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11 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↗
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↗
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↗
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↗
PubMed2026

Single-molecule tracking of RNA-DNA hybrid removal enzymes important for lagging-strand replication.

The formation of RNA-DNA hybrid (RDH) primers by primase is an essential step in the recruitment of DNA polymerase during replication initiation and for the synthesis of each Okazaki fragment on the lagging strand. In addition to primers, RDHs form through misincorporation of ribonucleotides by DNA polymerase during elongation and by formation of R loops during transcription. R loops are three-stranded structures that form when the nascent mRNA anneals to the template DNA strand, displacing the complementary DNA strand. The persistence of RDHs is deleterious to genome stability in all cells because they increase susceptibility to mutations, impaired replication fork progression, DNA double-stranded breaks, and genomic rearrangements. In many bacteria, it is well established that components of the replicative DNA polymerase form a macromolecular complex that can be imaged using single-molecule or ensemble fluorescence approaches. The spatiotemporal regulation of proteins involved in RDH removal during lagging-strand maturation is less clear. Here, we study three proteins that are involved in the removal of RDHs from the lagging strand during DNA replication in the Gram-positive bacterium Bacillus subtilis: DNA polymerase I (Pol I), FenA, and RNase HIII. We characterized the behavior of each PAmCherry-tagged lagging-strand enzyme in living cells using single-particle tracking photoactivated localization microscopy. We find that all three proteins are highly mobile, suggesting residence times at their target substrates are below our temporal resolution. We also find evidence that Pol I activity is modulated through interaction with the replisome, whereas FenA and RNase HIII are regulated through access to the nucleoid. Our results provide new insight into how enzymes are recruited to resolve RDHs during lagging-strand replication in vivo.

DNA Replication↗
PubMed2026

Mechanistic diversity of clamp loading at small DNA gaps.

DNA sliding clamps, including PCNA (proliferating cell nuclear antigen) and the 9-1-1 (RAD9-RAD1-HUS1 in humans) complex, are ring-shaped protein complexes that encircle DNA and serve as central interaction platforms in DNA replication, repair, and checkpoint signaling. While clamp loading at canonical primer-template junctions by AAA+ (ATPases associated with diverse cellular activities) clamp loaders is well established, how clamps are loaded onto physiologically relevant but geometrically constrained DNA intermediates, such as nicks and single-stranded gaps, has remained unclear. Recent cryo-electron microscopy studies reveal that clamp loaders have evolved distinct strategies to overcome these constraints and to specialize for different genomic contexts. At gapped DNA, the eukaryotic clamp loader RFC (replication factor C) engages both 3'- and 5'-recessed DNA ends and can locally unwind DNA, enabling PCNA loading across a wide range of gap sizes. In contrast, the bacterial DnaX clamp loader lacks a 5'-DNA-binding site and does not unwind DNA, instead loading the &#x3b2;-clamp at small gaps (<6 nt) by sharply bending DNA. The checkpoint clamp loader Rad24-RFC (RAD17-RFC in humans) similarly lacks DNA unwinding activity, restricting loading of 9-1-1 clamp to larger gaps (&#x2265;6 nt). In a distinct specialization, Ctf18-RFC interacts with the leading-strand DNA polymerase &#x3b5;, positioning it as a dedicated loader for leading-strand synthesis, whereas Elg1-RFC (ATAD5-RFC in humans) excludes DNA from its chamber and functions as a PCNA unloader. Together, these mechanisms illustrate how clamp loaders are diversified to accommodate DNA structure and replisome context, ensuring coordinated control of genome replication and maintenance.

9-1-1 clamp↗
PubMed2026

MCM10 and RECQL4 have cooperative and redundant roles in activating the CMG helicase during the replication initiation.

DNA replication initiation requires activation of the CMG helicase to establish the replisome. This process involves the extrusion of single-stranded DNA (ssDNA) from the central channel of MCM double hexamers, allowing the two CMG helicases to pass each other; however, the factors that mediate this process in human cells remain unclear. We show that degron-mediated depletion of either MCM10 or RECQL4 alone causes mild replication defects, whereas simultaneous depletion of both proteins severely impairs CMG activation. ChIP-seq analyses demonstrate that RECQL4 localizes to replication initiation zones (IZs) independently of MCM10, whereas MCM10 recruitment to IZs is enhanced upon RECQL4 depletion, consistent with partially redundant roles during CMG activation. Rescue experiments further indicate that RECQL4 cooperates with MCM10 through direct interaction, and that their ssDNA-binding activity underlies their functional overlap. We propose that MCM10 and RECQL4 act cooperatively and redundantly to promote CMG activation.

CMG activation↗