Mutations in ORC1, ORC4, ORC6, CDT1, and CDC6, which encode proteins required for DNA replication origin licensing, cause Meier-Gorlin syndrome (MGS), a disorder conferring microcephaly, primordial dwarfism, underdeveloped ears, and skeletal abnormalities. Mutations in ATR, which also functions during replication, can cause Seckel syndrome, a clinically related disorder. These findings suggest that impaired DNA replication could underlie the developmental defects characteristic of these disorders. Here, we show that although origin licensing capacity is impaired in all patient cells with mutations in origin licensing component proteins, this does not correlate with the rate of progression through S phase. Thus, the replicative capacity in MGS patient cells does not correlate with clinical manifestation. However, ORC1-deficient cells from MGS patients and siRNA–mediated depletion of origin licensing proteins also have impaired centrosome and centriole copy number. As a novel and unexpected finding, we show that they also display a striking defect in the rate of formation of primary cilia. We demonstrate that this impacts sonic hedgehog signalling in ORC1-deficient primary fibroblasts. Additionally, reduced growth factor-dependent signaling via primary cilia affects the kinetics of cell cycle progression following cell cycle exit and re-entry, highlighting an unexpected mechanism whereby origin licensing components can influence cell cycle progression. Finally, using a cell-based model, we show that defects in cilia function impair chondroinduction. Our findings raise the possibility that a reduced efficiency in forming cilia could contribute to the clinical features of MGS, particularly the bone development abnormalities, and could provide a new dimension for considering developmental impacts of licensing deficiency.

Recent studies have shown that homologous recombination (HR) requires chromatin repression as well as relaxation at DNA double strand breaks (DSBs). HP1 and SUV39H1/2 are repressive factors essential for HR. Here, we identify SETDB1 as an additional compacting factor promoting HR. Depletion of HP1, SUV39, SETDB1 or BRCA1 confer identical phenotypes. The repressive factors, like BRCA1, are dispensable for the initiation of resection but promote the extension step causing diminished RPA or RAD51 foci and HR in irradiated G2 cells. Depletion of the compacting factors does not inhibit BRCA1 recruitment but at 8 h post IR, BRCA1 foci are smaller and aberrantly positioned compared to control cells. BRCA1 promotes 53BP1 repositioning to the periphery of enlarged foci and formation of a devoid core with BRCA1 becoming enlarged and localized internally to 53BP1. Depletion of the compacting factors precludes these changes at irradiation-induced foci. Thus, the repressive factors are required for BRCA1 function in promoting the repositioning of 53BP1 during HR. Additionally, depletion of these repressive factors in undamaged cells causes diminished sister chromatid association at centromeric sequences. We propose a model for how these findings may be functionally linked.

Accumulation of peptide-linked DNA breaks contributes to neurodegeration in humans. This is typified by defects in tyrosyl DNA phosphodiesterase 1 (TDP1) and human hereditary ataxia. TDP1 primarily operates at single-strand breaks (SSBs) created by oxidative stress or by collision of transcription machinery with topoisomerase I intermediates (Top1-CCs). Cellular and cell-free studies have shown that Top1 at stalled Top1-CCs is first degraded to a small peptide resulting in Top1-SSBs, which are the primary substrates for TDP1. Here we established an assay to directly compare Top1-SSBs and Top1-CCs. We subsequently employed this assay to reveal an increased steady state level of Top1-CCs in neural cells lacking Atm; the protein mutated in ataxia telangiectasia. Our data suggest that the accumulation of endogenous Top1-CCs in Atm-/- neural cells is primarily due to elevated levels of reactive oxygen species. Biochemical purification of Top1-CCs from neural cell extract and the use of Top1 poisons further confirmed a role for Atm during the formation/resolution of Top1-CCs. Finally, we report that global transcription is reduced in Atm-/- neural cells and fails to recover to normal levels following Top1-mediated DNA damage. Together, these data identify a distinct role for ATM during the formation/resolution of neural Top1-CCs and suggest that their accumulation contributes to the neuropathology of ataxia telangiectasia.

Base damage and topoisomerase I (Top1)-linked DNA breaks are abundant forms of endogenous DNA breakage, contributing to hereditary ataxia and underlying the cytotoxicity of a wide range of anti-cancer agents. Despite their frequency, the overlapping mechanisms that repair these forms of DNA breakage are largely unknown. Here, we report that depletion of Tyrosyl DNA phosphodiesterase 1 (TDP1) sensitizes human cells to alkylation damage and the additional depletion of apurinic/apyrimidinic endonuclease I (APE1) confers hypersensitivity above that observed for TDP1 or APE1 depletion alone. Quantification of DNA breaks and clonogenic survival assays confirm a role for TDP1 in response to base damage, independently of APE1. The hypersensitivity to alkylation damage is partly restored by depletion of Top1, illustrating that alkylating agents can trigger cytotoxic Top1-breaks. Although inhibition of PARP activity does not sensitize TDP1-deficient cells to Top1 poisons, it confers increased sensitivity to alkylation damage, highlighting partially overlapping roles for PARP and TDP1 in response to genotoxic challenge. Finally, we demonstrate that cancer cells in which TDP1 is inherently deficient are hypersensitive to alkylation damage and that TDP1 depletion sensitizes glioblastoma-resistant cancer cells to the alkylating agent temozolomide.

