Microhomology-mediated end joining (MMEJ) is a Ku- and ligase IV-independent mechanism for the repair of DNA double-strand breaks that contributes to chromosome rearrangements. Here we used a ...chromosomal end-joining assay to determine the genetic requirements for MMEJ in Saccharomyces cerevisiae. We found that end resection influences the ability to expose microhomologies; however, it is not rate limiting for MMEJ in wild-type cells. The frequency of MMEJ increased by up to 350-fold in rfa1 hypomorphic mutants, suggesting that replication protein A (RPA) bound to the single-stranded DNA (ssDNA) overhangs formed by resection prevents spontaneous annealing between microhomologies. In vitro, the mutant RPA complexes were unable to fully extend ssDNA and were compromised in their ability to prevent spontaneous annealing. We propose that the helix-destabilizing activity of RPA channels ssDNA intermediates from mutagenic MMEJ to error-free homologous recombination, thus preserving genome integrity.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
We explored the presence of extrachromosomal circular DNA (eccDNA) in the plasma of pregnant women. Through sequencing following either restriction enzyme or Tn5 transposase treatment, we identified ...eccDNA molecules in the plasma of pregnant women. These eccDNA molecules showed bimodal size distributions peaking at ∼202 and ∼338 bp with distinct 10-bp periodicity observed throughout the size ranges within both peaks, suggestive of their nucleosomal origin. Also, the predominance of the 338-bp peak of eccDNA indicated that eccDNA had a larger size distribution than linear DNA in human plasma. Moreover, eccDNA of fetal origin were shorter than the maternal eccDNA. Genomic annotation of the overall population of eccDNA molecules revealed a preference of these molecules to be generated from 5′-untranslated regions (5′-UTRs), exonic regions, and CpG island regions. Two sets of trinucleotide repeat motifs flanking the junctional sites of eccDNA supported multiple possible models for eccDNA generation. This work highlights the topologic analysis of plasma DNA, which is an emerging direction for circulating nucleic acid research and applications.
Mammalian polymerase theta (Polθ) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses ...that reveal that, in addition to the polymerase domain, Polθ-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Polθ-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Polθ and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.
Foldback priming at DNA double-stranded breaks is one mechanism proposed to initiate palindromic gene amplification, a common feature of cancer cells. Here, we show that small (5–9 bp) inverted ...repeats drive the formation of large palindromic duplications, the major class of chromosomal rearrangements recovered from yeast cells lacking Sae2 or the Mre11 nuclease. RPA dysfunction increased the frequency of palindromic duplications in Sae2 or Mre11 nuclease-deficient cells by ∼1,000-fold, consistent with intra-strand annealing to create a hairpin-capped chromosome that is subsequently replicated to form a dicentric isochromosome. The palindromic duplications were frequently associated with duplication of a second chromosome region bounded by a repeated sequence and a telomere, suggesting the dicentric chromosome breaks and repairs by recombination between dispersed repeats to acquire a telomere. We propose secondary structures within single-stranded DNA are potent instigators of genome instability, and RPA and Mre11-Sae2 play important roles in preventing their formation and propagation, respectively.
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•Short inverted repeats drive formation of large palindromic duplications•RPA inhibits annealing of inverted repeats to restrict hairpin formation•Mre11-Sae2 opens hairpins to prevent formation of palindromic duplications•The hairpin-capped chromosome is replicated to form an unstable dicentric chromosome
In this study, Deng et al. present evidence in support of a foldback priming mechanism to generate palindromic duplications in yeast. This class of chromosome rearrangements is normally prevented by RPA, which inhibits intra-strand annealing between short repeats, and by the hairpin-cleaving activity of the Mre11 nuclease and Sae2.
Replication protein A (RPA) is the main eukaryotic single‐stranded DNA (ssDNA) binding protein, having essential roles in all DNA metabolic reactions involving ssDNA. RPA binds ssDNA with high ...affinity, thereby preventing the formation of secondary structures and protecting ssDNA from the action of nucleases, and directly interacts with other DNA processing proteins. Here, we discuss recent results supporting the idea that one function of RPA is to prevent annealing between short repeats that can lead to chromosome rearrangements by microhomology‐mediated end joining or the formation of hairpin structures that are substrates for structure‐selective nucleases. We suggest that replication fork catastrophe caused by depletion of RPA could result from cleavage of secondary structures by nucleases, and that failure to cleave hairpin structures formed at DNA ends could lead to gene amplification. These studies highlight the important role RPA plays in maintaining genome integrity.
