A mechanism to minimize errors during non-homologous end joining, bioRxiv, 2019-03-01
SUMMARYEnzymatic processing of DNA underlies all DNA repair, yet inappropriate DNA processing must be avoided. In vertebrates, double-strand breaks are repaired predominantly by non-homologous end-joining (NHEJ), which directly ligates DNA ends. NHEJ has the potential to be highly mutagenic because it employs DNA polymerases, nucleases, and other enzymes that modify incompatible DNA ends to allow their ligation. Using a biochemical system that recapitulates key features of cellular NHEJ, we show that end-processing requires formation of a “short-range synaptic complex” in which DNA ends are closely aligned in a ligation-competent state. Furthermore, single-molecule imaging directly demonstrates that processing occurs within the short-range complex. This confinement of end processing to a ligation-competent complex ensures that DNA ends undergo ligation as soon as they become compatible, thereby minimizing mutagenesis. Our results illustrate how the coordination of enzymatic catalysis with higher-order structural organization of substrate maximizes the fidelity of DNA repair.
biorxiv molecular-biology 0-100-users 2019Non-homologous end joining minimizes errors by coordinating DNA processing with ligation, bioRxiv, 2019-03-01
Genome stability requires efficient and faithful repair of DNA double-strand breaks (DSBs). The predominant DSB repair pathway in human cells is non-homologous end-joining (NHEJ), which directly ligates DNA ends1–5. Broken DNA ends at DSBs are chemically diverse, and many are not compatible for direct ligation by the NHEJ-associated DNA Ligase IV (Lig4). To solve this problem, NHEJ end-processing enzymes including polymerases and nucleases modify ends until they are ligatable. How cells regulate end processing to minimize unnecessary genomic alterations6 during repair of pathological DSBs remains unknown. Using a biochemical system that recapitulates key features of cellular NHEJ, we previously demonstrated that DNA ends are initially tethered at a distance, followed by Lig4-mediated formation of a “short-range synaptic complex” in which DNA ends are closely aligned for ligation7. Here, we show that a wide variety of end-processing activities all depend on formation of the short-range complex. Moreover, using real-time single molecule imaging, we find that end processing occurs within the short-range complex. Confining end processing to the Lig4-dependent short-range synaptic complex promotes immediate ligation of compatible ends and ensures that incompatible ends are ligated as soon as they become compatible, thereby minimizing end processing. Our results elucidate how NHEJ exploits end processing to achieve versatility while minimizing errors that cause genome instability.
biorxiv molecular-biology 0-100-users 2019Localization microscopy at doubled precision with patterned illumination, bioRxiv, 2019-02-20
MINFLUX offers a breakthrough in single molecule localization precision, but suffers from a tiny field of-view and a lack of practical parallelism. Here, we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micron sized field-of-view. We show a near twofold improvement in precision over standard localization with the same photon count on DNA-origami nano-structures.
biorxiv molecular-biology 0-100-users 2019Exercise twice-a-day potentiates skeletal muscle signalling responses associated with mitochondrial biogenesis in humans, which are independent of lowered muscle glycogen content, bioRxiv, 2019-02-12
Endurance exercise begun with reduced muscle glycogen stores seems to potentiate skeletal muscle protein abundance and gene expression. However, it is unknown whether this greater signalling responses is due to low muscle glycogen per se or to performing two exercise sessions in close proximity - as a first exercise session is necessary to reduce the muscle glycogen stores. In the present study, we manipulated the recovery duration between a first muscle glycogen-depleting exercise and a second exercise session, such that the second exercise session started with reduced muscle glycogen in both approaches but was performed either two or 15 h after the first exercise session (so-called twice-a-day and once-daily approaches, respectively). We found that exercise twice-a-day increased the nuclear abundance of transcription factor EB (TFEB) and nuclear factor of activated T cells (NFAT) and potentiated the transcription of peroxisome proliferator-activated receptor-ɣ coactivator 1 alpha (PGC-1a), peroxisome proliferator-activated receptor alpha (PPARa;) and peroxisome proliferator-activated receptor betadelta (PPARbd) genes, in comparison with the once-daily exercise. These results suggest that the elevated molecular signalling reported with previous train-low approaches can be attributed to performing two exercise sessions in close proximity rather than the reduced muscle glycogen content per se. The twice-a-day approach might be an effective strategy to induce adaptations related to mitochondrial biogenesis and fat oxidation.
