Did an early version of the eukaryal replisome enable the emergence of chromatin?
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Animals↗
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Polymerases and the replisome: machines within machines.
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DNA Helicases↗
Building a replisome from interacting pieces: sliding clamp complexed to a peptide from DNA polymerase and a polymerase editing complex.
We have solved the crystal structures of the bacteriophage RB69 sliding clamp, its complex with a peptide essential for DNA polymerase interactions, and the DNA polymerase complexed with primer-template DNA. The editing complex structure shows a partially melted duplex DNA exiting from the exonuclease domain at an unexpected angle and significant changes in the protein structure. The clamp complex shows the C-terminal 11 residues of polymerase bound in a hydrophobic pocket, and it allows docking of the editing and clamp structures together. The peptide binds to the sliding clamp at a position identical to that of a replication inhibitor peptide bound to PCNA, suggesting that the replication inhibitor protein p21CIP1 functions by competing with eukaryotic polymerases for the same binding pocket on the clamp.
Amino Acid Sequence↗
Eukaryotic DNA replication: a model for a fixed double replisome.
To duplicate their genomes, eukaryotic cells have to overcome some formidable chemical and topological hurdles, considering the number of nucleotides that have to be polymerized faithfully and the sheer physical size of the DNA molecules that have to be disentangled and partitioned in an orderly way. This article tackles one particular aspect of the process: the organization of the apparatus that advances the replicative growing forks along the DNA molecule. Here, I suggest a solution to the difficulty of separating the daughter molecules in an orderly way and propose an alternative to the current models, which reconciles the use of a single polarity of synthesis by the DNA polymerases with the need for parallel polymerization of two strands of opposite polarity.
DNA↗
Replisome assembly at oriC, the replication origin of E. coli, reveals an explanation for initiation sites outside an origin.
This study outlines the events downstream of origin unwinding by DnaA, leading to assembly of two replication forks at the E. coli origin, oriC. We show that two hexamers of DnaB assemble onto the opposing strands of the resulting bubble, expanding it further, yet helicase action is not required. Primase cannot act until the helicases move 65 nucleotides or more. Once primers are formed, two molecules of the large DNA polymerase III holoenzyme machinery assemble into the bubble, forming two replication forks. Primer locations are heterogeneous; some are even outside oriC. This observation generalizes to many systems, prokaryotic and eukaryotic. Heterogeneous initiation sites are likely explained by primase functioning with a moving helicase target.
Bacterial Proteins↗
Replisome assembly and the direct restart of stalled replication forks.
Failure to reactivate either stalled or collapsed replication forks is a source of genomic instability in both prokaryotes and eukaryotes. In prokaryotes, dedicated fork repair systems that involve both recombination and replication proteins have been identified genetically and characterized biochemically. Replication conflicts are solved through several pathways, some of which require recombination and some of which operate directly at the stalled fork. Some recent biochemical observations support models of direct fork repair in which the removal of the blocking template lesion is not always required for replication restart.
Animals↗
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.
Archaea↗
Building a replisome solution structure by elucidation of protein-protein interactions in the bacteriophage T4 DNA polymerase holoenzyme.
Assembly of DNA replication systems requires the coordinated actions of many proteins. The multiprotein complexes formed as intermediates on the pathway to the final DNA polymerase holoenzyme have been shown to have distinct structures relative to the ground-state structures of the individual proteins. By using a variety of solution-phase techniques, we have elucidated additional information about the solution structure of the bacteriophage T4 holoenzyme. Photocross-linking and mass spectrometry were used to demonstrate interactions between I107C of the sliding clamp and the DNA polymerase. Fluorescence resonance energy transfer, analytical ultracentrifugation, and isothermal titration calorimetry measurements were used to demonstrate that the C terminus of the DNA polymerase can interact at two distinct locations on the sliding clamp. Both of these binding modes may be used during holoenzyme assembly, but only one of these binding modes is found in the final holoenzyme. Present and previous solution interaction data were used to build a model of the holoenzyme that is consistent with these data.
