Plx1 is required for chromosomal DNA replication under stressful conditions

Polo-like kinase (Plk)1 is required for mitosis progression. However, although Plk1 is expressed throughout the cell cycle, its function during S-phase is unknown. Using Xenopus laevis egg extracts, we demonstrate that Plx1, the Xenopus orthologue of Plk1, is required for DNA replication in the presence of stalled replication forks induced by aphidicolin, etoposide or reduced levels of DNA-bound Mcm complexes. Plx1 binds to chromatin and suppresses the ATM/ATR-dependent intra-S-phase checkpoint that inhibits origin firing. This allows Cdc45 loading and derepression of DNA replication initiation. Checkpoint activation increases Plx1 binding to the Mcm complex through its Polo box domain. Plx1 recruitment to chromatin is independent of checkpoint mediators Tipin and Claspin. Instead, ATR-dependent phosphorylation of serine 92 of Mcm2 is required for the recruitment of Plx1 to chromatin and for the recovery of DNA replication under stress. Depletion of Plx1 leads to accumulation of chromosomal breakage that is prevented by the addition of recombinant Plx1. These data suggest that Plx1 promotes genome stability by regulating DNA replication under stressful conditions

Charakterisierung der Mutagensensitivität von Lymphozyten und lymphoblastoiden Zelllinien mit BRCA-Mutationen

Mutagensensitivität ist ein Merkmal vieler Krebserkrankungen. Auch bei einigen Brustkrebspatienten wurde eine erhöhte chromosomale Strahlensensitivität detektiert. In meiner Arbeit sollte im Mikronukleustest und im Comet-Assay untersucht werden, ob ein Zusammenhang zwischen BRCA1- oder BRCA2-Mutationen und Mutagensensitivität besteht. Des Weiteren sollten lymphoblastoide Zelllinien auf ihre Eignung als Modell zu Untersuchungen der Mutagensensitivität und der zugrunde liegenden Mechanismen untersucht werden. Die Untersuchung von Lymphozyten BRCA1- und BRCA2-heterozygoter Frauen im Mikronukleustest ließ eine erhöhte chromosomale Sensitivität erkennen. Sowohl gegenüber ionisierender Strahlung wie auch nach Behandlung mit H2O2 wurde dieser Effekt deutlich, was auf eine Funktion in der DNA-DSB-Reparatur wie auch in der Reparatur oxidativer Schäden hindeutet. Mit dem Comet-Assay konnte weder in der alkalischen Version noch in der neutralen Version ein Unterschied zwischen Zellen mit und ohne BRCA-Mutation hinsichtlich Induktion und Reparatur strahleninduzierter Schäden festgestellt werden. Die mit verschiedenen Cytostatika durchgeführten Experimente im MNT ließen eine erhöhte chromosomale Mutagensensitivität der BRCA1-heterozygoten Lymphozyten nach Behandlung mit DNA-schädigenden Substanzen erkennen. Diese Resultate unterstützen die kürzlich entdeckte Assoziation von BRCA1 mit dem BASC-Komplex. Die detaillierte Charakterisierung der 6 verwendeten Zelllinien wie auch die Analyse von DNA-Strangbrüchen und der DNA-DSB-Reparatur (neutraler Comet-Assay, Pulsfeldgelelektrophorese) konnten keine signifikanten Unterschiede zwischen Zelllinien mit und ohne BRCA1-Mutation feststellen. Die Verwendung lymphoblastoider Zelllinien mit BRCA1-Mutation als Modellsystem für Untersuchungen zur Mutagensensitivität scheint aufgrund der hier erhobenen Daten limitiert zu sein, da eine Transformation von Lymphozyten zum Verlust der Mutagensensitivität führen kann

Gene expression changes induced by the human carcinogen aristolochic acid I in renal and hepatic tissue of mice

