Although MTBP is essential for replication origin firing, we show here that strong depletion of MTBP can have minor effects on DNA replication rates. This suggests an adaptive process in the DNA replication program, so we examined mechanisms underlying this plasticity. Using an auxin-inducible degron to deplete MTBP, we found that acute suppression of MTBP blocked DNA replication, but that replication rates recovered over time. The timing of this recovery paralleled S phase expression of Cyclin B1, and inhibition of CDK1-Cyclin B1 prevented the recovery. Recovery did not involve restoration of origin firing; instead, replication recovered through accelerated fork progression. Consistent with CDK1 driving this acceleration, ATR inhibition, which activates CDK1, stimulated DNA replication in MTBP-depleted cells through CDK1-dependent increased fork progression rather than increased origin firing. Knockdown of RIF1, a known CDK1 target, phenocopied this effect. Although RIF1 is best known for opposing DDK-dependent MCM phosphorylation at origins, we find that RIF1 knockdown stimulates replication even when DDK is inhibited. Furthermore, RIF1 loss increased replication by accelerating fork progression rather than increasing origin firing. Together, these findings reveal a CDK1-RIF1-dependent mechanism that promotes fork speed during S phase and defines a form of replication plasticity in which fork rate compensates for reduced origin firing. Accurate genome duplication requires thousands of replication origins to fire and replication forks to complete DNA synthesis on schedule. When origin firing is compromised, it is unclear how cells avoid replication failure. We show that cells adapt to persistent loss of the origin-firing factor MTBP by accelerating replication fork progression through a CDK1-RIF1-dependent mechanism, partially compensating for reduced initiation. This adaptive response defines a form of replication plasticity in which cells rebalance origin usage and fork speed to sustain DNA synthesis. This mechanism may be especially relevant in cancer cells or other contexts where replication initiation is chronically stressed.
Replication fork collapse at single-strand DNA breaks threatens genome stability but how such forks are repaired and resolved has remained unclear. Here we replicate site-specific nicks with single or converging replication forks in Xenopus laevis egg extracts. Collapse of a single fork generates a single-ended double-strand break (DSB) that undergoes homologous recombination to yield stable D-loops and end-to-end fusions, yet does not restart DNA synthesis. Single collapsed forks can also undergo extensive nucleolytic degradation, appearing to disassemble the sister fork through 'secondary collapse' events that resolve single-ended DSBs without engaging DSB repair. In contrast, semisynchronous convergent collapse generates a double-ended DSB that is primarily repaired through annealing-dependent DSB repair, completing DNA synthesis but generating precise deletions and templated insertions. These error-prone products are not detected following single-fork collapse. Our findings demonstrate that single and semisynchronous convergent collapsed forks elicit distinct repair outcomes.
Accurate genome duplication requires tight-regulation of replication fork progression, and disruptions to this process are a major source of genomic instability, yet how fork dynamics are controlled during unperturbed S-phase remains unclear. We found replication forks elongate slowly in early S (ES) and faster in late S, independent of transcription or nucleotide availability. Elevated origin firing coupled with low TOP2A in ES generates torsional stress, causing replisome uncoupling, reduced fork speed, and basal ATR-CHK1 activation. Overexpression of TOP2A enhances fork speed and reduces replication stress in ES. Thus, TOP2A is a limiting replication factor during unperturbed ES, and basal ATR-CHK1 signaling is driven by transient replisome uncoupling. Also, TOP2A overexpression suppresses oncogene-driven replication stress. Given that TOP2A is frequently upregulated in cancers, it may function as a compensatory response to oncogene-induced replication stress. Together, these findings establish TOP2A as a central regulator of replication fork dynamics.
Replication fork reversal alleviates DNA replication stress and maintains genome stability. We previously showed that FBH1 and RAD54L cooperate to promote fork reversal in human cells, and that FBH1-dependent fork reversal requires the branch migration activity of RAD54L. However, the molecular basis of this cooperation remained unclear. Here, we identify both a physical and functional interaction between FBH1 and RAD54L. We demonstrate that purified FBH1 and RAD54L interact directly and form a complex at stalled replication forks in cells. Mapping studies revealed that RAD54L Lobe 1 is critical for interaction with the FBH1 2B subdomain. In cells, FBH1-RAD54L complex formation is enhanced in the absence of RAD51. Consistently, purified RAD54L displays a stronger affinity for RAD51 than for FBH1. Using biochemical reconstitution assays, we further show that FBH1 and RAD54L promote fork reversal more efficiently together than either protein alone, with maximal reversal observed when FBH1 acts before RAD54L. Collectively, our findings establish RAD54L as an essential functional partner of FBH1 in replication fork reversal and provide mechanistic insight into the sequential coordination of their activities.
