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At least 19 recordsLinked to original sources

PubMed2003

Dissociative properties of the proteins within the bacteriophage T4 replisome.

DNA replication is a highly processive and efficient process that involves the coordination of at least eight proteins to form the replisome in bacteriophage T4. Replication of DNA occurs in the 5' to 3' direction resulting in continuous replication on the leading strand and discontinuous replication on the lagging strand. A key question is how a continuous and discontinuous replication process is coordinated. One solution is to avoid having the completion of one Okazaki fragment to signal the start of the next but instead to have a key step such as priming proceed in parallel to lagging strand replication. Such a mechanism requires protein elements of the replisome to readily dissociate during the replication process. Protein trapping experiments were performed to test for dissociation of the clamp loader and primase from an active replisome in vitro whose template was both a small synthetic DNA minicircle and a larger DNA substrate. The primase, clamp, and clamp loader are found to dissociate from the replisome and are continuously recruited from solution. The effect of varying protein concentrations (dilution) on the size of Okazaki fragments supported the protein trapping results. These findings are in accord with previous results for the accessory proteins but, importantly now, identify the primase as dissociating from an active replisome. The recruitment of the primase from solution during DNA synthesis has also been found for Escherichia coli but not bacteriophage T7. The implications of these results for RNA priming and extension during the repetitive synthesis of Okazaki fragments are discussed.

Adenosine Triphosphatases↗

PubMed2026

Direct visualization of MCM helicase activation and replisome coupling in situ.

Deciphering the spatial organization of molecular machines that copy the genome remains a fundamental challenge in biology. Essential for eukaryotic DNA replication, Mini-Chromosome Maintenance (MCM2-7) helicases are loaded during G1 as double hexamers (DHs) to license replication origins. Upon activation in S phase, each DH is thought to split into two single hexamers (SHs) that form the active CMG helicases and travel bidirectionally. However, the field has long been divided: biochemical and structural studies define CMG helicases as autonomous, independent motors, while genomic and cellular imaging assays suggest sister replisomes remain physically coupled within replication factories. Here, we use MINFLUX nanoscopy to localize individual MCM complexes down to nanometer precision in situ, directly resolving DHs in human cells and capturing their separation into SHs upon origin firing. We find that the resulting sister replisomes do not diffuse apart: they remain coupled at a characteristic distance of ~40 nm throughout S phase. Depletion experiments identify two distinct contributions to this coupling: local, protein-mediated tethering by the AND1 scaffold, and higher-order spatial confinement dependent on cohesin, which is dispensable for MCM loading in G1 but required to maintain coupling in S phase. By linking the nanometer-scale architecture of the replisome to the genome-wide topology of replication fountains, these findings provide direct spatial evidence that sister forks are coupled during DNA synthesis and define the molecular forces that organize replisomes within their native nuclear context.

DNA replication↗

PubMed2000

Movement of replicating DNA through a stationary replisome.

We found that DNA is replicated at a central stationary polymerase, and each replicated region moves away from the replisome. In Bacillus subtilis, DNA polymerase is predominantly located at or near midcell. When replication was blocked in a specific chromosomal region, that region was centrally located with DNA polymerase. Upon release of the block, each copy of the duplicated region was located toward opposite cell poles, away from the central replisome. In a roughly synchronous population of cells, a region of chromosome between origin and terminus moved to the replisome prior to duplication. Thus, the polymerase at the replication forks is stationary, and the template is pulled in and released outward during duplication. We propose that B. subtilis, and probably many bacteria, harness energy released during nucleotide condensation by a stationary replisome to facilitate chromosome partitioning.

ATP-Binding Cassette Transporters↗

PubMed1999

Duplex opening by primosome protein PriA for replisome assembly on a recombination intermediate.

