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.     

Loading clamps for DNA replication and repair

Sliding clamps and clamp loaders were initially identified as DNA polymerase processivity factors. Sliding clamps are ring-shaped protein complexes that encircle and slide along duplex DNA, and clamp loaders are enzymes that load these clamps onto DNA. When bound to a sliding clamp, DNA polymerases remain tightly associated with the template being copied, but are able to translocate along DNA at rates limited by rates of nucleotide incorporation. Many different enzymes required for DNA replication and repair use sliding clamps. Clamps not only increase the processivity of these enzymes, but may also serve as an attachment point to coordinate the activities of enzymes required for a given process. Clamp loaders are members of the AAA+ family of ATPases and use energy from ATP binding and hydrolysis to catalyze the mechanical reaction of loading clamps onto DNA. Many structural and functional features of clamps and clamp loaders are conserved across all domains of life. Here, the mechanism of clamp loading is reviewed by comparing features of prokaryotic and eukaryotic clamps and clamp loaders.     

Replisome dynamics and use of DNA trombone loops to bypass replication blocks

Replisomes are dynamic multiprotein machines capable of simultaneously replicating both strands of the DNA duplex. This review focuses on the structure and function of the E. coli replisome, many features of which generalize to other bacteria and eukaryotic cells. For example, the bacterial replisome utilizes clamps and clamp loaders to coordinate the actions required of the trombone model of lagging strand synthesis made famous by Bruce Alberts. All cells contain clamps and clamp loaders and this review summarizes their structure and function. Clamp loaders are pentameric spirals that bind DNA in a structure specific fashion and thread it through the ring shaped clamp. The recent structure of the E. coli beta clamp in complex with primed DNA has implications for how multiple polymerases function on sliding clamps and how the primed DNA template is exchanged between them. Recent studies reveal a remarkable fluidity in replisome function that enables it to bypass template lesions on either DNA strand. During these processes the polymerases within the replisome functionally uncouple from one another. Mechanistic processes that underlie these actions may involve DNA looping, similar to the trombone loops that mediate the lagging strand Okazaki fragment synthesis cycle.     

Motors and switches: AAA+ machines within the replisome

Clamp loaders are required to load the ring-shaped clamps that tether replicative DNA polymerases onto DNA. Recently solved crystal structures, along with a series of biochemical studies, have provided a detailed understanding of the clamp loading reaction. In particular, studies of the Escherichia coli clamp loader--an AAA+ machine--have provided insights into the architecture of clamp loaders from eukaryotes, bacteriophage T4 and archaea. Other AAA+ proteins are also involved in the initiation of DNA replication, and studies of the E. coli clamp loader indicate mechanisms by which these proteins might function.     

Dynamics of Loading the Escherichia coli DNA Polymerase Processivity Clamp

ABSTRACT

Sliding clamps and clamp loaders are processivity factors required for efficient DNA replication. Sliding clamps are ring-shaped complexes that tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp loaders assemble these ring-shaped clamps onto DNA in an ATP-dependent reaction. The overall process of clamp loading is dynamic in that protein–protein and protein–DNA interactions must actively change in a coordinated fashion to complete the mechanical clamp-loading reaction cycle. The clamp loader must initially have a high affinity for both the clamp and DNA to bring these macromolecules together, but then must release the clamp on DNA for synthesis to begin. Evidence is presented for a mechanism in which the clamp-loading reaction comprises a series of binding reactions to ATP, the clamp, DNA, and ADP, each of which promotes some change in the conformation of the clamp loader that alters interactions with the next component of the pathway. These changes in interactions must be rapid enough to allow the clamp loader to keep pace with replication fork movement. This review focuses on the measurement of dynamic and transient interactions required to assemble the Escherichia coli sliding clamp on DNA.     