TIMELESS, together with TIPIN and CLASPIN, forms the Fork Protection Complex (FPC), an essential regulator of DNA replication that possesses multiple functions in genome stability including the regulation of fork progression and replication checkpoint signaling. Structural studies place TIMELESS at the leading edge of the CMG helicase, which is inconsistent with FPC functions at the lagging strand and on single-stranded DNA. Our observation that FPC chromatin loading during replication initiation started in G1 phase cells, but was also enhanced by DNA synthesis, led us to propose a model of a step-wise loading of the FPC with two TIMELESS molecules per replication fork. Split-TurboID proximity labelling supported this model, placing the second FPC in proximity to the lagging strand. Using an auxin-inducible degron, we show that TIMELESS depletion compromised chromatin loading of TIPIN and CLASPIN, but the TIMELESS mutant unable to bind MCM still supported CLASPIN and TIPIN chromatin loading. This mutant was proficient in replication checkpoint activation, but failed to regulate fork speed under both unperturbed and oxidative-stress conditions. We propose that two distinct FPC instances at each replication fork: one at the leading edge, regulating fork progression, and one at the lagging strand mediating checkpoint signaling, - together execute the essential functions of TIMELESS.
Replication fork reversal is a DNA damage tolerance mechanism important for genome stability that entails annealing of parental DNA to push the fork backwards. F-box helicase 1 (FBH1) is a 3'-5' ssDNA translocase and SCF (SKP-CUL1-F box) E3 ubiquitin ligase that catalyzes fork reversal and limits aberrant recombination, yet how its helicase activity drives strand annealing is unknown. Here, using single-molecule and biochemical assays, we show that SCF FBH1 reverses forks through a two-stage reaction in which translocation on the lagging strand template while remaining affixed at the junction destabilizes the leading strand duplex to ultimately displace the nascent leading strand. Reversal is force-sensitive and does not generate a four-way junction, revealing an annealing-independent mechanism distinct from those of SMARCAL1, HLTF, and ZRANB3. These results establish the importance of nascent strand unwinding to fork reversal and suggest the existence of distinct pathways that produce unique DNA structures, which has implications for fork restart and its measurement in cells.
DNA replication is a strictly regulated process during cell proliferation to ensure faithful duplication of the genome. Its firing and elongation can be arrested or temporally inhibited in response to a variety of internal and external causes. Inside cells numerous factors including cell cycle checkpoints, protein kinases, and others are involved in the control of this process to maintain genome integrity. Here, we describe that NuMA, a nuclear scaffolding protein, plays an important role in regulating DNA replication. We show that NuMA is present at active replication forks, and its deficiency impairs cell viability, reduces the replication fork speed and increases origin firing, leading to increased level of γH2AX and the activation of the ATM-CHK2 DNA damage response pathway. Mechanistically, our results show that NuMA depletion reduced the association of multiple key replisome proteins to replication forks, suggesting that NuMA is essential for efficient replisome proteins binding to ongoing forks. Our study uncovers a novel function of NuMA in maintaining genome stability, providing new insights into the important role of nuclear structural proteins in safeguarding DNA replication.
Intentional ingestion of sharp foreign bodies in adults is uncommon and often associated with self-harm behavior. Multiple sharp objects located in different gastrointestinal segments significantly increase the risk of perforation and bleeding. We report a 27-year-old man who intentionally ingested nine razor blades and a full-length metallic fork. Radiography demonstrated multiple radiopaque objects without free air. Non-contrast computed tomography revealed a 185-mm fork extending from the distal esophagus into the stomach and razor blades within the proximal small bowel. Given the length of the fork, the multiplicity of sharp objects, and their distribution beyond the pylorus, endoscopic retrieval was deemed unsafe. The patient underwent urgent exploratory laparotomy with combined enterotomy and gastrotomy, enabling complete removal under intraoperative fluoroscopic guidance. Recovery was uneventful, and psychiatric follow-up was arranged. This case highlights the importance of early imaging, timely surgical intervention, and multidisciplinary care in managing complex sharp foreign body ingestion.
