ABSTRACT
The faithful and timely copying of DNA by molecular machines known as replisomes depends on a disparate suite of enzymes and scaffolding factors working together in a highly orchestrated manner. Large, dynamic protein-nucleic acid assemblies that selectively morph between distinct conformations and compositional states underpin this critical cellular process. In this article, we discuss recent progress outlining the physical basis of replisome construction and progression in eukaryotes.
Subject(s)
DNA Replication , DNA/biosynthesis , Eukaryota/genetics , Origin Recognition Complex/metabolism , Animals , DNA/chemistry , DNA Polymerase III/chemistry , DNA Polymerase III/metabolism , Humans , Origin Recognition Complex/chemistry , Origin Recognition Complex/genetics , Proliferating Cell Nuclear Antigen/chemistry , Proliferating Cell Nuclear Antigen/metabolismABSTRACT
Recent studies of bacterial DNA replication have led to a picture of the replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the replisome, these observations suggest a role of the helicase as the central organizing hub. We show here that the molecular origin of this newly found plasticity lies in the 500-fold increase in strength of the interaction between the polymerase holoenzyme and the replicative helicase upon association of the primase with the replisome. By combining in vitro ensemble-averaged and single-molecule assays, we demonstrate that this conformational switch operates during replication and promotes recruitment of multiple holoenzymes at the fork. Our observations provide a molecular mechanism for polymerase exchange and offer a revised model for the replication reaction that emphasizes its stochasticity.
Subject(s)
DNA Primase/metabolism , DNA Replication , DNA-Directed DNA Polymerase/metabolism , DnaB Helicases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Holoenzymes/chemistry , DNA Primase/genetics , DNA, Bacterial , DNA-Directed DNA Polymerase/genetics , DnaB Helicases/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Holoenzymes/genetics , Holoenzymes/metabolism , Molecular Conformation , Protein Binding , Protein ConformationABSTRACT
The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded onto DNA by a clamp loader, e.g., the well-studied pentameric ATPase complex RFC (RFC1-5). The CTF18-RFC complex is an alternative clamp loader found recently to bind the leading strand DNA polymerase ε and load PCNA onto leading strand DNA, but its structure and the loading mechanism have been unknown. By cryo-EM analysis of in vitro assembled human CTF18-RFC-DNA-PCNA complex, we have captured seven loading intermediates, revealing a detailed PCNA loading mechanism onto a 3'-ss/dsDNA junction by CTF18-RFC. Interestingly, the alternative loader has evolved a highly mobile CTF18 AAA+ module likely to lower the loading activity, perhaps to avoid competition with the RFC and to limit its role to leading strand clamp loading. To compensate for the lost stability due to the mobile AAA+ module, CTF18 has evolved a unique ß-hairpin motif that reaches across RFC2 to interact with RFC5, thereby stabilizing the pentameric complex. Further, we found that CTF18 also contains a separation pin to locally melt DNA from the 3'-end of the primer; this ensures its ability to load PCNA to any 3'-ss/dsDNA junction, facilitated by the binding energy of the E-plug to the major groove. Our study reveals unique structural features of the human CTF18-RFC and contributes to a broader understanding of PCNA loading by the alternative clamp loaders.