Biochemical and mutational analyses of a unique clamp loader complex in the archaeon Methanosarcina acetivorans

Clamp loaders orchestrate the switch from distributive to processive DNA synthesis. Their importance in cellular processes is underscored by their conservation across all forms of life. Here, we describe a new form of clamp loader from the archaeon Methanosarcina acetivorans. Unlike previously described archaeal clamp loaders, which are composed of one small subunit and one large subunit, the M. acetivorans clamp loader comprises two similar small subunits (M. acetivorans replication factor C small subunit (MacRFCS)) and one large subunit (MacRFCL). The relatedness of the archaeal and eukaryotic clamp loaders (which are made up of four similar small subunits and one large subunit) suggests that the M. acetivorans clamp loader may be an intermediate form in the archaeal/eukaryotic sister lineages. The clamp loader complex reconstituted from the three subunits MacRFCS1, MacRFCS2, and MacRFCL stimulated DNA synthesis by a cognate DNA polymerase in the presence of its sliding clamp. We used site-directed mutagenesis in the Walker A and SRC motifs to examine the contribution of each subunit to the function of the M. acetivorans clamp loader. Although mutations in MacRFCL and MacRFCS2 did not impair clamp loading activity, any mutant clamp loader harboring a mutation in MacRFCS1 was devoid of the clamp loading property. Mac-RFCS1 is therefore critical to the clamp loading activity of the M. acetivorans clamp loader. It is our anticipation that the discovery of this unique replication factor C homolog will lead to critical insights into the evolution of more complex clamp loaders from simpler ones as more complex organisms evolved in the archaeal/eukaryotic sister lineages.     

跨损伤合成的DNA聚合酶——一类新的DNA聚合酶

细胞虽然拥有多种修复途径,但有些DNA损伤仍不可避免地会逃避修复而在基因组上保留下来,细胞跨损伤DNA合成的分子机制一直是DNA修复中主要的未解决问题之一.最近通过对一类结构相关性UmuC/DinB蛋白质超家族成员的研究发现它们具有DNA聚合酶功能.这类新发现的DNA聚合酶不同于经典的复制性DNA聚合酶,它们能以易误/突变(error-prone/mutagenic)或无误(error-free)方式进行跨损伤(translesion)DNA合成,并且从细菌到人在进化上功能保守.     

The interplay of primer-template DNA phosphorylation status and single-stranded DNA binding proteins in directing clamp loaders to the appropriate polarity of DNA

Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3′OH (3′DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3′DNA, fluorescent β and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5′phosphate (5′P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5′DNA). The 5′P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3′DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5′DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3′DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role.     

Dynamics of loading the Escherichia coli DNA polymerase processivity clamp

Sliding clamps and clamp loaders are processivity factors required for efficient DNA replication. Sliding clamps are ring-shaped complexes that tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp loaders assemble these ring-shaped clamps onto DNA in an ATP-dependent reaction. The overall process of clamp loading is dynamic in that protein-protein and protein-DNA interactions must actively change in a coordinated fashion to complete the mechanical clamp-loading reaction cycle. The clamp loader must initially have a high affinity for both the clamp and DNA to bring these macromolecules together, but then must release the clamp on DNA for synthesis to begin. Evidence is presented for a mechanism in which the clamp-loading reaction comprises a series of binding reactions to ATP, the clamp, DNA, and ADP, each of which promotes some change in the conformation of the clamp loader that alters interactions with the next component of the pathway. These changes in interactions must be rapid enough to allow the clamp loader to keep pace with replication fork movement. This review focuses on the measurement of dynamic and transient interactions required to assemble the Escherichia coli sliding clamp on DNA.     

