Early events in DNA double strand break repair after damage induction with a laser microbeam

In the present thesis DNA double strand break (DSB) induction and repair are analysed after laser-microbeam irradiation. Live cell imaging of DNA repair proteins fused to Green Fluorescent Protein (GFP) as well as immunofluorescent detection of endogenous protein are used. Laser induced DNA damage, detected by gamma-H2AX foci staining depend on a subtle combination of used laser pulse wavelength, pulse energy and dose. The recruitment times of repair proteins depend inverse linearly on laser pulse energy. By extrapolation to zero, the recruitment time at biological relevant conditions is calculated. Interestingly, considerable spatial dynamics of the foci is found. Two neighbouring foci even can fuse within ~20 min. Recruitment time comparison of molecules representing early and late Non-Homologous end Joining (NHEJ), Homologous Recombination Repair (HRR) and the Mre11-Rad50-NBS1 (MRN) complex reveals that the whole NHEJ machinery is assembled to DSBs within 1 min. Recruitment of latest NHEJ factor (XRCC4) is faster than NBS1 and is not directly influenced by the absence of NBS1. XRCC4 persists at DSBs longer in the G1 cell cycle phase than in G2 where the replacement of NHEJ by HRR molecules occurs. Rad51 is recruited when XRCC4 is released with the complementary kinetics. DNA-PKcs phosphorylation at two sites, known to facilitate DNA end processing, occurs between the recruitment of NHEJ and HRR. A new modification of Comet-assay technique the Immunofluorescent Comet-assay (IFCA) is developed in this work for direct visualisation of DSBs in single cells. IFCA uses the immunofluorescent detection of histone H1 in neutral and alkaline Comet-assay, and enables simple and clear visualisation of details in the comet tail, which are hardly detectable by conventional DNA staining dyes such as SYBR Green. Using IFCA, the fragment size at the end of the neutral comet tail is determined for the first time

TRIP6 functions in brain ciliogenesis

TRIP6, a member of the ZYXIN-family of LIM domain proteins, is a focal adhesion compo- nent. Trip6 deletion in the mouse, reported here, reveals a function in the brain: ependymal and choroid plexus epithelial cells are carrying, unexpectedly, fewer and shorter cilia, are poorly differentiated, and the mice develop hydrocephalus. TRIP6 carries numerous protein interaction domains and its functions require homodimerization. Indeed, TRIP6 disruption in vitro (in a choroid plexus epithelial cell line), via RNAi or inhibition of its homodimerization, confirms its function in ciliogenesis. Using super-resolution microscopy, we demonstrate TRIP6 localization at the pericentriolar material and along the ciliary axoneme. The requirement for homodimerization which doubles its interaction sites, its punctate localiza- tion along the axoneme, and its co-localization with other cilia components suggest a scaf- fold/co-transporter function for TRIP6 in cilia. Thus, this work uncovers an essential role of a LIM-domain protein assembly factor in mammalian ciliogenesis

Epidermal Nbn deletion causes premature hair loss and a phenotype resembling psoriasiform dermatitis

Nijmegen Breakage Syndrome is a disease caused by NBN mutations. Here, we report a novel function of Nbn in skin homeostasis. We found that Nbn deficiency in hair follicle (HF) progenitors promoted increased DNA damage signaling, stimulating p16Ink4a up-regulation, Trp53 stabilization and cytokines secretion leading to HF-growth arrest and hair loss. At later stages, the basal keratinocytes layer exhibited also enhanced DNA damage response but in contrast to the one in HF progenitor was not associated with pro-inflammatory cytokines expression, but rather increased proliferation, lack of differentiation and immune response resembling psoriasiform dermatitis. Simultaneous Nbn and Trp53 inactivation significantly exacerbated this phenotype, due to the lack of inhibition of pro-inflammatory cytokines secretion by Trp53. Altogether, we demonstrated novel functions of Nbn in HF maintenance and prevention of skin inflammation and we provide a mechanistic explanation that links cell intrinsic DNA maintenance with large scale morphological tissue alterations

HAT cofactor TRRAP modulates microtubule dynamics via SP1 signaling to prevent neurodegeneration