Subject(s)
ATPases Associated with Diverse Cellular Activities , Cryoelectron Microscopy , Nuclear Proteins , Proliferating Cell Nuclear Antigen , Replication Protein C , Humans , Cryoelectron Microscopy/methods , DNA/metabolism , DNA/chemistry , DNA Replication , Models, Molecular , Proliferating Cell Nuclear Antigen/metabolism , Proliferating Cell Nuclear Antigen/chemistry , Protein Binding , Replication Protein C/metabolism , Replication Protein C/chemistryABSTRACT
Clamp loaders are pentameric ATPases that place circular sliding clamps onto DNA, where they function in DNA replication and genome integrity. The central activity of a clamp loader is the opening of the ring-shaped sliding clamp and the subsequent binding to primer-template (p/t)-junctions. The general architecture of clamp loaders is conserved across all life, suggesting that their mechanism is retained. Recent structural studies of the eukaryotic clamp loader replication factor C (RFC) revealed that it functions using a crab-claw mechanism, where clamp opening is coupled to a massive conformational change in the loader. Here we investigate the clamp loading mechanism of the Escherichia coli clamp loader at high resolution using cryo-electron microscopy. We find that the E. coli clamp loader opens the clamp using a crab-claw motion at a single pivot point, whereas the eukaryotic RFC loader uses motions distributed across the complex. Furthermore, we find clamp opening occurs in multiple steps, starting with a partly open state with a spiral conformation, and proceeding to a wide open clamp in a surprising planar geometry. Finally, our structures in the presence of p/t-junctions illustrate how the clamp closes around p/t-junctions and how the clamp loader initiates release from the loaded clamp. Our results reveal mechanistic distinctions in a macromolecular machine that is conserved across all domains of life.
Subject(s)
DNA Replication , Escherichia coli , Cryoelectron Microscopy , Escherichia coli/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Protein Conformation , Replication Protein C/metabolism , Replication Protein C/chemistry , Replication Protein C/genetics , Models, Molecular , Protein Structure, QuaternaryABSTRACT
The τ-subunit of the clamp loader complex (CLC) physically interacts with both the DnaB helicase and the polymerase III (Pol III) core α-subunit through Domains IV and V, respectively. This interaction is proposed to help maintain rapid and efficient DNA synthesis rates with high genomic fidelity and plasticity, facilitating enzymatic coupling within the replisome. To test this hypothesis, CRISPR-Cas9 editing was used to create site-directed genomic mutations within the dnaX gene at the C-terminus of τ predicted to interact with the α-subunit of Pol III. Perturbation of the α-τ binding interaction in vivo resulted in cellular and genomic stress markers that included reduced growth rates, fitness, and viabilities. Specifically, dnaX:mut strains showed increased cell filamentation, mutagenesis frequencies, and activated SOS. In situ fluorescence flow cytometry and microscopy quantified large increases in the amount of single-stranded DNA (ssDNA) gaps present. Removal of the C-terminus of τ (I618X) still maintained its interactions with DnaB and stimulated unwinding but lost its interaction with Pol III, resulting in significantly reduced rolling circle DNA synthesis. Intriguingly, dnaX:L635P/D636G had the largest induction of SOS, high mutagenesis, and the most prominent ssDNA gaps, which can be explained by an impaired ability to regulate the unwinding speed of DnaB resulting in a faster rate of in vitro rolling circle DNA replication, inducing replisome decoupling. Therefore, τ stimulated DnaB unwinding and physical coupling with Pol III acts to enforce replisome plasticity to maintain an efficient rate of synthesis and prevent genomic instability.
ABSTRACT
Proliferating cell nuclear antigen (PCNA) is a homo-trimeric clamp complex that serves as the molecular hub for various DNA transactions, including DNA synthesis and post-replicative mismatch repair. Its timely loading and unloading are critical for genome stability. PCNA loading is catalyzed by Replication factor C (RFC) and the Ctf18 RFC-like complex (Ctf18-RLC), and its unloading is catalyzed by Atad5/Elg1-RLC. However, RFC, Ctf18-RLC, and even some subcomplexes of their shared subunits are capable of unloading PCNA in vitro, leaving an ambiguity in the division of labor in eukaryotic clamp dynamics. By using a system that specifically detects PCNA unloading, we show here that Atad5-RLC, which accounts for only approximately 3% of RFC/RLCs, nevertheless provides the major PCNA unloading activity in Xenopus egg extracts. RFC and Ctf18-RLC each account for approximately 40% of RFC/RLCs, while immunodepletion of neither Rfc1 nor Ctf18 detectably affects the rate of PCNA unloading in our system. PCNA unloading is dependent on the ATP-binding motif of Atad5, independent of nicks on DNA and chromatin assembly, and inhibited effectively by PCNA-interacting peptides. These results support a model in which Atad5-RLC preferentially unloads DNA-bound PCNA molecules that are free from their interactors.
