replication Factor C Complex

Many proteins involved in DNA replication and repair undergo post-translational modifications such as phosphorylation and ubiquitylation. Proliferating cell nuclear antigen (PCNA; a homotrimeric protein that encircles double-stranded DNA to function as a sliding clamp for DNA polymerases) is monoubiquitylated by the RAD6-RAD18 complex and further polyubiquitylated by the RAD5-MMS2-UBC13 complex in response to various DNA-damaging agents. PCNA mono- and polyubiquitylation activate an error-prone translesion synthesis pathway and an error-free pathway of damage avoidance, respectively. Here we show that replication factor C (RFC; a heteropentameric protein complex that loads PCNA onto DNA) was also ubiquitylated in a RAD18-dependent manner in cells treated with alkylating agents or H(2)O(2). A mutant form of RFC2 with a D228A substitution (corresponding to a yeast Rfc4 mutation that reduces an interaction with replication protein A (RPA), a single-stranded DNA-binding protein) was heavily ubiquitylated in cells even in the absence of DNA damage. Furthermore RFC2 was ubiquitylated by the RAD6-RAD18 complex in vitro, and its modification was inhibited in the presence of RPA. The inhibitory effect of RPA on RFC2 ubiquitylation was relatively specific because RAD6-RAD18-mediated ubiquitylation of PCNA was RPA-insensitive. Our findings suggest that RPA plays a regulatory role in DNA damage responses via repression of RFC2 ubiquitylation in human cells.

Site-directed Mutations of T4 Helicase Loading Protein (gp59) Reveal Multiple Modes of DNA Polymerase Inhibition and the Mechanism of Unlocking by gp41 Helicase

The T4 helicase loading protein (gp59) interacts with a multitude of DNA replication proteins. In an effort to determine the functional consequences of these protein-protein interactions, point mutations were introduced into the gp59 protein. Mutations were chosen based on the available crystal structure and focused on hydrophobic residues with a high degree of solvent accessibility. Characterization of the mutant proteins revealed a single mutation, Y122A, which is defective in polymerase binding and has weakened affinity for the helicase. The interaction between single-stranded DNA-binding protein and Y122A is unaffected, as is the affinity of Y122A for DNA substrates. When standard concentrations of helicase are employed, Y122A is unable to productively load the helicase onto forked DNA substrates. As a result of the loss of polymerase binding, Y122A cannot inhibit the polymerase during nucleotide idling or prevent it from removing the primer strand of a D-loop. However, Y122A is capable of inhibiting strand displacement synthesis by polymerase. The retention of strand displacement inhibition by Y122A, even in the absence of a gp59-polymerase interaction, indicates that there are two modes of polymerase inhibition by gp59. Inhibition of the polymerase activity only requires gp59 to bind to the replication fork, whereas inhibition of the exonuclease activity requires an interaction between the polymerase and gp59. The inability of Y122A to interact with both the polymerase and the helicase suggests a mechanism for polymerase unlocking by the helicase based on a direct competition between the helicase and polymerase for an overlapping binding site on gp59.

Phosphorylation of replication protein A by S-phase checkpoint kinases

The major eukaryotic single-stranded DNA (ssDNA) binding protein, replication protein A (RPA), is a heterotrimer with subunits of 70, 32 and 14 kDa (RPA70, RPA32 and RPA14). RPA-coated ssDNA has been implicated as one of the triggers for intra-S-phase checkpoint activation. Phosphorylation of RPA occurs in cells with damaged DNA or stalled replication forks. Here we show that human RPA70 and RPA32 can be phosphorylated by purified S-phase checkpoint kinases, ATR and Chk1. While ATR phosphorylates the N-terminus of RPA70, Chk1 preferentially phosphorylates RPA's major ssDNA binding domain. Chk1 phosphorylated RPA70 shows reduced ssDNA binding activity, and binding of RPA to ssDNA blocks Chk1 phosphorylation, suggesting that Chk1 and ssDNA compete for RPA's major ssDNA binding domain. ssDNA stimulates RPA32 phosphorylation by ATR in a length dependent manner. Furthermore, 3′-, but not 5′-, recessed single strand/double strand DNA junctions produce an even stronger stimulatory effect on RPA32 phosphorylation by ATR. This stimulation occurs for both RNA and DNA recessed ends. RPA's DNA binding polarity and its interaction to 3′-primer-template junctions contribute to efficient RPA32 phosphorylation. Progression of DNA polymerase is able to block the accessibility of the 3′-recessed ends and prevent the stimulatory effects of primer-template junctions on RPA phosphorylation by ATR. We propose models for the role of RPA phosphorylation by Chk1 in S-phase checkpoint pathways, and the possible regulation of ATR activity by different nucleic acid structures.

Analysis of Interactions Between Mismatch Repair Initiation Factors and the Replication Processivity Factor PCNA

In eukaryotes, the DNA replication factor PCNA is loaded onto primer-template junctions to act as a processivity factor for DNA polymerases. Genetic and biochemical studies suggest that PCNA also functions in early steps in mismatch repair (MMR) to facilitate the repair of misincorporation errors generated during DNA replication. These studies have shown that PCNA interacts directly with several MMR components, including MSH3, MSH6, MLH1, and EXO1. At present, little is known about how these interactions contribute to the mismatch repair mechanism. The interaction between MLH1 and PCNA is of particular interest because MLH1-PMS1 is thought to act as a matchmaker to signal mismatch recognition to downstream repair events; in addition, PCNA has been hypothesized to act in strand discrimination steps in MMR. Here, we utilized both genetic and surface plasmon resonance techniques to characterize the MLH1-PMS1-PCNA interaction. These analyses enabled us to determine the stability of the complex (K(D) = 300 nM) and to identify residues (572-579) in MLH1 and PCNA (126,128) that appear important to maintain this stability. We favor a model in which PCNA acts as a scaffold for consecutive protein-protein interactions that allow for the coordination of MMR steps.

Open clamp structure in the clamp-loading complex visualized by electron microscopic image analysis

Ring-shaped sliding clamps and clamp loader ATPases are essential factors for rapid and accurate DNA replication. The clamp ring is opened and resealed at the primer-template junctions by the ATP-fueled clamp loader function. The processivity of the DNA polymerase is conferred by its attachment to the clamp loaded onto the DNA. In eukarya and archaea, the replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) play crucial roles as the clamp loader and the clamp, respectively. Here, we report the electron microscopic structure of an archaeal RFC-PCNA-DNA complex at 12-A resolution. This complex exhibits excellent fitting of each atomic structure of RFC, PCNA, and the primed DNA. The PCNA ring retains an open conformation by extensive interactions with RFC, with a distorted spring washer-like conformation. The complex appears to represent the intermediate, where the PCNA ring is kept open before ATP hydrolysis by RFC.

DNA polymerase clamp loaders and DNA recognition

Clamp loaders are heteropentameric ATPase assemblies that load sliding clamps onto DNA and are critical for processive DNA replication. The DNA targets for clamp loading are double-stranded/single-stranded junctions with recessed 3′ ends (primer-template junctions). Here, we briefly review the crystal structures of clamp loader complexes and the insights they have provided into the mechanism of the clamp loading process.