DNA damage stabilizes interaction of CSB with the transcription elongation machinery

The Cockayne syndrome B (CSB) protein is essential for transcription-coupled DNA repair (TCR), which is dependent on RNA polymerase II elongation. TCR is required to quickly remove the cytotoxic transcription-blocking DNA lesions. Functional GFP-tagged CSB, expressed at physiological levels, was homogeneously dispersed throughout the nucleoplasm in addition to bright nuclear foci and nucleolar accumulation. Photobleaching studies showed that GFP-CSB, as part of a high molecular weight complex, transiently interacts with the transcription machinery. Upon (DNA damage-induced) transcription arrest CSB binding these interactions are prolonged, most likely reflecting actual engagement of CSB in TCR. These findings are consistent with a model in which CSB monitors progression of transcription by regularly probing elongation complexes and becomes more tightly associated to these complexes when TCR is active

Different SWI/SNF complexes coordinately promote R-loop- and RAD52-dependent transcription-coupled homologous recombination

The SWI/SNF family of ATP-dependent chromatin remodeling complexes is implicated in multiple DNA damage response mechanisms and frequently mutated in cancer. The BAF, PBAF and ncBAF complexes are three major types of SWI/SNF complexes that are functionally distinguished by their exclusive subunits. Accumulating evidence suggests that double-strand breaks (DSBs) in transcriptionally active DNA are preferentially repaired by a dedicated homologous recombination pathway. We show that different BAF, PBAF and ncBAF subunits promote homologous recombination and are rapidly recruited to DSBs in a transcription-dependent manner. The PBAF and ncBAF complexes promote RNA polymerase II eviction near DNA damage to rapidly initiate transcriptional silencing, while the BAF complex helps to maintain this transcriptional silencing. Furthermore, ARID1A-containing BAF complexes promote RNaseH1 and RAD52 recruitment to facilitate R-loop resolution and DNA repair. Our results highlight how multiple SWI/SNF complexes perform different functions to enable DNA repair in the context of actively transcribed genes.</p

A very brief description of LOFAR - the Low Frequency Array

LOFAR (Low Frequency Array) is an innovative radio telescope optimized for the frequency range 30-240 MHz. The telescope is realized as a phased aperture array without any moving parts. Digital beam forming allows the telescope to point to any part of the sky within a second. Transient buffering makes retrospective imaging of explosive short-term events possible. The scientific focus of LOFAR will initially be on four key science projects (KSPs): 1) detection of the formation of the very first stars and galaxies in the universe during the so-called epoch of reionization by measuring the power spectrum of the neutral hydrogen 21-cm line (Shaver et al. 1999) on the ~5' scale; 2) low-frequency surveys of the sky with of order $10^8$ expected new sources; 3) all-sky monitoring and detection of transient radio sources such as gamma-ray bursts, x-ray binaries, and exo-planets (Farrell et al. 2004); and 4) radio detection of ultra-high energy cosmic rays and neutrinos (Falcke & Gorham 2003) allowing for the first time access to particles beyond 10^21 eV (Scholten et al. 2006). Apart from the KSPs open access for smaller projects is also planned. Here we give a brief description of the telescope.Comment: 2 pages, IAU GA 2006, Highlights of Astronomy, Volume 14, K.A. van der Hucht, e

Nuclear Dynamics of PCNA in DNA Replication and Repair

The DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA) is central to both DNA replication and repair. The ring-shaped homotrimeric PCNA encircles and slides along double-stranded DNA, acting as a “sliding clamp” that localizes proteins to DNA. We determined the behavior of green fluorescent protein-tagged human PCNA (GFP-hPCNA) in living cells to analyze its different engagements in DNA replication and repair. Photobleaching and tracking of replication foci revealed a dynamic equilibrium between two kinetic pools of PCNA, i.e., bound to replication foci and as a free mobile fraction. To simultaneously monitor PCNA action in DNA replication and repair, we locally inflicted UV-induced DNA damage. A surprisingly longer residence time of PCNA at damaged areas than at replication foci was observed. Using DNA repair mutants, we showed that the initial recruitment of PCNA to damaged sites was dependent on nucleotide excision repair. Local accumulation of PCNA at damaged regions was observed during all cell cycle stages but temporarily disappeared during early S phase. The reappearance of PCNA accumulation in discrete foci at later stages of S phase likely reflects engagements of PCNA in distinct genome maintenance processes dealing with stalled replication forks, such as translesion synthesis (TLS). Using a ubiquitination mutant of GFP-hPCNA that is unable to participate in TLS, we noticed a significantly shorter residence time in damaged areas. Our results show that changes in the position of PCNA result from de novo assembly of freely mobile replication factors in the nucleoplasmic pool and indicate different binding affinities for PCNA in DNA replication and repair