Irinotecan and topotecan are derivatives of the naturally occurring cytotoxic compound camptothecin that are used in the treatment of patients with colorectal cancer, either as single agents or in combination with radiotherapy and/or other chemotherapy drugs. They are inhibitors of DNA topoisomerase I (Top I) and exert their cytotoxic effects in replicating cells by inducing DNA strand breaks. A wide range of DNA repair proteins is involved in the recognition and repair of these breaks, and depletion or inhibition of some of these proteins increases the cytotoxic effects of Top I inhibitors. Building on these laboratory observations, ongoing translational research is aiming to establish whether this mechanistic information can be used to improve the treatment of patients with certain types of cancer. Two potential strategies are under investigation: (1) individualising treatment by evaluating levels and/or patterns of expression of DNA repair proteins that predict clinical response to Top I inhibitors, and (2) developing small molecule inhibitors of these repair enzymes to overcome tumour resistance and improve outcomes. This review summarises the current status of these research endeavours, focusing on the key roles of tyrosyl DNA phosphodiesterase 1 (Tdp1) and poly(ADP-ribose) polymerase (PARP), and examines the pre-clinical and clinical data that support the potential value of these and other DNA repair proteins as predictive markers and therapeutic targets. Since irinotecan is increasingly being combined with radiotherapy, the potential for these proteins to act as predictive biomarkers for both Top I inhibitors and radiation is proposed, and the possibility of synergistic potentiation of chemoradiation regimes by Tdp1 and/or PARP inhibitors is considered.

Summary Forty-nine ovarian tumours were examined for loss of heterozygosity (LOH) on chromosome 5 using eight microsatellite markers spanning both arms, including one at the APC locus. LOH on Sq was a frequent event, detectable in 23 of 49 (47%) tumours, whereas 5p LOH was detected in only 1 of 22 tumours (5%). Six tumours showed partial LOH on 5q, enabling the candidate region to be localised to a 22 cM region proximal to APC, flanked by D5S424 and D5S644. An association was found between 5q LOH and TP53 mutation, with 18 of 23 (78%) tumours with LOH on Sq also harbouring a TP53 mutation. LOH on Sq was observed in 6 of 18 (33%) stage I tumours, suggesting that it may be an early event in the molecular pathogenesis of certain ovarian carcinomas.

Purpose. Early infantile epileptic encephalopathy (EIEE) 57 belongs to a group of encephalopathies with early-onset and characterised by severe electroencephalogram abnormalities, seizures, developmental delay and intellectual disability. Method. We carried out Whole Exome analysis using Next Generation Sequencing (NGS) and bioinformatic analysis performed to find mutation associated with the patient phenotypes. The effect of the mutation on protein structure analysed by PolyPhen2 and Swissmodel ExPASy. Results. In this study, we evaluated two unrelated Turkish males diagnosed with EIEE type 57 to investigate the genetic cause of this disease. Whole exome sequencing revealed mutations in KCN2 gene, which is a member of Potassium channels (KCN) gene family associated with epileptic encephalopathies. Two mutations, c.545A>T (p.Asn182Ile and c.2638C>A (p.Leu880Met) were reported here as a novel mutation. Conclusions. Our findings implicate the genotype-phenotype correlation of these mutations. Furthermore, the computational analysis showed their effect on protein binding site and function suggesting their role in the development of early infantile epileptic encephalopathy 57.

With the invention of RNA sequencing over a decade ago, diagnosis and identification of the gene-related diseases entered a new phase that enabled more accurate analysis of the diseases that are difficult to approach and analyze. RNA sequencing has availed in-depth study of transcriptomes in different species and provided better understanding of rare diseases and taxonomical classifications of various eukaryotic organisms. Development of single-cell, short-read, long-read and direct RNA sequencing using both blood and biopsy specimens of the organism together with recent advancement in computational analysis programs has made the medical professional’s ability in identifying the origin and cause of genetic disorders indispensable. Altogether, such advantages have evolved the treatment design since RNA sequencing can detect the resistant genes against the existing therapies and help medical professions to take a further step in improving methods of treatments towards higher effectiveness and less side effects. Therefore, it is of essence to all researchers and scientists to have deeper insight in all available methods of RNA sequencing while taking a step-in therapy design.