Subject(s)
ATPases Associated with Diverse Cellular Activities , DNA-Binding Proteins , Proliferating Cell Nuclear Antigen , Animals , DNA , DNA Replication , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Replication Protein C/genetics , Replication Protein C/metabolism , Xenopus laevis/metabolism , Oocytes , ATPases Associated with Diverse Cellular Activities/genetics , ATPases Associated with Diverse Cellular Activities/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolismABSTRACT
The ability of mutations to facilitate adaptation is central to evolution. To understand how mutations can lead to functional adaptation in a complex molecular machine, we created a defective version of the T4 clamp-loader complex, which is essential for DNA replication. This variant, which is â¼5,000-fold less active than the wild type, was made by replacing the catalytic domains with those from another phage. A directed-evolution experiment revealed that multiple substitutions to a single negatively charged residue in the chimeric clamp loader-Asp 86-restore fitness to within â¼20-fold of wild type. These mutations remove an adventitious electrostatic repulsive interaction between Asp 86 and the sliding clamp. Thus, the fitness decrease of the chimeric clamp loader is caused by a reduction in affinity between the clamp loader and the clamp. Deep mutagenesis shows that the reduced fitness of the chimeric clamp loader is also compensated for by lysine and arginine substitutions of several DNA-proximal residues in the clamp loader or the sliding clamp. Our results demonstrate that there is a latent capacity for increasing the affinity of the clamp loader for DNA and the sliding clamp, such that even single-point mutations can readily compensate for the loss of function due to suboptimal interactions elsewhere.
Subject(s)
Adenosine Triphosphatases , Adenosine Triphosphate , Adenosine Triphosphatases/genetics , Adenosine Triphosphate/chemistry , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , DNA Replication , DNAABSTRACT
DNA replication is a tightly coordinated event carried out by a multiprotein replication complex. An essential factor in the bacterial replication complex is the ring-shaped DNA sliding clamp, ß-clamp, ensuring processive DNA replication and DNA repair through tethering of polymerases and DNA repair proteins to DNA. ß -clamp is a hub protein with multiple interaction partners all binding through a conserved clamp binding sequence motif. Due to its central role as a DNA scaffold protein, ß-clamp is an interesting target for antimicrobial drugs, yet little effort has been put into understanding the functional interactions of ß-clamp. In this review, we scrutinize the ß-clamp structure and dynamics, examine how its interactions with a plethora of binding partners are regulated through short linear binding motifs and discuss how contexts play into selection. We describe the dynamic process of clamp loading onto DNA and cover the recent advances in drug development targeting ß-clamp. Despite decades of research in ß-clamps and recent landmark structural insight, much remains undisclosed fostering an increased focus on this very central protein.
Subject(s)
Bacterial Proteins , DNA Replication , DNA, Bacterial , Drug Discovery , DNA, Bacterial/metabolism , DNA, Bacterial/chemistry , Drug Discovery/methods , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Protein Binding , DNA Polymerase III/metabolism , DNA Polymerase III/chemistry , Models, Molecular , Bacteria/metabolism , Bacteria/genetics , DNA RepairABSTRACT
The RAD9-RAD1-HUS1 complex (9-1-1) is a eukaryotic DNA clamp with a crucial role at checkpoints for DNA damage. The ring-like structure of 9-1-1 is opened for loading onto 5' recessed DNA by the clamp loader RAD17 RFC-like complex (RAD17-RLC), in which the RAD17 subunit is responsible for specificity to 9-1-1. Loading of 9-1-1 is required for activation of the ATR-CHK1 checkpoint pathway and the activation is stimulated by a 9-1-1 interacting protein, RHINO, which interacts with 9-1-1 via a recently identified RAD1-binding motif. This discovery led to the hypothesis that other interacting proteins may contain a RAD1-binding motif as well. Here, we show that vertebrate RAD17 proteins also have a putative RAD1-binding motif in their N-terminal regions, and we report the crystal structure of human 9-1-1 bound to a human RAD17 peptide incorporating the motif at 2.1 Å resolution. Our structure confirms that the N-terminal region of RAD17 binds to the RAD1 subunit of 9-1-1 via specific interactions. Furthermore, we show that the RAD1-binding motif of RHINO disturbs the interaction of the N-terminal region of RAD17 with 9-1-1. Our results provide deeper understanding of how RAD17-RLC specifically recognizes 9-1-1 and imply that RHINO has a functional role in 9-1-1 loading/unloading and checkpoint activation.