The recent success of mRNA therapeutics against pathogenic infections has increased interest in their use for other human diseases including cancer. However, the precise delivery of the genetic cargo ...to cells and tissues of interest remains challenging. Here, we show an adaptive strategy that enables the docking of different targeting ligands onto the surface of mRNA-loaded small extracellular vesicles (sEVs). This is achieved by using a microfluidic electroporation approach in which a combination of nano- and milli-second pulses produces large amounts of IFN-γ mRNA-loaded sEVs with CD64 overexpressed on their surface. The CD64 molecule serves as an adaptor to dock targeting ligands, such as anti-CD71 and anti-programmed cell death-ligand 1 (PD-L1) antibodies. The resulting immunogenic sEVs (imsEV) preferentially target glioblastoma cells and generate potent antitumour activities in vivo, including against tumours intrinsically resistant to immunotherapy. Together, these results provide an adaptive approach to engineering mRNA-loaded sEVs with targeting functionality and pave the way for their adoption in cancer immunotherapy applications.
Significance Chromosomal double-strand breaks (DSBs) are cytotoxic forms of DNA damage that must be accurately repaired to maintain genome integrity. The conserved Mre11–Rad50–Xrs2/NBS1 ...nuclease/ATPase complex plays an important role in repair by functioning as a damage sensor and by regulation of DNA end processing to ensure repair by the most appropriate mechanism. Yeast Sae2 is known to function with Mre11 to process DNA ends, but its precise role is poorly understood. Here we show that it is the failure to remove Mre11 from DNA ends, leading to persistent DNA damage signaling and cell cycle arrest, that causes sensitivity of Sae2-deficient cells to DNA damaging agents.
The Mre11–Rad50–Xrs2/NBS1 (MRX/N) nuclease/ATPase complex plays structural and catalytic roles in the repair of DNA double-strand breaks (DSBs) and is the DNA damage sensor for Tel1/ATM kinase activation. Saccharomyces cerevisiae Sae2 can function with MRX to initiate 5′-3′ end resection and also plays an important role in attenuation of DNA damage signaling. Here we describe a class of mre11 alleles that suppresses the DNA damage sensitivity of sae2 Δ cells by accelerating turnover of Mre11 at DNA ends, shutting off the DNA damage checkpoint and allowing cell cycle progression. The mre11 alleles do not suppress the end resection or hairpin-opening defects of the sae2 Δ mutant, indicating that these functions of Sae2 are not responsible for DNA damage resistance. The purified M ᴾ¹¹⁰ᴸRX complex shows reduced binding to single- and double-stranded DNA in vitro relative to wild-type MRX, consistent with the increased turnover of Mre11 from damaged sites in vivo. Furthermore, overproduction of Mre11 causes DNA damage sensitivity only in the absence of Sae2. Together, these data suggest that it is the failure to remove Mre11 from DNA ends and attenuate Rad53 kinase signaling that causes hypersensitivity of sae2 Δ cells to clastogens.
Borderline personality disorder (BPD) is thought to involve aberrant social learning, including impaired revision of social interpretations with new evidence (social interpretation inflexibility). ...However, this topic has received little empirical attention outside of specific literatures, such as moral inference or behavioral economics. Further, the contribution of comorbid depression to BPD-related interpretation inflexibility has not yet been assessed.
In two independent samples (Study 1: N = 213; Study 2: N = 210, oversampled for BPD features), we assessed the associations between BPD symptoms, depressive symptoms, and task-based measures of social interpretation flexibility.
We found that BPD symptoms, particularly volatility of identity and relationships, were associated with less revision of social interpretations with both positive and negative evidence. Meanwhile, depressive symptoms were associated with a pattern of less revision of social interpretations with positive versus negative information.
The use of cross-sectional, crowdsourced samples limits causal interpretations. Translation to clinical populations should be assessed in future studies.
Results suggest that inflexible social interpretations across valences may be a feature of BPD-related pathology, and could be connected to symptoms involving volatility in social contexts. Future studies should investigate whether treatments geared toward increasing the flexibility of social interpretations are effective in treating BPD symptoms, especially those involving interpersonal difficulties.
Nanomedicines have been approved to treat multiple human diseases. However, clinical adoption of nanoformulated agents is often hindered by concerns about hepatic uptake and clearance, a process that ...is not fully understood. Here we show that the antitumour efficacy of cancer nanomedicine exhibits an age-associated disparity. Tumour delivery and treatment outcomes are superior in old versus young mice, probably due to an age-related decline in the ability of hepatic phagocytes to take up and remove nanoparticles. Transcriptomic- and protein-level analysis at the single-cell and bulk levels reveals an age-associated decrease in the numbers of hepatic macrophages that express the scavenger receptor MARCO in mice, non-human primates and humans. Therapeutic blockade of MARCO is shown to decrease the phagocytic uptake of nanoparticles and improve the antitumour effect of clinically approved cancer nanotherapeutics in young but not aged mice. Together, these results reveal an age-associated disparity in the phagocytic clearance of nanotherapeutics that affects their antitumour response, thus providing a strong rationale for an age-appropriate approach to cancer nanomedicine.
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