The tumour suppressor complex BRCA1-BARD1 functions in the repair of DNA double-stranded breaks by homologous recombination. During this process, BRCA1-BARD1 facilitates the nucleolytic resection of ...DNA ends to generate a single-stranded template for the recruitment of another tumour suppressor complex, BRCA2-PALB2, and the recombinase RAD51. Here, by examining purified wild-type and mutant BRCA1-BARD1, we show that both BRCA1 and BARD1 bind DNA and interact with RAD51, and that BRCA1-BARD1 enhances the recombinase activity of RAD51. Mechanistically, BRCA1-BARD1 promotes the assembly of the synaptic complex, an essential intermediate in RAD51-mediated DNA joint formation. We provide evidence that BRCA1 and BARD1 are indispensable for RAD51 stimulation. Notably, BRCA1-BARD1 mutants with weakened RAD51 interactions show compromised DNA joint formation and impaired mediation of homologous recombination and DNA repair in cells. Our results identify a late role of BRCA1-BARD1 in homologous recombination, an attribute of the tumour suppressor complex that could be targeted in cancer therapy.
Central to homologous recombination in eukaryotes is the RAD51 recombinase, which forms helical nucleoprotein filaments on single-stranded DNA (ssDNA) and catalyzes strand invasion with homologous ...duplex DNA. Various regulatory proteins assist this reaction including the RAD51 paralogs. We recently discovered that a RAD51 paralog complex from C. elegans, RFS-1/RIP-1, functions predominantly downstream of filament assembly by binding and remodeling RAD-51-ssDNA filaments to a conformation more proficient for strand exchange. Here, we demonstrate that RFS-1/RIP-1 acts by shutting down RAD-51 dissociation from ssDNA. Using stopped-flow experiments, we show that RFS-1/RIP-1 confers this dramatic stabilization by capping the 5′ end of RAD-51-ssDNA filaments. Filament end capping propagates a stabilizing effect with a 5′→3′ polarity approximately 40 nucleotides along individual filaments. Finally, we discover that filament capping and stabilization are dependent on nucleotide binding, but not hydrolysis by RFS-1/RIP-1. These data define the mechanism of RAD51 filament remodeling by RAD51 paralogs.
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•A nematode RAD51 paralog complex binds the 5′ end of RAD51 pre-synaptic filaments•Filament binding drives pre-synaptic complex remodeling with 5′→3′ polarity•Pre-synaptic complex remodeling propagates up to 40 nucleotides from the 5′ end•Filament binding is dependent on a nucleotide co-factor, but not ATP hydrolysis
RAD51 paralogs promote homologous recombination by remodeling RAD51-ssDNA filaments. Here, Taylor et al. demonstrate that RAD51 paralogs mediate remodeling by specifically binding the 5′ end of RAD51 filaments in a nucleotide-dependent manner. This propagates a stabilizing effect with 5′→3′ polarity distal to their binding site, slowing RAD51 dissociation from DNA.
Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded ...DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair. Here, we use single-molecule imaging to visualize the interplay between human RPA (hRPA) and human RAD51 during presynaptic complex assembly and disassembly. We demonstrate that ssDNA-bound hRPA can undergo facilitated exchange, enabling hRPA to undergo rapid exchange between free and ssDNA-bound states only when free hRPA is present in solution. Our results also indicate that the presence of free hRPA inhibits RAD51 filament nucleation, but has a lesser impact upon filament elongation. This finding suggests that hRPA exerts important regulatory influence over RAD51 and may in turn affect the properties of the assembled RAD51 filament. These experiments provide an important basis for further investigations into the regulation of human presynaptic complex assembly.
Variants in the ABCA4 gene are causal for a variety of retinal dystrophy phenotypes, including Stargardt disease (STGD1). However, 15% of patients who present with symptoms compatible with ...STGD1/ABCA4 disease do not have identifiable causal ABCA4 variants. We hypothesized that a case-control collapsing analysis in ABCA4-negative patients with compatible symptoms would provide an objective measure to identify additional disease genes.
We performed a genome-wide enrichment analysis of "qualifying variants"-ultrarare variants predicted to impact protein function-in protein-coding genes in 79 unrelated cases and 9028 unrelated controls.
Despite modest sample size, two known retinal dystrophy genes, PRPH2 and CRX, achieved study-wide significance (p < 1.33 × 10
) under a dominant disease model, and eight additional known retinal dystrophy genes achieved nominal significance (p < 0.05). Across these ten genes, the excess of qualifying variants explained up to 36.8% of affected individuals. Furthermore, under a recessive model, the cone-rod dystrophy gene CERKL approached study-wide significance.
Our results indicate that case-control collapsing analyses can efficiently identify pathogenic variants in genes in non-ABCA4 retinal dystrophies. The genome-wide collapsing analysis framework is an objective discovery method particularly suitable in settings with overlapping disease phenotypes.
Abstract
Ginger (Zingiber officinale) is one of the most valued spice plants worldwide; it is prized for its culinary and folk medicinal applications and is therefore of high economic and cultural ...importance. Here, we present a haplotype-resolved, chromosome-scale assembly for diploid ginger anchored to 11 pseudochromosome pairs with a total length of 3.1 Gb. Remarkable structural variation was identified between haplotypes, and two inversions larger than 15 Mb on chromosome 4 may be associated with ginger infertility. We performed a comprehensive, spatiotemporal, genome-wide analysis of allelic expression patterns, revealing that most alleles are coordinately expressed. The alleles that exhibited the largest differences in expression showed closer proximity to transposable elements, greater coding sequence divergence, more relaxed selection pressure, and more transcription factor binding site differences. We also predicted the transcription factors potentially regulating 6-gingerol biosynthesis. Our allele-aware assembly provides a powerful platform for future functional genomics, molecular breeding, and genome editing in ginger.