Subject(s)
Carrier Proteins , Cell Cycle Proteins , Exonucleases , Humans , Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , DNA/metabolism , DNA Damage , DNA-Binding Proteins/metabolism , Exonucleases/metabolismABSTRACT
African swine fever virus (ASFV) is an important animal pathogen that is causing a current African swine fever pandemic and affecting pork industry globally. ASFV encodes at least 150 proteins, and the functions of many of them remain to be clarified. The ASFV protein E301R (pE301R) was predicted to be a DNA sliding clamp protein homolog working as a DNA replication processivity factor. However, structural evidence was lacking to support the existence of a ring-shaped sliding clamp in large eukaryotic DNA viruses. Here, we have solved a high-resolution crystal structure of pE301R and identified a canonical ring-shaped clamp comprising a pE301R trimer. Interestingly, this complete-toroidal structure is different from those of the monomeric clamp protein homolog, herpes simplex virus UL42, and the C-shaped dimeric human cytomegalovirus UL44, but highly homologous to that of the eukaryotic clamp homolog proliferating cell nuclear antigen. Moreover, pE301R has a unique N-terminal extension that is important in maintaining the trimeric form of the protein in solution, while specific features in length and surface electrostatic potential of its interdomain connector implies specificity in interactions with binding partners such as the viral DNA polymerase. Thus, our data pave the way for further dissection of the processivity clamp protein structural and functional diversity and ASFV DNA replication mechanisms.
Subject(s)
African Swine Fever Virus , African Swine Fever , Swine , Humans , Animals , African Swine Fever Virus/genetics , Protein Conformation , DNA-Directed DNA Polymerase/chemistry , DNA, Viral/geneticsABSTRACT
Clamp loaders are pentameric AAA+ assemblies that use ATP to open and close circular DNA sliding clamps around DNA. Clamp loaders show homology in all organisms, from bacteria to human. The eukaryotic PCNA clamp is loaded onto 3' primed DNA by the replication factor C (RFC) hetero-pentameric clamp loader. Eukaryotes also have three alternative RFC-like clamp loaders (RLCs) in which the Rfc1 subunit is substituted by another protein. One of these is the yeast Rad24-RFC (Rad17-RFC in human) that loads a 9-1-1 heterotrimer clamp onto a recessed 5' end of DNA. Recent structural studies of Rad24-RFC have discovered an unexpected 5' DNA binding site on the outside of the clamp loader and reveal how a 5' end can be utilized for loading the 9-1-1 clamp onto DNA. In light of these results, new studies reveal that RFC also contains a 5' DNA binding site, which functions in gap repair. These studies also reveal many new features of clamp loaders. As reviewed herein, these recent studies together have transformed our view of the clamp loader mechanism.