Accessory proteins assist exonuclease-deficient bacteriophage T4 DNA polymerase in replicating past an abasic site

Replicative DNA polymerases, such as T4 polymerase, possess both elongation and 3'-5' exonuclease proofreading catalytic activities. They arrest at the base preceding DNA damage on the coding DNA strand and specialized DNA polymerases have evolved to replicate across the lesion by a process known as TLS (translesion DNA synthesis). TLS is considered to take place in two steps that often require different enzymes, insertion of a nucleotide opposite the damaged template base followed by extension from the inserted nucleotide. We and others have observed that inactivation of the 3'-5' exonuclease function of T4 polymerase enables TLS across a single site-specific abasic [AP (apurinic/apyrimidinic)] lesion. In the present study we report a role for auxiliary replicative factors in this reaction. When replication is performed with a large excess of DNA template over DNA polymerase in the absence of auxiliary factors, the exo- polymerase (T4 DNA polymerase deficient in the 3'-5' exonuclease activity) inserts one nucleotide opposite the AP site but does not extend past the lesion. Addition of the clamp processivity factor and the clamp loader complex restores primer extension across an AP lesion on a circular AP-containing DNA substrate by the exo- polymerase, but has no effect on the wild-type enzyme. Hence T4 DNA polymerase exhibits a variety of responses to DNA damage. It can behave as a replicative polymerase or (in the absence of proofreading activity) as a specialized DNA polymerase and carry out TLS. As a specialized polymerase it can function either as an inserter or (with the help of accessory proteins) as an extender. The capacity to separate these distinct functions in a single DNA polymerase provides insight into the biochemical requirements for translesion DNA synthesis.     

Replication of damaged DNA by translesion synthesis in human cells

Most types of DNA damage block the passage of the replication machinery. In order to bypass these blocks, cells employ special translesion synthesis (TLS) DNA polymerases, which have lower stringency than replicative polymerases. DNA polymerase eta is the major polymerase responsible for bypassing UV lesions in DNA and its absence results in the variant form of the genetic disorder, xeroderma pigmentosum. Other TLS polymerases have specificities for different types of damage, but their precise roles inside the cell have not yet been established. These polymerases are located in replication factories during DNA replication and the polymerase sliding clamp PCNA plays an important role in mediating switching between different polymerases.     

Clamp loaders and replication initiation

Clamp loaders are ATP-driven multiprotein machines that couple ATP hydrolysis to the opening and closing of a circular protein ring around DNA. This ring-shaped clamp slides along DNA, and interacts with numerous proteins involved in DNA replication, DNA repair and cell cycle control. Recently determined structures of clamp loader complexes from prokaryotic and eukaryotic sources have revealed exciting new details of how these complex AAA+ machines perform this essential clamp loading function.     

Dual functions, clamp opening and primer-template recognition, define a key clamp loader subunit

Clamp loader proteins catalyze assembly of circular sliding clamps on DNA to enable processive DNA replication. During the reaction, the clamp loader binds primer-template DNA and positions it in the center of a clamp to form a topological link between the two. Clamp loaders are multi-protein complexes, such as the five protein Escherichia coli, Saccharomyces cerevisiae, and human clamp loaders, and the two protein Pyrococcus furiosus and Methanobacterium thermoautotrophicum clamp loaders, and thus far the site(s) responsible for binding and selecting primer-template DNA as the target for clamp assembly remain unknown. To address this issue, we analyzed the interaction between the E.coli gamma complex clamp loader and DNA using UV-induced protein-DNA cross-linking and mass spectrometry. The results show that the delta subunit in the gamma complex makes close contact with the primer-template junction. Tryptophan 279 in the delta C-terminal domain lies near the 3'-OH primer end and may play a key role in primer-template recognition. Previous studies have shown that delta also binds and opens the beta clamp (hydrophobic residues in the N-terminal domain of delta contact beta. The clamp-binding and DNA-binding sites on delta appear positioned for facile entry of primer-template into the center of the clamp and exit of the template strand from the complex. A similar analysis of the S.cerevisiae RFC complex suggests that the dual functionality observed for delta in the gamma complex may be true also for clamp loaders from other organisms.     