Aristolochic acid (AA) is the causative agent of urothelial tumors associated with AA nephropathy and is also implicated in the development of Balkan endemic nephropathy-associated urothelial tumors. These tumors contain AA-characteristic TP53 mutations. We examined gene expression changes in Hupki (human TP53 knock-in) mice after treatment with aristolochic acid I (AAI) by gavage (5 mg/kg body weight). After 3, 12 and 21 days of treatment gene expression profiles were investigated using Agilent Whole Mouse 44K Genome Oligo Array. Expression profiles were significantly altered by AAI treatment in both target (kidney) and nontarget (liver) tissue. Renal pathology and DNA adduct analysis confirmed kidney as the target tissue of AAI-induced toxicity. Gene ontology for functional analysis revealed that processes related to apoptosis, cell cycle, stress response, immune system, inflammatory response and kidney development were altered in kidney. Canonical pathway analysis indicated Nf?b, aryl hydrocarbon receptor, Tp53 and cell cycle signaling as the most important pathways modulated in kidney. Expression of Nf?b1 and other Nf?b-target genes was confirmed by quantitative real-time PCR (qRT-PCR) and was consistent with the induction of Nf?b1 protein. Myc oncogene, frequently overexpressed in urothelial tumors, was upregulated by AAI on the microarrays and confirmed by qRT-PCR and protein induction. Collectively we found that microarray gene expression analysis is a useful tool to define tissue-specific responses in AAI-induced toxicity. Several genes identified such as TP53, Rb1, Mdm2, Cdkn2a and Myc are frequently affected in human urothelial cancer, and may be valuable prognostic markers in future clinical studies. © 2010 UICC.SCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe

AOP Report: Development of an Adverse Outcome Pathway for Oxidative DNA Damage Leading to Mutations and Chromosomal Aberrations.

The Genetic Toxicology Technical Committee (GTTC) of the Health and Environmental Sciences Institute (HESI) is developing adverse outcome pathways (AOPs) that describe modes of action leading to potentially heritable genomic damage. The goal was to enhance the use of mechanistic information in genotoxicity assessment by building empirical support for the relationships between relevant molecular initiating events (MIEs) and regulatory endpoints in genetic toxicology. Herein, we present an AOP network that links oxidative DNA damage to two adverse outcomes (AOs): mutations and chromosomal aberrations. We collected empirical evidence from the literature to evaluate the key event relationships between the MIE and the AOs, and assessed the weight of evidence using the modified Bradford‐Hill criteria for causality. Oxidative DNA damage is constantly induced and repaired in cells given the ubiquitous presence of reactive oxygen species and free radicals. However, xenobiotic exposures may increase damage above baseline levels through a variety of mechanisms and overwhelm DNA repair and endogenous antioxidant capacity. Unrepaired oxidative DNA base damage can lead to base substitutions during replication and, along with repair intermediates, can also cause DNA strand breaks that can lead to mutations and chromosomal aberrations if not repaired adequately. This AOP network identifies knowledge gaps that could be filled by targeted studies designed to better define the quantitative relationships between key events, which could be leveraged for quantitative chemical safety assessment. We anticipate that this AOP network will provide the building blocks for additional genotoxicity‐associated AOPs and aid in designing novel integrated testing approaches for genotoxicity

ATM and ATR promote Mre11 dependent restart of collapsed replication forks and prevent accumulation of DNA breaks

Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia Rad3-related (ATR) and the Mre11/Rad50/Nbs1 complex ensure genome stability in response to DNA damage. However, their essential role in DNA metabolism remains unknown. Here we show that ATM and ATR prevent accumulation of DNA double-strand breaks (DSBs) during chromosomal replication. Replicating chromosomes accumulate DSBs in Xenopus laevis egg extracts depleted of ATM and ATR. Addition of ATM and ATR proteins to depleted extracts prevents DSB accumulation by promoting restart of collapsed replication forks that arise during DNA replication. We show that collapsed forks maintain MCM complex but lose Pol ɛ, and that Pol ɛ reloading requires ATM and ATR. Replication fork restart is abolished in Mre11 depleted extracts and is restored by supplementation with recombinant human Mre11/Rad50/Nbs1 complex. Using a novel fluorescence resonance energy transfer-based technique, we demonstrate that ATM and ATR induce Mre11/Rad50/Nbs1 complex redistribution to restarting forks. This study provides direct biochemical evidence that ATM and ATR prevent accumulation of chromosomal abnormalities by promoting Mre11/Rad50/Nbs1 dependent recovery of collapsed replication forks