To determine if baseline diabetic retinopathy (DR) severity mediates the relationship between health insurance status and DR progression.
Retrospective cohort study.
Seven hundred sixteen patients ...aged ≥ 18 years with a diagnosis of type 1 or 2 diabetes mellitus, and a diagnosis of nonproliferative DR (NPDR) were identified from the electronic health record of a tertiary academic center between June 2012 and February 2022.
NPDR severity at baseline was the proposed mediator in the relationship between insurance status and proliferative DR (PDR) progression. Logistic regression was used to determine the association between insurance status and NPDR severity at baseline, and Cox proportional hazards regression was used to assess the association between insurance status and time to PDR progression. To analyze the mediation effect of NPDR severity at baseline, a counterfactual approach, which decomposes a total effect into a natural direct effect and a natural indirect effect was applied.
Time to progression from first NPDR diagnosis to first PDR diagnosis.
Of the 716 patients, 581 (81%) had Medicare or private insurance, 107 (15%) had Medicaid, and 28 (4.0%) were uninsured at their baseline eye visit. Uninsured or Medicaid patients had a higher proportion of moderate or severe NPDR at their baseline eye visit and a higher proportion of progression to PDR. After adjusting for confounders and NPDR severity at baseline, patients who were uninsured had significantly greater risk of progression to PDR compared with that of patients with Medicare/private insurance (hazard ratio HR: 2.63; 95% confidence interval CI: 1.10-6.25). Patients with Medicaid also had an increased risk of progression to PDR compared with that of patients with Medicare/private insurance, although not statistically significant (HR: 1.53; 95% CI: 0.81-2.89). NPDR severity at baseline mediated 41% of the effect of insurance status (uninsured vs. Medicare/private insurance) on PDR progression.
Patients who were uninsured were more likely to have an advanced stage of NPDR at their baseline eye visit and were at significantly greater risk of progression to PDR compared with patients who had Medicare or were privately insured. Mediation analysis revealed that differences in baseline NPDR severity by insurance explained a significant proportion of the relationship between insurance status and DR progression.
Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
To develop models for progression of nonproliferative diabetic retinopathy (NPDR) to proliferative diabetic retinopathy (PDR) and determine if incorporating updated information improves model ...performance.
Retrospective cohort study.
Electronic health record (EHR) data from a tertiary academic center, University of California San Francisco (UCSF), and a safety-net hospital, Zuckerberg San Francisco General (ZSFG) Hospital were used to identify patients with a diagnosis of NPDR, age ≥ 18 years, a diagnosis of type 1 or 2 diabetes mellitus, ≥ 6 months of ophthalmology follow-up, and no prior diagnosis of PDR before the index date (date of first NPDR diagnosis in the EHR).
Four survival models were developed: Cox proportional hazards, Cox with backward selection, Cox with LASSO regression and Random Survival Forest. For each model, three variable sets were compared to determine the impact of including updated clinical information: Static0 (data up to the index date), Static6m (data updated 6 months after the index date), and Dynamic (data in Static0 plus data change during the 6-month period). The UCSF data were split into 80% training and 20% testing (internal validation). The ZSFG data were used for external validation. Model performance was evaluated by the Harrell’s concordance index (C-Index).
Time to PDR.
The UCSF cohort included 1130 patients and 92 (8.1%) patients progressed to PDR. The ZSFG cohort included 687 patients and 30 (4.4%) patients progressed to PDR. All models performed similarly (C-indices ∼ 0.70) in internal validation. The random survival forest with Static6m set performed best in external validation (C-index 0.76). Insurance and age were selected or ranked as highly important by all models. Other key predictors were NPDR severity, diabetic neuropathy, number of strokes, mean Hemoglobin A1c, and number of hospital admissions.
Our models for progression of NPDR to PDR achieved acceptable predictive performance and validated well in an external setting. Updating the baseline variables with new clinical information did not consistently improve the predictive performance.
Proprietary or commercial disclosure may be found after the references.
Megakaryocytes (MKs) are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this ...investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL) and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in the SP600125-induced polyploidization of these cell lines synergistically with other signaling pathways.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In response to genotoxic stress, a transient arrest in cell-cycle progression enforced by the DNA-damage checkpoint (DDC) signalling pathway positively contributes to genome maintenance. Because ...hyperactivated DDC signalling can lead to a persistent and detrimental cell-cycle arrest, cells must tightly regulate the activity of the kinases involved in this pathway. Despite their importance, the mechanisms for monitoring and modulating DDC signalling are not fully understood. Here we show that the DNA-repair scaffolding proteins Slx4 and Rtt107 prevent the aberrant hyperactivation of DDC signalling by lesions that are generated during DNA replication in Saccharomyces cerevisiae. On replication stress, cells lacking Slx4 or Rtt107 show hyperactivation of the downstream DDC kinase Rad53, whereas activation of the upstream DDC kinase Mec1 remains normal. An Slx4-Rtt107 complex counteracts the checkpoint adaptor Rad9 by physically interacting with Dpb11 and phosphorylated histone H2A, two positive regulators of Rad9-dependent Rad53 activation. A decrease in DDC signalling results from hypomorphic mutations in RAD53 and H2A and rescues the hypersensitivity to replication stress of cells lacking Slx4 or Rtt107. We propose that the Slx4-Rtt107 complex modulates Rad53 activation by a competition-based mechanism that balances the engagement of Rad9 at replication-induced lesions. Our findings show that DDC signalling is monitored and modulated through the direct action of DNA-repair factors.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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