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At least 19 recordsLinked to original sources
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↗
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↗
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↗
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↗
PubMed1976

[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↗
PubMed1985

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↗
PubMed1991

A possible interaction of single-strand binding protein and RecA protein during post-ultraviolet DNA synthesis.

The mechanism of DNA replication in ultraviolet (UV)-irradiated Escherichia coli is proposed. Immediately after UV exposure, the replisome aided by single-strand DNA-binding protein (SSB) can proceed past UV-induced pyrimidine dimers without insertion of nucleotides. Polymerisation eventually resumes somewhere downstream of the dimer sites. Due to the limited supply of SSB, only a few dimers can be bypassed in this way. Nevertheless, this early DNA synthesis is of great biological importance because it generates single-stranded DNA regions. Single-stranded DNA can bind and activate RecA protein, thus leading to induction of the SOS response. During the SOS response, the cellular level of RecA protein increases dramatically. Due to the simultaneous increase in the concentration of ATP, RecA protein achieves the high-affinity state for single-stranded DNA. Therefore it is able to displace DNA-bound SSB. The cycling of SSB on and off DNA enables the replisome to bypass a large number of dimers at late post-UV times. During this late replication, the stoichiometric amounts of RecA protein needed for recombination are involved in the process of postreplication repair.

DNA Replication↗
PubMed1989

Post-ultraviolet DNA synthesis in the absence of repair: role of the single-strand DNA-binding protein.

Post-ultraviolet DNA synthesis kinetics were investigated in the Escherichia coli uvrA recA strain and its isogenic counterpart, overproducing single-strand DNA-binding protein (SSB). It was demonstrated that large quantities of SSB enhance the capacity of the unmodified replisome to use the UV-damaged template for DNA synthesis. DNA thus synthesized is of low molecular weight, as shown by sedimentation in alkaline sucrose gradients. It is therefore suggested that SSB actively participates in the replisome translocation past dimers and/or the initiation of new DNA chains downstream of these lesions.

DNA Repair↗
PubMed2020

Human NK cell deficiency as a result of biallelic mutations in MCM10.

Human natural killer cell deficiency (NKD) arises from inborn errors of immunity that lead to impaired NK cell development, function, or both. Through the understanding of the biological perturbations in individuals with NKD, requirements for the generation of terminally mature functional innate effector cells can be elucidated. Here, we report a cause of NKD resulting from compound heterozygous mutations in minichromosomal maintenance complex member 10 (MCM10) that impaired NK cell maturation in a child with fatal susceptibility to CMV. MCM10 has not been previously associated with monogenic disease and plays a critical role in the activation and function of the eukaryotic DNA replisome. Through evaluation of patient primary fibroblasts, modeling patient mutations in fibroblast cell lines, and MCM10 knockdown in human NK cell lines, we have shown that loss of MCM10 function leads to impaired cell cycle progression and induction of DNA damage-response pathways. By modeling MCM10 deficiency in primary NK cell precursors, including patient-derived induced pluripotent stem cells, we further demonstrated that MCM10 is required for NK cell terminal maturation and acquisition of immunological system function. Together, these data define MCM10 as an NKD gene and provide biological insight into the requirement for the DNA replisome in human NK cell maturation and function.

Alleles↗
PubMed1992

Activation in vivo of the minimal replication origin beta of plasmid R6K requires a small target sequence essential for DNA looping.