The Escherichia coli clamp loader can actively pry open the β-sliding clamp

Clamp loaders load ring-shaped sliding clamps onto DNA. Once loaded onto DNA, sliding clamps bind to DNA polymerases to increase the processivity of DNA synthesis. To load clamps onto DNA, an open clamp loader-clamp complex must form. An unresolved question is whether clamp loaders capture clamps that have transiently opened or whether clamp loaders bind closed clamps and actively open clamps. A simple fluorescence-based clamp opening assay was developed to address this question and to determine how ATP binding contributes to clamp opening. A direct comparison of real time binding and opening reactions revealed that the Escherichia coli γ complex binds β first and then opens the clamp. Mutation of conserved "arginine fingers" in the γ complex that interact with bound ATP decreased clamp opening activity showing that arginine fingers make an important contribution to the ATP-induced conformational changes that allow the clamp loader to pry open the clamp.     

DNA Replication-Coupled PCNA Mono-Ubiquitination and Polymerase Switching in a Human InVitro System

Translesion DNA synthesis is a mechanism of DNA damage tolerance, and mono-ubiquitination of proliferating cell nuclear antigen (PCNA) is considered to play a key role in regulating the switch from replicative to translesion DNA polymerases (pols). In this study, we analyzed effects of a replicative pol δ on PCNA mono-ubiquitination with the ubiquitin-conjugating enzyme and ligase UBE2A/HHR6A/RAD6A-RAD18. The results revealed that PCNA interacting with pol δ is a better target for ubiquitination, and PCNA mono-ubiquitination could be coupled with DNA replication. Consequently, we could reconstitute replication-coupled switching between pol δ and a translesion pol, pol η, on an ultraviolet-light-irradiated template. With this system, we obtained direct evidence that polymerase switching reactions are stimulated by mono-ubiquitination of PCNA, depending on a function of the ubiquitin binding zinc finger domain of pol η. This study provides a framework for detailed analyses of molecular mechanisms of human pol switching and regulation of translesion DNA synthesis.     

The ATP Sites of AAA+ Clamp Loaders Work Together as a Switch to Assemble Clamps on DNA

Clamp loaders belong to a family of proteins known as ATPases associated with various cellular activities (AAA+). These proteins utilize the energy from ATP binding and hydrolysis to perform cellular functions. The clamp loader is required to load the clamp onto DNA for use by DNA polymerases to increase processivity. ATP binding and hydrolysis are coordinated by several key residues, including a conserved Lys located within the Walker A motif (or P-loop). This residue is required for each subunit to bind ATP. The specific function of each ATP molecule bound to the Saccharomyces cerevisiae clamp loader is unknown. A series of point mutants, each lacking a single Walker A Lys residue, was generated to study the effects of abolishing ATP binding in individual clamp loader subunits. A variety of biochemical assays were used to analyze the function of ATP binding during discrete steps of the clamp loading reaction. All mutants reduced clamp binding/opening to different degrees. Decreased clamp binding activity was generally correlated with decreases in the population of open clamps, suggesting that differences in the binding affinities of Walker A mutants stem from differences in stabilization of proliferating cell nuclear antigen in an open conformation. Walker A mutations had a smaller effect on DNA binding than clamp binding/opening. Our data do not support a model in which each ATP site functions independently to regulate a different step in the clamp loading cycle to coordinate these steps. Instead, the ATP sites work in unison to promote conformational changes in the clamp loader that drive clamp loading.     

Structure of a sliding clamp on DNA

The structure of the E. coli beta clamp polymerase processivity factor has been solved in complex with primed DNA. Interestingly, the clamp directly binds the DNA duplex and also forms a crystal contact with the ssDNA template strand, which binds into the protein-binding pocket of the clamp. We demonstrate that these clamp-DNA interactions function in clamp loading, perhaps by inducing the ring to close around DNA. Clamp binding to template ssDNA may also serve to hold the clamp at a primed site after loading or during switching of multiple factors on the clamp. Remarkably, the DNA is highly tilted as it passes through the beta ring. The pronounced 22 degrees angle of DNA through beta may enable DNA to switch between multiple factors bound to a single clamp simply by alternating from one protomer of the ring to the other.     