Biotin↗
Replisome architecture and dynamics in Escherichia coli.
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Bacterial Physiological Phenomena↗
Protein--protein interactions in the eubacterial replisome.
Replication of genomic DNA is a universal process that proceeds in distinct stages, from initiation to elongation and finally to termination. Each stage involves multiple stable or transient interactions between protein subunits with functions that are more or less conserved in all organisms. In Escherichia coli, initiation of bidirectional replication at the origin (oriC) occurs through the concerted actions of the DnaA replication initiator protein, the hexameric DnaB helicase, the DnaC?helicase loading partner and the DnaG primase, leading to establishment of two replication forks. Elongation of RNA primers at each fork proceeds simultaneously on both strands by actions of the multimeric replicase, DNA polymerase III holoenzyme. The fork that arrives first in the terminus region is halted by its encounter with a correctly-oriented complex of the Tus replication terminator protein bound at one of several Ter sites, where it is trapped until the other fork arrives. We summarize current understanding of interactions among the various proteins that act in the different stages of replication of the chromosome of E. coli, and make some comparisons with the analogous proteins in Bacillus subtilis and the coliphages T4 and T7.
Adenosine Triphosphatases↗
Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C.
Chromatin assembly and DNA replication are temporally coupled, and DNA replication in the absence of histone synthesis causes inviability. Here we demonstrate that chromatin assembly factor Asf1 also affects DNA replication. In budding yeast cells lacking Asf1, the amounts of several DNA replication proteins, including replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and DNA polymerase epsilon (Pol epsilon), are reduced at stalled replication forks. In contrast, DNA polymerase alpha (Pol alpha) accumulates to higher than normal levels at stalled forks in asf1Delta cells. Using purified, recombinant proteins, we demonstrate that RFC directly binds Asf1 and can recruit Asf1 to DNA molecules in vitro. We conclude that histone chaperone protein Asf1 maintains a subset of replication elongation factors at stalled replication forks and directly interacts with the replication machinery.
Cell Cycle Proteins↗
Pre-replication assembly of E. coli replisome components.
The localization of SeqA, thymidylate synthase, DnaB (helicase) and the DNA polymerase components alpha and tau, has been studied by immunofluorescence microscopy. The origin has been labelled through GFP-LacI bound near oriC. SeqA was located in the cell centre for one replication factory (RF) and at 1/4 and 3/4 positions in pre-divisional cells harbouring two RFs. The transition of central to 1/4 and 3/4 positions of SeqA appeared abrupt. Labelled thymidylate synthetase was found all over the cell, thus not supporting the notion of a dNTP-synthesizing complex exclusively localized near the RF. More DnaB, alpha and tau foci were found than expected. We have hypothesized that extra foci arise at pre-replication assembly sites, where the number of sites equals the number of origins, i.e. the number of future RFs. A reasonable agreement was found between predicted and found foci. In the case of multifork replication the number of foci appeared consistent with the assumption that three RFs are grouped into a higher-order structure. The RF is probably separate from the foci containing SeqA and the hemi-methylated SeqA binding sites because these foci did not coincide significantly with DnaB as marker of the RF. Co-labelling of DnaB and oriC revealed limited colocalization, indicating that DnaB did not yet become associated with oriC at a pre-replication assembly site. DnaB and tau co-labelled in the cell centre, though not at presumed pre-replication assembly sites. By contrast, alpha and tau co-labelled consistently suggesting that they are already associated before replication starts.
Bacterial Outer Membrane Proteins↗
Characterization of the putative replisome organizer of the lactococcal bacteriophage r1t.