PriA and other primosome assembly proteins of Escherichia coli recruit the major replicative helicase DnaB for replisome assembly during bacteriophage Mu transposition and replication. MuA transposase catalyzes the transfer of Mu ends to target DNA, forming a potential replication fork that provides the assembly site for the replisome. However, this fork lacks the single-stranded DNA needed to load DnaB. Although no pre-existing primosome assembly sites that bind PriA were found within the Mu end sequences, PriA was able to bind to the forked DNA structure created by MuA. The helicase activity of PriA could then open the duplex to create the DnaB binding site. In a tightly coupled reaction on synthetic forked substrates, PriA promoted both the unwinding of the lagging strand arm and preprimosome assembly to load DnaB onto the lagging strand template. PriA apparently translocated 3' to 5' along the lagging strand template until sufficient single-stranded DNA was exposed for binding of DnaB, which then translocated 5' to 3' in the opposite direction. Mutant PriA lacking helicase activity was unable to promote this process, and loss of PriA helicase impaired Mu DNA replication in vivo and in vitro. This suggests that the opening of the duplex by PriA helicase is a critical step in the initiation of Mu DNA replication. Concerted helicase and primosome assembly functions would allow PriA to act as initiator on recombination intermediates and stalled replication forks. As part of the replisome, PriA may act as a mobile initiator that minimizes interruptions in chromosomal replication.

Bacterial Proteins↗

PubMed2026

Structure and operating principles of a monkeypox virus replisome.

Poxviruses are double-stranded DNA viruses with large genomes. Among them, monkeypox virus (MPXV) has been responsible for two recent public health emergencies as declared by the World Health Organization1. The MPXV polymerase comprises three subunits-a catalytic subunit (F8) and a heterodimeric processivity factor (A22 and E4). The viral polymerase must coordinate activities with the hexameric helicase-primase (E5) to initiate replication of the viral genome2. Although structures of MPXV E5 (refs. 3,4) and the polymerase5-7 in isolation are available, how they assemble into a functional replisome remains unclear. In isolation, E5 is in an autoinhibited conformation and has very weak helicase activity3,4, and the mechanism for helicase activation is unclear. Here we used cryo-electron microscopy to determine the structures of DNA-bound MPXV replisomes comprising the polymerase holoenzyme (F8, A22 and E4) and the E5 helicase hexamer. We show that, during replisome assembly, E5 undergoes large-scale conformational changes that allow two of its primase domains to interact with the polymerase F8 thumb and A22 subunit. Biochemical assays and single-molecule experiments reveal that this E5 conformational change is coupled to helicase activation and enhances primase activity. Taken together, these findings identify fundamental mechanisms governing coordinated helicase and polymerase activities during DNA replication for an important class of viral pathogens.

Journal Article↗

PubMed2004

Checkpoint-mediated control of replisome-fork association and signalling in response to replication pausing.

The replication checkpoint controls the integrity of replicating chromosomes by stabilizing stalled forks, thus preventing the accumulation of abnormal replication and recombination intermediates that contribute to genome instability. Checkpoint-defective cells are susceptible to rearrangements at chromosome fragile sites when replication pauses, and certain human cancer prone diseases suffer checkpoint abnormalities. It is unclear as to how the checkpoint stabilizes stalled forks and how cells sense replication blocks. We have analysed the checkpoint contribution in controlling replisome-fork association when replication pauses. We show that in yeast wild-type cells, stalled forks exhibit stable replisome complexes and the checkpoint sensors Ddc1 and Ddc2, thus activating Rad53 checkpoint kinase. Ddc1/Ddc2 recruitment on stalled forks and Rad53 activation are influenced by the single-strand-binding protein replication factor A (RFA). rad53 forks exhibit a defective association with DNA polymerases alpha, epsilon and delta. Further, in rad53 mutants, stalled forks progressively generate abnormal structures that turn into checkpoint signals by accumulating RFA, Ddc1 and Ddc2. We suggest that, following replication blocks, checkpoint activation mediated by RFA-ssDNA filaments stabilizes stalled forks by controlling replisome-fork association, thus preventing unscheduled recruitment of recombination enzymes that could otherwise cause the pathological processing of the forks.