Clamp loaders and sliding clamps

A coherent view of the structure and function of DNA polymerase processivity factors (sliding clamps and clamp loaders) is emerging from recent structural studies. Crystal structures of sliding clamps from the T4 and RB69 bacteriophages, and from an archaebacterium expand the gallery of ring-shaped processivity factors and clarify how the clamp interacts with the DNA polymerase. Crystallographic and electron microscopic views of clamp loaders from bacteria, archaebacteria and eukaryotes emphasize their common architecture and have produced models of how ATPbinding might be coupled to clamp opening/loading.     

Replication Clamps and Clamp Loaders

To achieve the high degree of processivity required for DNA replication, DNA polymerases associate with ring-shaped sliding clamps that encircle the template DNA and slide freely along it. The closed circular structure of sliding clamps necessitates an enzyme-catalyzed mechanism, which not only opens them for assembly and closes them around DNA, but specifically targets them to sites where DNA synthesis is initiated and orients them correctly for replication. Such a feat is performed by multisubunit complexes known as clamp loaders, which use ATP to open sliding clamp rings and place them around the 3′ end of primer–template (PT) junctions. Here we discuss the structure and composition of sliding clamps and clamp loaders from the three domains of life as well as T4 bacteriophage, and provide our current understanding of the clamp-loading process.During each round of DNA replication, thousands to billions of nucleotides must be faithfully copied in a short period of time. However, by themselves, replicative DNA polymerases are distributive, synthesizing only ten or so nucleotides of complementary DNA before dissociating. To achieve the high degree of processivity required for efficient DNA replication, replicative DNA polymerases associate with ring-shaped sliding clamps that encircle the template DNA and slide freely along it. Such an association effectively tethers the polymerase to DNA, substantially increasing the amount of continuous replication. The closed circular structure of sliding clamps necessitates an enzyme-catalyzed mechanism, which not only opens them for assembly and closes them around DNA, but specifically targets them to sites where DNA synthesis is initiated and orients them correctly for interaction with DNA polymerases. Such a feat is performed by multisubunit complexes known as clamp loaders, which use ATP to open sliding clamp rings and place them around the 3′ end of primer–template (PT) junctions. Here we discuss the structure and composition of sliding clamps and clamp loaders from the three domains of life as well as T4 bacteriophage, and provide our current understanding of the clamp-loading process.     

DNA structure requirements for the Escherichia coli gamma complex clamp loader and DNA polymerase III holoenzyme

The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ring-shaped protein, the beta clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The beta clamp is assembled onto DNA by the multiprotein gamma complex clamp loader that opens and closes the beta ring around DNA in an ATP-dependent manner. This study examines the DNA structure required for clamp loading action. We found that the gamma complex assembles beta onto supercoiled DNA (replicative form I), but only at very low ionic strength, where regions of unwound DNA may exist in the duplex. Consistent with this, the gamma complex does not assemble beta onto relaxed closed circular DNA even at low ionic strength. Hence, a 3'-end is not required for clamp loading, but a single-stranded DNA (ssDNA)/double-stranded DNA (dsDNA) junction can be utilized as a substrate, a result confirmed using synthetic oligonucleotides that form forked ssDNA/dsDNA junctions on M13 ssDNA. On a flush primed template, the gamma complex exhibits polarity; it acts specifically at the 3'-ssDNA/dsDNA junction to assemble beta onto the DNA. The gamma complex can assemble beta onto a primed site as short as 10 nucleotides, corresponding to the width of the beta ring. However, a protein block placed closer than 14 base pairs (bp) upstream from the primer 3' terminus prevents the clamp loading reaction, indicating that the gamma complex and its associated beta clamp interact with approximately 14-16 bp at a ssDNA/dsDNA junction during the clamp loading operation. A protein block positioned closer than 20-22 bp from the 3' terminus prevents use of the clamp by the polymerase in chain elongation, indicating that the polymerase has an even greater spatial requirement than the gamma complex on the duplex portion of the primed site for function with beta. Interestingly, DNA secondary structure elements placed near the 3' terminus impose similar steric limits on the gamma complex and polymerase action with beta. The possible biological significance of these structural constraints is discussed.