Analysis of the nucleotide sequence of the genome of the lactococcal bacteriophage r1t showed that it may encode at least two proteins involved in DNA replication. On the basis of its similarity with the G38P protein encoded by the Bacillus subtilis phage SPP1, the product of orf11 (Pro11) is thought to be involved in the initiation of phage DNA replication. This protein was overexpressed in Lactococcus lactis and partially purified. Gel retardation analysis using various r1t DNA fragments indicates that Pro11 specifically binds to a sequence located within its cognate gene. DNase I footprinting showed that Pro11 protects a stretch of DNA of 47 bp. This region spans four 6-bp short direct repeats, which suggests that the region contains four binding sites for Pro11. 1,10-Phenanthroline-copper footprinting confirmed the protection of the hexamers. An asymmetric protection pattern of each strand was observed, suggesting that Pro11 contacts each DNA strand separately at contiguous hexamers. We propose a model for the binding of Pro11 to its target sites that may account for the torsion strain required for strand opening at the origin of replication.
Bacteriophages↗
Replisome-mediated DNA replication.
The elaborate process of genomic replication requires a large collection of proteins properly assembled at a DNA replication fork. Several decades of research on the bacterium Escherichia coli and its bacteriophages T4 and T7 have defined the roles of many proteins central to DNA replication. These three different prokaryotic replication systems use the same fundamental components for synthesis at a moving DNA replication fork even though the number and nature of some individual proteins are different and many lack extensive sequence homology. The components of the replication complex can be grouped into functional categories as follows: DNA polymerase, helix destabilizing protein, polymerase accessory factors, and primosome (DNA helicase and DNA primase activities). The replication of DNA derives from a multistep enzymatic pathway that features the assembly of accessory factors and polymerases into a functional holoenzyme; the separation of the double-stranded template DNA by helicase activity and its coupling to the primase synthesis of RNA primers to initiate Okazaki fragment synthesis; and the continuous and discontinuous synthesis of the leading and lagging daughter strands by the polymerases. This review summarizes and compares and contrasts for these three systems the types, timing, and mechanism of reactions and of protein-protein interactions required to initiate, control, and coordinate the synthesis of the leading and lagging strands at a DNA replication fork and comments on their generality.
Bacterial Proteins↗
Spatial and temporal organization of the Bacillus subtilis replication cycle.
DNA replication occurs at discrete sites in the cell. To gain insight into the spatial and temporal organization of the Bacillus subtilis replication cycle, we simultaneously visualized replication origins and the replication machinery (replisomes) inside live cells. We found that the origin of replication is positioned near midcell prior to replication. After initiation, the replisome colocalizes with the origin, confirming that replication initiates near midcell. The replisome remains near midcell after duplicated origins separate. Artificially mispositioning the origin region leads to mislocalization of the replisome indicating that the location of the origin at the time of initiation establishes the position of the replisome. Time-lapse microscopy revealed that a single replisome focus reversibly splits into two closely spaced foci every few seconds in many cells, including cells that recently initiated replication. Thus, sister replication forks are likely not intimately associated with each other throughout the replication cycle. Fork dynamics persisted when replication elongation was halted, and is thus independent of the relative movement of DNA through the replisome. Our results provide new insights into how the replisome is positioned in the cell and refine our current understanding of the spatial and temporal events of the B. subtilis replication cycle.
Bacillus subtilis↗
[Detailed model of semi-conservative DNA replication. Possible role of DNA-polymerase errors in fragmented copying of both matrix strands].