PARPi are effective therapy for BRCA1/2 mutant cancers, yet recurrent PARPi resistance frequently develops. The underlying mechanism of PARPi resistance remains largely unresolved. Here, we identify STN1, a component of the CTC1/STN1/TEN1 (CST) complex, as a modulator of PARPi resistance in BRCA2-deficient cells. RNA-seq analysis of PARPi-resistant cancer cells from BRCA2-mutated backgrounds shows largely distinct transcriptomic profiles with limited overlap, suggesting multiple routes to resistance. Notably, STN1 is consistently upregulated in resistant cells. We observe that overexpression of STN1 enhances Olaparib resistance in multiple BRCA2-deficient cell lines and alleviates DNA damage under replication stress. Mechanistically, we find that STN1 overexpression increases RAD51 loading to stalled replication forks while restricting MRE11 recruitment in BRCA2-deficient cells, thereby protecting stalled forks from nascent-strand degradation. Furthermore, STN1 overexpression rescues the accumulation of ssDNA gaps, a major determinant of PARPi sensitivity in BRCA2-deficient cells. Taken together, these findings suggest that elevated STN1 levels can partially compensate for BRCA2 loss by stabilizing stalled replication forks and limiting ssDNA gap accumulation. Our study uncovers a STN1-dependent pathway of replication stress tolerance that promotes PARPi resistance independently of homologous recombination restoration, highlighting STN1 as a potential biomarker and mechanistic contributor to therapeutic resistance in BRCA2-mutated cancers.
PDS5B (Precocious Dissociation of Sisters 5B) functions in sister chromatid cohesion and genome organization. Interestingly, PDS5B also associates with RAD51, the recombinase required for DNA damage repair by homologous recombination (HR) and the preservation of stressed DNA replication forks against nucleolytic attrition. We show that PDS5B binds dsDNA preferentially over ssDNA and that it enhances RAD51-mediated DNA strand exchange via the capture of dsDNA. PDS5B also acts synergistically with BRCA2-DSS1 to help overcome the interference of RPA in DNA strand exchange and works in conjunction with RAD51 to protect dsDNA against digestion by MRE11-RAD50-NBS1. DNA binding activity resides within the disordered C-terminal region of PDS5B, and testing of a DNA binding mutant provides evidence that this PDS5B attribute underpins protein functions in vitro and in HR and replication fork protection in cells. Our findings thus reveal distinct functions of PDS5B in genome repair and maintenance.
By promoting replication through DNA lesions, translesion synthesis (TLS) DNA polymerases protect against chromosomal instability and tumorigenesis. However, it is not known whether TLS in mammalian cells operates in conjunction with the replisome or in postreplicational gaps and how that impacts genomic stability. Here we show that TLS in human cells operates in close coordination with the replisome and that ATR stabilizes the replisome at the stalled replication fork (RF). In ATR-inhibited cells, the CMG helicase and DNA synthesis components of the replisome disassemble from RFs stalled at DNA lesions, and the composition of TLS and DNA synthesis components and the ensuing TLS and replication mechanisms at the stalled RFs are altered drastically from those in ATR-proficient cells. These alterations include the lack of requirement for Rad18-dependent PCNA ubiquitination for TLS by Polη and primer synthesis by the newly identified PrimPol--PolA1/PolA2 Polα complex. These results reveal the coupling of TLS to DNA replication, thus providing a means for protection against chromosome instability.
While the application of tuning fork Weber (TFW) test in adult is well-documented, its accuracy in the paediatric population remains uncertain although conductive hearing loss is frequently identified in children. Similarly, the effectiveness of the audiometric Weber (AW) test is ambiguous. Given evidence suggesting that age influences lateralization accuracy and understanding of instructions, this research specifically targets school-aged children to evaluate these diagnostic tools effectively. This study aims to explore the lateralization accuracy of TFW and AW tests in detecting conductive hearing loss in children and the agreement of these tests with the predicted lateralization responses based on audiogram. This study involved 75 children with conductive hearing loss aged between seven to twelve years old. Each subject underwent TFW (256 Hz and 512 Hz) and AW (250 Hz and 500 Hz) tests. All subjects achieved high accuracy (> 80%) in both TFW and AW tests. McNemar's test revealed there was no significant difference between the TFW and AW tests for both frequencies (p > 0.05). Using Cohen's Kappa, the lateralization responses in both tests demonstrate substantial to almost perfect agreement (k = 0.78-0.92). with the predicted lateralization responses for both frequencies. Large air-bone gap group also demonstrated higher accuracy for both tests compared to the small air-bone gap group, although the difference was not statistically significant (p > 0.05). Both the TFW and AW tests showed high lateralization accuracy in either unilateral or bilateral CHL supported by substantial to almost perfect agreement with the respective PTA in school-aged children with conductive hearing loss.