Cell Cycle Proteins↗

PubMed2001

Handoff from recombinase to replisome: insights from transposition.

Bacteriophage Mu replicates as a transposable element, exploiting host enzymes to promote initiation of DNA synthesis. The phage-encoded transposase MuA, assembled into an oligomeric transpososome, promotes transfer of Mu ends to target DNA, creating a fork at each end, and then remains tightly bound to both forks. In the transition to DNA synthesis, the molecular chaperone ClpX acts first to weaken the transpososome's interaction with DNA, apparently activating its function as a molecular matchmaker. This activated transpososome promotes formation of a new nucleoprotein complex (prereplisome) by yet unidentified host factors [Mu replication factors (MRF alpha 2)], which displace the transpososome in an ATP-dependent reaction. Primosome assembly proteins PriA, PriB, DnaT, and the DnaB--DnaC complex then promote the binding of the replicative helicase DnaB on the lagging strand template of the Mu fork. PriA helicase plays an important role in opening the DNA duplex for DnaB binding, which leads to assembly of DNA polymerase III holoenzyme to form the replisome. The MRF alpha 2 transition factors, assembled into a prereplisome, not only protect the fork from action by nonspecific host enzymes but also appear to aid in replisome assembly by helping to activate PriA's helicase activity. They consist of at least two separable components, one heat stable and the other heat labile. Although the MRF alpha 2 components are apparently not encoded by currently known homologous recombination genes such as recA, recF, recO, and recR, they may fulfill an important function in assembling replisomes on arrested replication forks and products of homologous strand exchange.

Adenosine Triphosphatases↗

PubMed2002

Protein-protein interactions in the bacteriophage T4 replisome. The leading strand holoenzyme is physically linked to the lagging strand holoenzyme and the primosome.

The bacteriophage T4 replication complex is composed of eight proteins that function together to replicate DNA. This replisome can be broken down into four basic units: a primosome composed of gp41, gp61, and gp59; a leading strand holoenzyme composed of gp43, gp44/62, and gp45; a lagging strand holoenzyme; and a single strand binding protein polymer. These units interact further to form the complete replisome. The leading and lagging strand polymerases are physically linked in the presence of DNA or an active replisome. The region of interaction was mapped to an extension of the finger domain, such that Cys-507 of one subunit is in close proximity to Cys-507 of a second subunit. The leading strand polymerase and the primosome also associate, such that gp59 mediates the contact between the two complexes. Binding of gp43 to the primosome complex causes displacement of gp32 from the gp59.gp61.gp41 primosome complex. The resultant species is a complex of proteins that may allow coordinated leading and lagging strand synthesis, helicase DNA unwinding activity, and polymerase nucleotide incorporation.

Bacteriophage T4↗

PubMed2026

Replication of DNA Containing Trinucleotide Repeats by the Bacteriophage T7 Replisome.

Trinucleotide repeats in the human genome are implicated in various neurodegenerative diseases. The tendency of these repetitive DNA sequences to form non-B DNA structures can cause abnormal replication, leading to genomic instability. This instability contributes to disease progression, though the underlying mechanisms are not fully understood. We investigated the replication of DNA containing CAG and CTG trinucleotide repeats using individual components of the T7 bacteriophage replication machinery, as well as the complete replisome. Our results show that repeats in linear single-stranded DNA (ssDNA) inhibit the activity of T7 DNA polymerase and ssDNA-binding proteins, with a more pronounced effect observed in CTG repeats compared to CAG repeats. Direct unwinding assays showed that the T7 gene 4 helicase unwound forked substrates containing CAG or CTG repeats at least as efficiently as random-sequence substrates; however, the displaced repeat strands were recovered predominantly as compact, structured species rather than as unstructured single-stranded DNA, providing direct evidence that secondary structure forms immediately upon unwinding. Minicircle templates containing CTG repeats exhibited robust DNA synthesis on both the leading and lagging strands, though synthesis was not enhanced by the T7 gene 2.5 ssDNA-binding protein. The lagging strand products generated from the CTG repeat minicircle were significantly longer than those from random sequence templates, and their lengths were not extended by the presence of T7 gene 2.5 protein. When the repeated sequences were incorporated into the T7 phage genome, heterogeneity was observed downstream of the repeats, depending on their length. We propose that aberrant extension occurs predominantly in the lagging strand, driven by dynamic interactions between the repeated sequences and the DNA replisome. This study may provide a foundation for understanding the mechanisms underlying the extension or deletion of repetitive genomic regions.