biorxiv molecular-biology 100-200-users 2019Environmental DNA (eDNA) metabarcoding of pond water as a tool to survey conservation and management priority mammals, bioRxiv, 2019-02-11
Abstract<jatslist list-type=order><jatslist-item>Environmental DNA (eDNA) metabarcoding is largely used to survey aquatic communities, but can also provide data on terrestrial taxa utilising aquatic habitats. However, the entry, dispersal, and detection of terrestrial species’ DNA within waterbodies is understudied.<jatslist-item><jatslist-item>We evaluated eDNA metabarcoding of pond water for monitoring semi-aquatic, ground-dwelling, and arboreal mammals, and examined spatiotemporal variation in mammal eDNA signals using experiments in captive and wild conditions.<jatslist-item><jatslist-item>We selected nine focal species of conservation and management concern European water vole, European otter, Eurasian beaver, European hedgehog, European badger, red deer, Eurasian lynx, red squirrel, and European pine marten. We hypothesised that eDNA signals (i.e. proportional read counts) would be stronger for semi-aquatic than terrestrial species, and at sites where mammals exhibited behaviours (e.g. swimming, urination). We tested this by sampling waterbodies in enclosures of captive focal species at specific sites where behaviours had been observed (‘directed’ sampling) and at equidistant intervals along the shoreline (‘stratified’ sampling). We then surveyed natural ponds (N = 6) where focal species were present using stratified water sampling, camera traps, and field signs. eDNA samples were metabarcoded using vertebrate-specific primers.<jatslist-item><jatslist-item>All focal species were detected in captivity. eDNA signal strength did not differ between directed and stratified samples across or within species, between species lifestyles (i.e. semi-aquatic, ground-dwelling, arboreal), or according to behaviours. Therefore, eDNA was evenly distributed within artificial waterbodies. Conversely, eDNA was unevenly distributed in natural ponds. eDNA metabarcoding, camera trapping, and field signs detected beaver, red deer, and roe deer. Badger and red fox were recorded with cameras and field signs, but not eDNA metabarcoding. However, eDNA metabarcoding detected small mammals missed by cameras and field signs, e.g. water vole. Terrestrial mammal eDNA signals were weaker and detected in fewer samples than semi-aquatic mammal eDNA signals.<jatslist-item><jatslist-item>eDNA metabarcoding has potential for inclusion in mammal monitoring schemes by enabling large-scale, multi-species distribution assessment for priority and difficult to survey species, and could provide early indication of range expansions or contractions. However, eDNA surveys need high spatiotemporal resolution and metabarcoding biases require further investigation before this tool is routinely implemented.<jatslist-item>
biorxiv molecular-biology 0-100-users 2019Ensuring meiotic DNA break formation in the mouse pseudoautosomal region Data File S1, bioRxiv, 2019-01-31
Sex chromosomes in males share only a diminutive homologous segment, the pseudoautosomal region (PAR), wherein meiotic double-strand breaks (DSBs), pairing, and crossing over must occur for correct segregation. How cells ensure PAR recombination is unknown. Here we delineate cis- and trans-acting factors that control PAR ultrastructure and make the PAR the hottest area of DSB formation in the male mouse genome. Prior to DSB formation, PAR chromosome axes elongate, sister chromatids separate, and DSB-promoting factors hyperaccumulate. These phenomena are linked to mo-2 minisatellite arrays and require ANKRD31 protein. We propose that the repetitive PAR sequence confers unique chromatin and higher order structures crucial for DSB formation, X-Y pairing, and recombination. Our findings establish a mechanistic paradigm of mammalian sex chromosome segregation during spermatogenesis.
biorxiv molecular-biology 100-200-users 2019