Dynamics of Relative Chromosome Position during the Cell Cycle

The position of chromosomal neighborhoods in living cells was followed using three different methods for marking chromosomal domains occupying arbitrary locations in the nucleus; photobleaching of GFP-labeled histone H2B, local UV-marked DNA, and photobleaching of fluorescently labeled DNA. All methods revealed that global chromosomal organization can be reestablished through one cell division from mother to daughters. By simultaneously monitoring cell cycle stage in the cells in which relative chromosomal domain positions were tracked, we observed that chromosomal neighborhood organization is apparently lost in the early G1 phase of the cell cycle. However, the daughter cells eventually regain the general chromosomal organization pattern of their mothers, suggesting an active mechanism could be at play to reestablish chromosomal neighborhoods

Recruitment of the nucleotide excision repair endonuclease XPG to sites of UV-induced DNA damage depends on functional TFIIH

The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3′ side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5′ incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process. Copyrigh

LRP10 interacts with SORL1 in the intracellular vesicle trafficking pathway in non-neuronal brain cells and localises to Lewy bodies in Parkinson’s disease and dementia with Lewy bodies

Loss-of-function variants in the low-density lipoprotein receptor-related protein 10 (LRP10) gene have been associated with autosomal-dominant Parkinson’s disease (PD), PD dementia, and dementia with Lewy bodies (DLB). Moreover, LRP10 variants have been found in individuals diagnosed with progressive supranuclear palsy and amyotrophic lateral sclerosis. Despite this genetic evidence, little is known about the expression and function of LRP10 protein in the human brain under physiological or pathological conditions. To better understand how LRP10 variants lead to neurodegeneration, we first performed an in-depth characterisation of LRP10 expression in post-mortem brains and human-induced pluripotent stem cell (iPSC)-derived astrocytes and neurons from control subjects. In adult human brain, LRP10 is mainly expressed in astrocytes and neurovasculature but undetectable in neurons. Similarly, LRP10 is highly expressed in iPSC-derived astrocytes but cannot be observed in iPSC-derived neurons. In astrocytes, LRP10 is present at trans-Golgi network, plasma membrane, retromer, and early endosomes. Interestingly, LRP10 also partially co-localises and interacts with sortilin-related receptor 1 (SORL1). Furthermore, although LRP10 expression and localisation in the substantia nigra of most idiopathic PD and DLB patients and LRP10 variant carriers diagnosed with PD or DLB appeared unchanged compared to control subjects, significantly enlarged LRP10-positive vesicles were detected in a patient carrying the LRP10 p.Arg235Cys variant. Last, LRP10 was detected in Lewy bodies (LB) at late maturation stages in brains from idiopathic PD and DLB patients and in LRP10 variant carriers. In conclusion, high LRP10 expression in non-neuronal cells and undetectable levels in neurons of control subjects indicate that LRP10-mediated pathogenicity is initiated via cell non-autonomous mechanisms, potentially involving the interaction of LRP10 with SORL1 in vesicle trafficking pathways. Together with the specific pattern of LRP10 incorporation into mature LBs, these data support an important mechanistic role for disturbed vesicle trafficking and loss of LRP10 function in neurodegenerative diseases.</p

The Apertif Radio Transient System (ARTS): Design, commissioning, data release, and detection of the first five fast radio bursts

Fast radio bursts (FRBs) must be powered by uniquely energetic emission mechanisms. This requirement has eliminated a number of possible source types, but several remain. Identifying the physical nature of FRB emitters arguably requires good localisation of more detections, as well as broad-band studies enabled by real-time alerting. In this paper, we present the Apertif Radio Transient System (ARTS), a supercomputing radio-telescope instrument that performs real-time FRB detection and localisation on the Westerbork Synthesis Radio Telescope (WSRT) interferometer. It reaches coherent-addition sensitivity over the entire field of the view of the primary-dish beam. After commissioning results verified that the system performed as planned, we initiated the Apertif FRB survey (ALERT). Over the first 5 weeks we observed at design sensitivity in 2019, we detected five new FRBs, and interferometrically localised each of them to 0.4–10 sq. arcmin. All detections are broad band, very narrow, of the order of 1 ms in duration, and unscattered. Dispersion measures are generally high. Only through the very high time and frequency resolution of ARTS are these hard-to-find FRBs detected, producing an unbiased view of the intrinsic population properties. Most localisation regions are small enough to rule out the presence of associated persistent radio sources. Three FRBs cut through the halos of M31 and M33. We demonstrate that Apertif can localise one-off FRBs with an accuracy that maps magneto-ionic material along well-defined lines of sight. The rate of one every ~7 days ensures a considerable number of new sources are detected for such a study. The combination of the detection rate and localisation accuracy exemplified by the first five ARTS FRBs thus marks a new phase in which a growing number of bursts can be used to probe our Universe