A model of semiconservative DNA synthesis is considered. The model is based on a suggestion that the synthesis proceeds in the course of one-directional stretching of both matrix strands through the replicase complex (replisome), or of one-directional movement of the replisome along the chromosome. Each nucleotide link of the matrix strand find itself successively in the section of RNA primer synthesis, in the section of DNA polymerase and in the control section of a proofreading 3' leads to 5' exonuclease. Deoxynucleoside incorrectly joined by DNA polymerase of the replisome to the 3'-OH end of growing chain are, as a rule, removed when transferred in the control section. Since the reverse movement is prohibited, the DNA polymerase loses the 3'-OH end of new DNA chain and proves to be incapable to continue the synthesis. It can be renewed only after a new RNA primer formation. For continuation of the DNA synthesis after an incorrect deoxynucleotide addition, replicase must take two more mistakes in succession: to retain the incorrect nucleotide and to join the next one to this unproperly arranged primer. Only in that case the non-complementary nucleotide in a new DNA chain can escape the action of proofreading exonucleases. Hence, according to the model under discussion, the frequency of mutations should be many times lower than that of incorrect nucleotide joinings. The latter results, as a rule, in DNA chain synthesis cessation and in fragments formation. Taking into consideration the frequency of spontaneous mutations in bacteria (about 10(-9) per base-pair), the frequency of non-complementary joinings during semiconservative synthesis can be suggested of the order 10(-3) in accordance with the average Okazaki fragments size of 1000 nucleotides. Another possible reason for periodical interruptions of semiconservative DNA synthesis and for fragments formation is a distortion of replisome structure, accompanying the release of the matrix strand being copied in the direction of already replicated chromosome region. Both mechanisms can participate concurrently in DNA fragments formation. If replisomes are attached to a cell membrane, the sites of chromosome that are also fastened in the membrane, in particular, the site of "origin", are to be replicated using some mobile replicase complex.
DNA Replication↗
Replication-induced protein synthesis and its importance to proteomics.
Replication-induced protein synthesis (RIPS) can occur following the passage of the replisome due to transcription initiated by RNA polymerase in association with: (i) negative supercoiling trailing the replisome / replication fork, (ii) hemimethylation prior to the action of dam methylase, (iii) transient derepression following passage of the replisome/replication fork and prior to renewed synthesis of the repressor gene-product, and (iv) 'sliding clamp' accessory DNA-binding proteins binding to the lagging strand DNA duplex to retard rotational upstream propagation of supercoils. The latter include subunits of DNA polymerase III in Escherichia coli and gp45 in T4 bacteriophage. By far the most convincing evidence for the existence of RIPS comes from the pulse of protein synthesis which follows the passage of the replisome in late T4 bacteriophage, the dynamics of replication in Escherichia coli, recent results from cDNA high-density expression arrays in yeast and the workings of the lac-operon. More circumstantial evidence is provided by 'leaky' or 'aberrant' protein expression in genetic systems where attempts have been made to turn off protein synthesis by molecular means. In higher vertebrates, RIPS may have a potentially important role in explaining the mechanisms by which thymic and peripheral immune self-tolerance is established, either directly through antigen presentation on dendritic cells or through the presentation of peptides derived from T-cells. The latter model is preferred, as young T-cells will have recently divided and will be dying in large numbers near the antigen-presenting dendritic cells in the thymus. The functional utility of RIPS would appear to be linked to both facilitating cellular metabolism and an improved survival during stress. RIPS, as a potentially universal molecular phenomenon, presents proteomics with numerous challenges and opportunities, both technical and commercial.
Animals↗
Mechanism of transient inhibition of DNA synthesis in ultraviolet-irradiated E. coli: inhibition is independent of recA whilst recovery requires RecA protein itself and an additional, inducible SOS function.
The mechanism of the inhibition and of the recovery of DNA synthesis in E. coli following UV-irradiation was analysed in several mutants defective in repair or in the regulation of the RecA-LexA dependent SOS response. Several lines of evidence indicated that inhibition is not an inducible function and is probably due to the direct effect of lesions in the template blocking replisome movement. Recovery of DNA synthesis after UV was largely unaffected by mutations in the uvrA, recB or umuC genes. Resumption of DNA synthesis does however require protein synthesis and the regulatory action of recA. Experiments with a recA constitutive mutant and recA 200 (temperature sensitive RecA) demonstrated that RecA protein itself is directly required but is not sufficient for recovery of DNA synthesis. We therefore propose that recovery of DNA synthesis depends upon the concerted activity of RecA and the synthesis of an inducible Irr (induced replisome reactivation) factor under RecA control. We suggest that the mechanism of recovery involves the action of Irr and RecA to promote movement of replisomes past non-instructive lesions, uncoupled from polymerisation and/or that Irr and RecA are required to promote re-initiation of a stalled replication complex downstream of a UV-lesion subsequent to such an uncoupling step.
Bacterial Proteins↗