This paper concerns the estimation of front telescopic fork suspension elongation speed through the use of Kalman-filtering techniques. A full-motorcycle model in the state-space domain is developed and subsequently used in the filter along with synthetic input data simulating two accelerometer measurements. In addition, the force of semi-active suspension is considered as an input, from which the value is estimated on the basis of a look-up table and the estimated elongation speed. The performance of the full-motorcycle filter is compared to that of a filter built considering the monocorner model, indicating superiority in performance. The ratio of the mean squared error of the suspension elongation speed to the mean square of the elongation speed originating from the non-linear model is used as a performance metric. For the proposed estimator, it is 6.54% with respect to the best class of road profile (A) and 7.07% for the worst (H). This is in contrast to the monocorner filter, displaying values of 57.46% and 94.47% for the best and worst road classes, respectively. The influence of system pitch dynamics is evidenced to have a marginal influence on the accuracy of speed estimation. However, it is the use of a larger set of states that adds the notable advantage of employing such a solution.
T cells live or die by their metabolism, yet one nutrient can serve very different ends. In this issue of Cell, Kelly et al. show that cysteine's sulfur is partitioned between glutathione and iron-sulfur cluster synthesis. This routing drives CD8+ T cell proliferation, effector function, and anti-tumor immunity.
Streptococcus suis (S. suis) is an emerging zoonotic agent that now rivals classical food-borne pathogens in its global clinical burden of meningitis and septic shock. Recent epidemiological syntheses covering more than 30 countries now list over 1600 laboratory-confirmed human cases with a pooled case-fatality of about 12%, climbing beyond 18% in East Asia. The widely cited pooled case-fatality estimate derives from a PRISMA-guided systematic review and meta-analysis that searched PubMed, Scopus, Web of Science, ScienceDirect, and Google Scholar through December 2012 and pooled study-level event rates using inverse-variance methods with random-effects models when heterogeneity was present. We critically synthesize recent molecular, cellular, and translational studies to define how this swine commensal breaches host epithelial and blood-brain barriers (BBBs), subverts innate immunity, and disseminates systemically. Newly identified virulence mechanisms include serine-threonine kinase-driven claudin-5 cleavage, vimentin-dependent transcytosis, quorum-sensing control of biofilm maturation, and metabolic reprogramming that fuels neutrophil evasion. We integrate multiomics signatures with structural data to map conserved targets such as IdeSui and capsular polysaccharide that underpin next-generation conjugate and nanoparticle vaccines. Diagnostic advances spanning CRISPR-based assays and high-resolution imaging are assessed for their capacity to enable point-of-care detection. We also highlight host transcriptomic signatures that can be integrated into microfluidic chips, allowing syndromic discrimination between S. suis and pneumococcal meningitis within 40 min, a critical window for targeted therapy. Finally, we present a prevention framework uniting farm biosecurity, rational antibiotic stewardship, probiotic and bacteriocin interventions, and community education. Collectively, the review delivers an up-to-date roadmap for mitigating S. suis transmission and disease, highlights outstanding knowledge gaps in host-pathogen interactions, and outlines translational priorities needed to transform bench discoveries into effective public health countermeasures.
Bacteriophage-based products are gaining attention as effective tools to reduce harmful germs in food and combat antimicrobial resistance throughout the food production process. However, in South America, their use is still limited because of complicated regulations and inconsistent evidence requirements. This review aims to (i) explore the current scientific and technological landscape of using bacteriophages in South American food systems, (ii) identify main regulatory challenges that impact their classification, approval, and use, and (iii) highlight the need for consistent international guidelines, especially from Codex Alimentarius, to help safely and effectively incorporate phage-based products in food. Research on phage-based products is growing, but it is not consistent across different regions. There are more patents and advancements in biotechnology, but they are limited to certain areas. Although progress is being made, the regulatory frameworks are still unclear, especially when it comes to how these products are classified, labeled, and monitored for safety. To address these gaps, risk-based guidelines are needed. These should define product categories and claims, set safety standards, and include rules for tracking products and monitoring them after they hit the market. Creating a new Codex Alimentarius project on phage-based products could help establish global guidelines that promote safe use, reduce uncertainty in regulations, and improve trade in food markets around the world.