Brain homeostasis is regulated by the viability and functionality of neurons. HAT (histone acetyltransferase) and HDAC (histone deacetylase) inhibitors have been applied to treat neurological deficits in humans; yet, the epigenetic regulation in neurodegeneration remains elusive. Mutations of HAT cofactor TRRAP (transformation/transcription domain-associated protein) cause human neuropathies, including psychosis, intellectual disability, autism, and epilepsy, with unknown mechanism. Here we show that Trrap deletion in Purkinje neurons results in neurodegeneration of old mice. Integrated transcriptomics, epigenomics, and proteomics reveal that TRRAP via SP1 conducts a conserved transcriptomic program. TRRAP is required for SP1 binding at the promoter proximity of target genes, especially microtubule dynamics. The ectopic expression of Stathmin3/4 ameliorates defects of TRRAP-deficient neurons, indicating that the microtubule dynamics is particularly vulnerable to the action of SP1 activity. This study unravels a network linking three well-known, but up-to-date unconnected, signaling pathways, namely TRRAP, HAT, and SP1 with microtubule dynamics, in neuroprotection

Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

Damaged replication forks activate poly(ADP-ribose) polymerase 1 (PARP1), which catalyses poly(ADP-ribose) (PAR) formation; however, how PARP1 or poly(ADP-ribosyl)ation is involved in the S-phase checkpoint is unknown. Here we show that PAR, supplied by PARP1, interacts with Chk1 via a novel PAR-binding regulatory (PbR) motif in Chk1, independent of ATR and its activity. iPOND studies reveal that Chk1 associates readily with the unperturbed replication fork and that PAR is required for efficient retention of Chk1 and phosphorylated Chk1 at the fork. A PbR mutation, which disrupts PAR binding, but not the interaction with its partners Claspin or BRCA1, impairs Chk1 and the S-phase checkpoint activation, and mirrors Chk1 knockdown-induced hypersensitivity to fork poisoning. We find that long chains, but not short chains, of PAR stimulate Chk1 kinase activity. Collectively, we disclose a previously unrecognized mechanism of the S-phase checkpoint by PAR metabolism that modulates Chk1 activity at the replication fork

Kinetics of poly(ADP-ribosyl)ation, but not PARP1 itself, determines the cell fate in response to DNA damage in vitro and in vivo

One of the fastest cellular responses to genotoxic stress is the formation of poly(ADP-ribose) polymers (PAR) by poly(ADP-ribose)polymerase 1 (PARP1, or ARTD1). PARP1 and its enzymatic product PAR regulate diverse biological processes, such as DNA repair, chromatin remodeling, transcription and cell death. However, the inter-dependent function of the PARP1 protein and its enzymatic activity clouds the mechanism underlying the biological response. We generated a PARP1 knock-in mouse model carrying a point mutation in the catalytic domain of PARP1 (D993A), which impairs the kinetics of the PARP1 activity and the PAR chain complexity in vitro and in vivo, designated as hypo-PARylation. PARP1D993A/D993A mice and cells are viable and show no obvious abnormalities. Despite a mild defect in base excision repair (BER), this hypo-PARylation compromises the DNA damage response during DNA replication, leading to cell death or senescence. Strikingly, PARP1D993A/D993A mice are hypersensitive to alkylation in vivo, phenocopying the phenotype of PARP1 knockout mice. Our study thus unravels a novel regulatory mechanism, which could not be revealed by classical loss-of-function studies, on how PAR homeostasis, but not the PARP1 protein, protects cells and organisms from acute DNA damage

Nbn and Atm Cooperate in a Tissue and Developmental Stage-Specific Manner to Prevent Double Strand Breaks and Apoptosis in Developing Brain and Eye

<div><p>Nibrin (NBN or NBS1) and ATM are key factors for DNA Double Strand Break (DSB) signaling and repair. Mutations in <i>NBN</i> or <i>ATM</i> result in Nijmegen Breakage Syndrome and Ataxia telangiectasia. These syndromes share common features such as radiosensitivity, neurological developmental defects and cancer predisposition. However, the functional synergy of Nbn and Atm in different tissues and developmental stages is not yet understood. Here, we show <i>in vivo</i> consequences of conditional inactivation of both genes in neural stem/progenitor cells using <i>Nestin-Cre</i> mice. Genetic inactivation of <i>Atm</i> in the central nervous system of Nbn-deficient mice led to reduced life span and increased DSBs, resulting in increased apoptosis during neural development. Surprisingly, the increase of DSBs and apoptosis was found only in few tissues including cerebellum, ganglionic eminences and lens. In sharp contrast, we showed that apoptosis associated with <i>Nbn</i> deletion was prevented by simultaneous inactivation of <i>Atm</i> in developing retina. Therefore, we propose that Nbn and Atm collaborate to prevent DSB accumulation and apoptosis during development in a tissue- and developmental stage-specific manner.</p></div