DNA repeats↗

PubMed2025

An orthogonal T7 replisome for continuous hypermutation and accelerated evolution in E. coli.

Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can substantially accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in Escherichia coli based on the controlled expression of the replisome of bacteriophage T7 (T7-ORACLE). The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 10-5 substitutions per base in vivo-100,000-fold above the genomic mutation rate. We demonstrated continuous evolution using the T7 replisome by expanding the substrate scope of TEM-1 β-lactamase and increasing activity 5000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than 1 week.

Bacteriophage T7↗

PubMed1991

An RNA replisome as the ancestor of the ribosome.

The ribosome is proposed to have evolved from an earlier RNA-replisome, which synthesized RNA. Ancestral tRNA molecules originally were loaded with trinucleotide sequences and donated them to growing RNA chains. The enzymatic addition of the C-C-A trinucleotide to presentday transfer RNA molecules is a carryover from this function. The strategies of reading RNA sequences by triplet codons and of housing information genetically in special repository molecules predates the origin of protein and DNA. These latter two polymers arose together at the time when the RNA replisome was converted to a ribosome.

Base Sequence↗

PubMed1992

Replisome pausing in mutagenesis.

E. coli cells containing a temperature-sensitive dnaE mutation, in the alpha-subunit of holoenzyme DNA polymerase III, do not survive at the restrictive temperature. Such cells may survive in the presence of the pcbA1 mutation, an allele of the gyrB gene. Such survival is dependent on an active DNA polymerase I. Evidence indicates that DNA polymerase I interacts directly in the replisome (REP.A). Despite normal survival for cells using the pcbA replication pathway after some type of DNA damage, we have noted a failure of damage-induced mutagenesis. Here we present evidence supporting a model of replisome pausing in cells dependent upon the pcbA replication pathway. The model argues that the (REP.A) complex pauses longer at the site of the lesion, allowing excision repair to occur completely. In the normal replication pathway (REP.E) bypass of the lesion occurs, fixing the mutation.

Alleles↗

PubMed2024

FANCJ DNA helicase is recruited to the replisome by AND-1 to ensure genome stability.

FANCJ, a DNA helicase linked to Fanconi anemia and frequently mutated in cancers, counteracts replication stress by dismantling unconventional DNA secondary structures (such as G-quadruplexes) that occur at the DNA replication fork in certain sequence contexts. However, how FANCJ is recruited to the replisome is unknown. Here, we report that FANCJ directly binds to AND-1 (the vertebrate ortholog of budding yeast Ctf4), a homo-trimeric protein adaptor that connects the CDC45/MCM2-7/GINS replicative DNA helicase with DNA polymerase α and several other factors at DNA replication forks. The interaction between FANCJ and AND-1 requires the integrity of an evolutionarily conserved Ctf4-interacting protein (CIP) box located between the FANCJ helicase motifs IV and V. Disruption of the CIP box significantly reduces FANCJ association with the replisome, causing enhanced DNA damage, decreased replication fork recovery and fork asymmetry in cells unchallenged or treated with Pyridostatin, a G-quadruplex-binder, or Mitomycin C, a DNA inter-strand cross-linking agent. Cancer-relevant FANCJ CIP box variants display reduced AND-1-binding and enhanced DNA damage, a finding that suggests their potential role in cancer predisposition.