Subject(s)
DNA Damage , Saccharomyces cerevisiae Proteins , Humans , Replication Protein C/chemistry , Replication Protein C/genetics , Replication Protein C/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , DNA Replication , DNA/metabolism , Adenosine Triphosphate/metabolism , DNA, Circular/metabolism , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The DNA clamp loader is critical to the processivity of the DNA polymerase and coordinating synthesis on the leading and lagging strands. In bacteria, the major subunit of the clamp loader, DnaX, has two forms: the essential full-length τ form and shorter γ form. These are conserved across bacterial species, and three distinct mechanisms have been found to create them: ribosomal frameshift, transcriptional slippage, and, in Caulobacter crescentus, partial proteolysis. This conservation suggests that DnaX processing is evolutionarily important, but its role remains unknown. Here we find a bias against switching from expression of a wild-type dnaX to a nonprocessable τ-only allele in Caulobacter. Despite this bias, cells are able to adapt to the τ-only allele with little effect on growth or morphology and only minor defects during DNA damage. Motivated by transposon sequencing, we find that loss of the gene sidA in the τ-only strain slows growth and increases filamentation. Even in the absence of exogenous DNA damage treatment, the ΔsidA τ-only double mutant shows induction of and dependence on recA, likely due to a defect in resolution of DNA damage or replication fork stalling. We find that some of the phenotypes of the ΔsidA τ-only mutant can be complemented by expression of γ but that an overabundance of τ-only dnaX is also detrimental. The data presented here suggest that DnaX processing is important during resolution of DNA damage events during DNA replication stress. Although the presence of DnaX τ and γ forms is conserved across bacteria, different species have developed different mechanisms to make these forms. This conservation and independent evolution of mechanisms suggest that having two forms of DnaX is important. Despite having been discovered more than 30 years ago, the purpose of expressing both τ and γ is still unclear. Here, we present evidence that expressing two forms of DnaX and controlling the abundance and/or ratio of the forms are important during the resolution of DNA replication stress. IMPORTANCE Though the presence of DnaX τ and γ forms is conserved across bacteria, different species have developed different mechanisms to make these forms. This conservation and independent evolution of mechanisms suggest that having two forms of DnaX is important. Despite having been discovered more than 30 years ago, the purpose of expressing both τ and γ is still unclear. Here, we present evidence that expressing two forms of DnaX and controlling the abundance and/or ratio of the forms is important during the resolution of DNA replication stress.
Subject(s)
Bacterial Proteins , Caulobacter crescentus , DNA Polymerase III , DNA Replication , Bacterial Proteins/genetics , DNA Polymerase III/genetics , Caulobacter crescentus/genetics , DNA, Bacterial/geneticsABSTRACT
DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader replication factor C (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryogenic electron microscopy to an overall resolution of â¼3.4 Å. The active sites of RFC are fully bound to adenosine 5'-triphosphate (ATP) analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation before PCNA opening, with the clamp loader ATPase modules forming an overtwisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a "limited change/induced fit" mechanism in which the clamp first opens, followed by DNA binding, inducing opening of the loader to release autoinhibition. The proposed change from an overtwisted to an active conformation reveals an additional regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health.
Subject(s)
ATPases Associated with Diverse Cellular Activities , DNA Replication/physiology , Proliferating Cell Nuclear Antigen , Replication Protein C , ATPases Associated with Diverse Cellular Activities/chemistry , ATPases Associated with Diverse Cellular Activities/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Cryoelectron Microscopy , Humans , Proliferating Cell Nuclear Antigen/chemistry , Proliferating Cell Nuclear Antigen/metabolism , Proliferating Cell Nuclear Antigen/ultrastructure , Replication Protein C/chemistry , Replication Protein C/metabolism , Replication Protein C/ultrastructureABSTRACT
In all cell types, a multi-protein machinery is required to accurately duplicate the large duplex DNA genome. This central life process requires five core replisome factors in all cellular life forms studied thus far. Unexpectedly, three of the five core replisome factors have no common ancestor between bacteria and eukaryotes. Accordingly, the replisome machines of bacteria and eukaryotes have important distinctions in the way that they are organized and function. This chapter outlines the major replication proteins that perform DNA duplication at replication forks, with particular attention to differences and similarities in the strategies used by eukaryotes and bacteria.