Forks in the Bitcoin network arise as a natural consequence of competition within its Proof-of-Work consensus protocol, but lead to resource inefficiencies and compromise network security. The frequency of forks, therefore, serves as a critical indicator of a distributed ledger's operational efficiency. We model the fork rate in a network of heterogeneous miners as a function of the number of miners, their hash rate distribution, and block propagation times within the peer-to-peer infrastructure. Empirical evidence demonstrates that fork rates are well-approximated by the ratio of the median block propagation time to mining time. Our model provides a theoretical foundation for this relationship while also capturing the fork rate's additional dependency on miner heterogeneity. Our work establishes a robust mathematical setting for investigating factors often unobservable from existing empirical data, such as power concentration, competition, and asymmetric propagation times in distributed networks. Using this as a null model, we can detect anomalies in the historical fork rate-e.g. around 2016-indicating either high concentration of mining power or strongly heterogeneous latency in the Bitcoin network. We also estimate the mining power wasted in mining blocks on top of a nonlatest block, which potentiates accidental forks. The wastage amounts to approximately 16,000 MW in the most recent year, equivalent to half of the power generated in the United Kingdom.
Pif1-family helicases are essential for proper nuclear and mitochondrial genome maintenance, yet the regulation of their activities remains incompletely understood. Here, we use single-molecule manipulation and visualization techniques to dissect the real-time mechanochemical behavior of Pfh1, the sole Pif1-family helicase in Schizosaccharomyces pombe. We systematically varied force, ATP concentration, fork composition, and the single-stranded DNA-binding protein spRim1, to quantify the unwinding and single-stranded DNA translocation properties of Pfh1. We find that Pfh1 operates through unwinding-rewinding cycles during which coordinated interactions with both DNA strands at the fork optimize ATP utilization. Contacts with the translocating strand modulate ATP affinity, while interactions with the displaced strand control maximum unwinding velocity. Binding of spRim1 to the displaced strand disrupts the latter interactions, increasing the unwinding velocity. Stable interactions of the helicase with both strands at the fork may limit unwinding processivity to ~20 bp, eventually triggering transition to rewinding. Rewinding proceeds through an ATP-dependent process that is incompatible with strand switching, in which ATP turnover modulates DNA contacts and rewinding rate. Binding of spRim1 to the displaced strand further accelerates rewinding, possibly by competing with helicase-DNA interactions, and facilitates recovery of the active unwinding conformation once the fork has rewound. Together, these findings suggest that Pfh1 balances unwinding and rewinding through coordinated ATP-dependent strand interactions, providing insight into how Pif1-family helicases are controlled at replication forks.
Mrc1 is a replication fork component that mediates communication between the replication checkpoint and fork progression. Here, we find that Mrc1 forms a dynamic mechanical linkage between CMG helicase and DNA Polymerase ε that balances fork stability and flexibility. In its coupled state, Mrc1 bridges CMG helicase and the Pol2 C-terminal domain of Polymerase ε to maintain helicase-polymerase coordination, promoting tolerance to replication stress but constraining fork remodeling. Upon checkpoint activation, Mec1/Rad53-dependent phosphorylation of Mrc1 shifts this equilibrium toward a loosened state, reducing coupling and enabling fork flexibility and lower mutation rate at the cost of reduced stress resistance.
Faithful genome duplication requires coordination between transcription and replication. Disruption of this coordination causes transcription-replication conflicts (TRCs), leading to replication stress and genome instability. How chromatin regulators modulate these processes remains unclear. Here, we show that the Rpd3L histone deacetylase complex dynamically modulates chromatin state to control replication fork progression and buffer TRCs in Saccharomyces cerevisiae. Rpd3L is targeted through both histone H3 lysine 4 methylation-dependent recruitment and methylation-independent mechanisms engaged under replication stress. Loss of H3K4 methylation or Rpd3L function promotes histone acetylation, accelerates fork progression through transcribed regions, and increases transcription-associated genome instability. Balanced acetylation at multiple histone lysines is required to stabilize replication forks under stress. While histone deacetylase complexes have been implicated in repairing damaged forks, our findings reveal that Rpd3L acts preemptively to modulate chromatin state and replication dynamics during TRCs, defining a chromatin-based mechanism that safeguards genome stability.