The plasmid R6K contains three distinct origins of replication: alpha, beta, and gamma. The gamma sequence is essential in cis and acts as an enhancer that activates the distant alpha and beta origins. R6K therefore represents a favorable procaryotic model system with which to unravel the biochemical mechanisms underlying selective origin activation, particularly activation involving distant sites on the same chromosome. We have discovered that plasmids containing the origins alpha and gamma required the Escherichia coli DnaA initiator protein in addition to the R6K-encoded initiator protein, Pi, and other host replisomal proteins for their maintenance in vivo. Plasmids initiating replication from origin beta required only the Pi initiator protein and other host replisomal proteins. We have exploited the differential requirement for the DnaA protein by origins gamma and beta to selectively study and localize the minimal origin beta sequences by deletion analysis as one test of a looping model of origin activation. A 64-bp region spanning the extreme -COOH terminal coding sequence of the Pi protein was found to be essential for replication in vivo in the absence of DnaA protein, consistent with the approximate physical location of the beta origin. Replication emanating from origin beta could be abolished in vivo by deletion of the 9-bp target site for Pi protein-mediated DNA looping between the gamma origin/enhancer and the distant beta origin. Electron microscopy of nascent replication intermediates generated in vivo directly confirmed our genetic localization of the beta origin. Our results strongly suggest that activation of the beta origin by a distant replication enhancer element requires a small target sequence essential for initiator protein-mediated DNA looping.

Amino Acid Sequence↗
PubMed2026

Maintenance of nucleosome organization through replication and transcription counteracts aberrant coalescence of active chromatin.

Nucleosomes with their associated modifications organize and regulate the genome. It is unclear how this is integrated with the requirement of replication and transcription to access the DNA template without jeopardizing chromatin function. Here, we reveal a unified requirement for the histone chaperone FACT in mediating nucleosome disruption and reassembly during mammalian replication and transcription. Upon acute FACT depletion, replisome and RNA polymerase progression is halted genome wide, and chromatin structure in their wake collapses, with reduced nucleosome occupancy, irregular spacing, and intermediate assemblies. Chromatin states deteriorate as modified histones are lost due to a lack of histone recycling. Chromatin fiber disorder further manifests in the 3D genome, triggering active genes to coalesce in aberrant microcompartments. Similarly, aberrant compartments form in cells failing to maintain chromatin fiber structure through replication. Nucleosome organization therefore dynamically regulates genome architecture, guarding against spurious chromatin aggregation.

Nucleosomes↗
PubMed1985

DNA damage and cell killing. Cause and effect?

The evidence supporting a cause and effect relationship between DNA damage and cell killing is examined in the light of what is currently known about the organization and replication of genomic DNA in eukaryotic cells and the radio-energetics of DNA breakage. A large disparity is identified between characteristic doses for cell killing and for the production of DNA lesions (i.e., single- or double-strand breaks). In contrast, the sensitive phase of the inhibition of DNA synthesis has a dependence on dose quantitatively similar to that of cell killing. A model is developed in which single- and double-strand breaks are associated with the inhibition of replicon initiation, whereas only double-strand breaks are primarily responsible for strand elongation. Furthermore, the model points to the replisome and the region of replicated DNA just downstream from the fork as the locus of radiation action.

Animals↗
PubMed1984

Regulation of a new bacteriophage T4 gene, 69, that spans an origin of DNA replication.

We have determined the DNA sequence and transcription patterns in a 3-kb segment (between 15 and 18 kb on the standard phage T4 map) spanning an origin of DNA replication. A new gene, 69, spans this origin. Gene 69 codes for two overlapping proteins that share a common C-terminal segment. Defective DNA replication in an appropriate amber mutant shows that at least the larger of the two proteins is required for efficient T4 DNA replication. The two proteins coded by gene 69 are expressed from different transcripts that are under different regulation. The smaller protein, gp69*, can be expressed immediately from an Escherichia coli-like promoter, whereas expression of the larger protein, gp69, must be delayed since its middle promoter requires T4 coded proteins, most likely gp mot, for activation. We discuss the possible significance of two overlapping proteins in the assembly of replisomes. Gene 69 is bracketed by the non-essential early gene dam (DNA adenine methylase) and the late gene soc (small outer capsid protein). Transcripts through this region are interdigitated in a complex pattern, which reveals all elements that are thought to be important in regulation of pre-replicative and post-replicative T4 genes.

Amino Acid Sequence↗