Subject(s)
DNA Replication , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/metabolism , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Bacteria/enzymology , Bacteria/genetics , Eukaryota/enzymology , Eukaryota/geneticsABSTRACT
The eukaryotic genome is primarily replicated by two DNA polymerases, Pol ε and Pol δ, that function on the leading and lagging strands, respectively. Previous studies have established recruitment mechanisms whereby Cdc45-Mcm2-7-GINS (CMG) helicase binds Pol ε and tethers it to the leading strand, and PCNA (proliferating cell nuclear antigen) binds tightly to Pol δ and recruits it to the lagging strand. The current report identifies quality control mechanisms that exclude the improper polymerase from a particular strand. We find that the replication factor C (RFC) clamp loader specifically inhibits Pol ε on the lagging strand, and CMG protects Pol ε against RFC inhibition on the leading strand. Previous studies show that Pol δ is slow and distributive with CMG on the leading strand. However, Saccharomyces cerevisiae Pol δ-PCNA is a rapid and processive enzyme, suggesting that CMG may bind and alter Pol δ activity or position it on the lagging strand. Measurements of polymerase binding to CMG demonstrate Pol ε binds CMG with a Kd value of 12 nM, but Pol δ binding CMG is undetectable. Pol δ, like bacterial replicases, undergoes collision release upon completing replication, and we propose Pol δ-PCNA collides with the slower CMG, and in the absence of a stabilizing Pol δ-CMG interaction, the collision release process is triggered, ejecting Pol δ on the leading strand. Hence, by eviction of incorrect polymerases at the fork, the clamp machinery directs quality control on the lagging strand and CMG enforces quality control on the leading strand.
Subject(s)
DNA Polymerase III/metabolism , DNA Polymerase II/metabolism , DNA Replication/genetics , Eukaryotic Cells/metabolism , Minichromosome Maintenance Proteins/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Quality Control , RNA-Dependent RNA Polymerase/metabolism , Replication Protein C/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The affinity tags in fusion proteins are extensively used in protein expression techniques. The most common affinity tags, such as glutathione S-transferase (GST), poly-histidine, maltose binding protein (MBP), and streptavidin tags, are routinely used for increasing expression, improving solubility, and facilitating protein purification. The large affinity tags (MBP, GST) are known to influence the conformational homogeneity and, therefore, the three-dimensional structure of in vivo folded proteins. The current study described in vivo effects of small affinity fusion 6 x His tag on the growth of cells. Hexa-histidine tagged full length ß-clamp and non-hexa-histidine tagged ß-clamp were over-expressed and co-expressed in possible combinations with truncated DnaE in E. coli expression strain. After the induction with IPTG, the protein expression was assessed by SDS PAGE. The comparative analysis of the growth curves generated for the induced and un-induced cells demonstrated a decrease in growth rates of the cells over-expressing non-6 x His tagged ß-clamp as compared to 6 x His tagged ß-clamp. Based on the analysis of the soluble and insoluble protein fractions by SDS PAGE gels and published His-tagged ß-clamp structure (PDB: 4K74) we propose that N-terminal 6 x His Tag on ß-clamp occludes its Gly66 to ultimately affect its ability to interact with the 8 subunit of the clamp loader.
Subject(s)
DNA Polymerase III/chemistry , Escherichia coli/growth & development , Glycine/chemistry , Histidine/chemistry , Chromatography, Affinity , Recombinant Fusion Proteins , SolubilityABSTRACT
The machines that decode and regulate genetic information require the translation, transcription and replication pathways essential to all living cells. Thus, it might be expected that all cells share the same basic machinery for these pathways that were inherited from the primordial ancestor cell from which they evolved. A clear example of this is found in the translation machinery that converts RNA sequence to protein. The translation process requires numerous structural and catalytic RNAs and proteins, the central factors of which are homologous in all three domains of life, bacteria, archaea and eukarya. Likewise, the central actor in transcription, RNA polymerase, shows homology among the catalytic subunits in bacteria, archaea and eukarya. In contrast, while some "gears" of the genome replication machinery are homologous in all domains of life, most components of the replication machine appear to be unrelated between bacteria and those of archaea and eukarya. This review will compare and contrast the central proteins of the "replisome" machines that duplicate DNA in bacteria, archaea and eukarya, with an eye to understanding the issues surrounding the evolution of the DNA replication apparatus.