Humans↗

PubMed2000

Leading and lagging strand DNA synthesis in vitro by a reconstituted herpes simplex virus type 1 replisome.

The synthesis of double-stranded DNA by a rolling circle mechanism was reconstituted in vitro with a replisome consisting of the DNA polymerase-UL42 complex and the heterotrimeric helicase-primase encoded by herpes simplex virus type 1. Okazaki fragments 3 kilobases in length and leading strands that may exceed 10 kilobases are produced. Lagging strand synthesis is stimulated by ribonucleoside triphosphates. DNA replication appears to be processive because it resists competition with an excess of (dT)(150)/(dA)(20). The single-strand DNA binding protein ICP8 is not required, and high concentrations of ICP8 can, in fact, inhibit lagging strand synthesis. The inhibition can, however, be overcome by the addition of an excess of the UL8 component of the helicase-primase. Rolling circle replication by the herpesvirus and bacteriophage T7 replisomes appears to proceed by a similar mechanism.

DNA↗

PubMed1997

The phiX174-type primosome promotes replisome assembly at the site of recombination in bacteriophage Mu transposition.

Initiation of Escherichia coli DNA synthesis primed by homologous recombination is believed to require the phiX174-type primosome, a mobile priming apparatus assembled without the initiator protein DnaA. We show that this primosome plays an essential role in bacteriophage Mu DNA replication by transposition. Upon promoting transfer of Mu ends to target DNA, the Mu transpososome undergoes transition to a pre-replisome that permits initiation of DNA synthesis only in the presence of primosome assembly proteins PriA, DnaT, DnaB and DnaC. These assembly proteins promote the engagement of primase and DNA polymerase III holoenzyme, initiating semi-discontinuous replication preferentially at the Mu left end. The results indicate that these proteins play a crucial role in promoting replisome assembly on a recombination intermediate.

Bacterial Proteins↗

PubMed2024

An Orthogonal T7 Replisome for Continuous Hypermutation and Accelerated Evolution in E. coli.

Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can dramatically accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in E. coli based on the controlled expression of the replisome of bacteriophage T7. The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 10 -5 substitutions per base in vivo - 100,000-fold above the genomic mutation rate. Continuous evolution using the mutagenic T7 replisome was demonstrated by expanding the substrate scope of TEM-1 β-lactamase and increase activity 1,000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than one week.

Journal Article↗

PubMed1996

Replisome assembly reveals the basis for asymmetric function in leading and lagging strand replication.

The E. coli replicase, DNA polymerase III holoenzyme, contains two polymerases for replication of duplex DNA. The DNA strands are antiparallel requiring different modes of replicating the two strands: one is continuous (leading) while the other is discontinuous (lagging). The two polymerases within holoenzyme are generally thought to have asymmetric functions for replication of these two strands. This report finds that the two polymerases have equal properties, both are capable of replicating the more difficult lagging strand. Asymmetric action is, however, imposed by the helicase that encircles the lagging strand. The helicase contact defines the leading polymerase constraining it to a subset of actions, while leaving the other to cycle on the lagging strand. The symmetric actions of the two polymerases free holoenzyme to assemble into the replisome in either orientation without concern for a correct match to one or the other strand.

Bacterial Proteins↗

PubMed2001

Dynamic protein interactions in the bacteriophage T4 replisome.

The bacteriophage T4 DNA replisome is a complex dynamic system employing a variety of proteins to orchestrate the synthesis of DNA on both the leading and lagging strands. Assembly of the protein complexes responsible for DNA synthesis and priming requires the coordination of transient biomolecular interactions. This interplay of proteins has been dissected through the use of small molecules including fluorescent probes and crosslinkers, enabling the development of a complex dynamic structural and kinetic model for DNA polymerase holoenzyme assembly and primosome formation.

Bacteriophage T4↗