Subject(s)
DNA Replication , Evolution, Molecular , Protein Biosynthesis , Transcription, Genetic , Animals , DNA/genetics , Genetic Variation , Humans , Models, Molecular , Proteins/genetics , RNA/geneticsABSTRACT
DNA polymerases depend on circular sliding clamps for processive replication. Clamps must be loaded onto primer-template DNA (ptDNA) by clamp loaders that open and close clamps around ptDNA in an ATP-fueled reaction. All clamp loaders share a core structure in which five subunits form a spiral chamber that binds the clamp at its base in a twisted open form and encloses ptDNA within, while binding and hydrolyzing ATP to topologically link the clamp and ptDNA. To understand how clamp loaders perform this complex task, here we focused on conserved arginines that might play a central coordinating role in the mechanism because they can alternately contact ptDNA or Walker B glutamate in the ATPase site and lie close to the clamp loader-clamp-binding interface. We mutated Arg-84, Arg-88, and Arg-101 in the ATPase-active B, C, and D subunits of Saccharomyces cerevisiae replication factor C (RFC) clamp loader, respectively, and assessed the impact on multiple transient events in the reaction: proliferating cell nuclear antigen (PCNA) clamp binding/opening/closure/release, ptDNA binding/release, and ATP hydrolysis/product release. The results show that these arginines relay critical information between the PCNA-binding, DNA-binding, and ATPase sites at all steps of the reaction, particularly at a checkpoint before RFC commits to ATP hydrolysis. Moreover, their actions are subunit-specific with RFC-C Arg-88 serving as an accelerator that enables rapid ATP hydrolysis upon contact with ptDNA and RFC-D Arg-101 serving as a brake that confers specificity for ptDNA as the correct substrate for loading PCNA.
Subject(s)
Biocatalysis , DNA, Fungal/chemistry , DNA, Fungal/metabolism , Replication Protein C/chemistry , Replication Protein C/metabolism , Adenosine Triphosphate/metabolism , DNA-Directed DNA Polymerase/metabolism , Hydrolysis , Models, Molecular , Proliferating Cell Nuclear Antigen/metabolism , Protein Binding , Protein Conformation , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Examples of dynamic polymerase exchange have been previously characterized in model systems provided by coliphages T4 and T7. Using a dominant negative D403E polymerase (Pol) III α that can form initiation complexes and sequester primer termini but not elongate, we investigated the possibility of exchange at the Escherichia coli replication fork on a rolling circle template. Unlike other systems, addition of polymerase alone did not lead to exchange. Only when D403E Pol III was bound to a τ-containing DnaX complex did exchange occur. In contrast, addition of Pol IV led to rapid exchange in the absence of bound DnaX complex. Examination of Pol III* with varying composition of τ or the alternative shorter dnaX translation product γ showed that τ-, τ2-, or τ3-DnaX complexes supported equivalent levels of synthesis, identical Okazaki fragment size, and gaps between fragments, possessed the ability to challenge pre-established replication forks, and displayed equivalent susceptibility to challenge by exogenous D403E Pol III*. These findings reveal that redundant interactions at the replication fork must stabilize complexes containing only one τ. Previously, it was thought that at least two τs in the trimeric DnaX complex were required to couple the leading and lagging strand polymerases at the replication fork. Possible mechanisms of exchange are discussed.
Subject(s)
Bacterial Proteins/metabolism , DNA Polymerase III/metabolism , DNA Polymerase beta/metabolism , DNA Replication , DNA, Bacterial/metabolism , Escherichia coli/enzymology , Bacterial Proteins/genetics , DNA Polymerase III/genetics , DNA Polymerase beta/genetics , DNA, Bacterial/genetics , Electrophoresis, Agar Gel , Escherichia coli/geneticsABSTRACT
Sliding clamps are opened and loaded onto primer template junctions by clamp loaders, and once loaded on DNA, confer processivity to replicative polymerases. Previously determined crystal structures of eukaryotic and T4 clamp loader-clamp complexes have captured the sliding clamps in either closed or only partially open interface conformations. In these solution structure studies, we have captured for the first time the clamp loader-sliding clamp complex from Escherichia coli using size exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS). The data suggests the sliding clamp is in an open conformation which is wide enough to permit duplex DNA binding. The data also provides information about spatial arrangement of the sliding clamp with respect to the clamp loader subunits and is compared to complex crystal structures determined from other organisms.