Stabilization of Dicentric Translocations through Secondary Rearrangements Mediated by Multiple Mechanisms in S. cerevisiae

The gross chromosomal rearrangements (GCRs) observed in S. cerevisiae mutants with increased rates of accumulating GCRs include predicted dicentric GCRs such as translocations, chromosome fusions and isoduplications. These GCRs resemble the genome rearrangements found as mutations underlying inherited diseases as well as in the karyotypes of many cancers exhibiting ongoing genome instabilityThe structures of predicted dicentric GCRs were analyzed using multiple strategies including array-comparative genomic hybridization, pulse field gel electrophoresis, PCR amplification of predicted breakpoints and sequencing. The dicentric GCRs were found to be unstable and to have undergone secondary rearrangements to produce stable monocentric GCRs. The types of secondary rearrangements observed included: non-homologous end joining (NHEJ)-dependent intramolecular deletion of centromeres; chromosome breakage followed by NHEJ-mediated circularization or broken-end fusion to another chromosome telomere; and homologous recombination (HR)-dependent non-reciprocal translocations apparently mediated by break-induced replication. A number of these GCRs appeared to have undergone multiple bridge-fusion-breakage cycles. We also observed examples of chromosomes with extensive ongoing end decay in mec1 tlc1 mutants, suggesting that Mec1 protects chromosome ends from degradation and contributes to telomere maintenance by HR.HR between repeated sequences resulting in secondary rearrangements was the most prevalent pathway for resolution of dicentric GCRs regardless of the structure of the initial dicentric GCR, although at least three other resolution mechanisms were observed. The resolution of dicentric GCRs to stable rearranged chromosomes could in part account for the complex karyotypes seen in some cancers

The impact of cyclin-dependent kinase 5 depletion on poly(ADP-ribose) polymerase activity and responses to radiation

Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5KD cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro-irradiated Cdk5KD cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5KD compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5-dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5KD cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5KD cells is due to a role of Cdk5 in other pathways or the altered polymer levels

A Genetic and Structural Study of Genome Rearrangements Mediated by High Copy Repeat Ty1 Elements

Ty elements are high copy number, dispersed repeated sequences in the Saccharomyces cerevisiae genome known to mediate gross chromosomal rearrangements (GCRs). Here we found that introduction of Ty912, a previously identified Ty1 element, onto the non-essential terminal region of the left arm of chromosome V led to a 380-fold increase in the rate of accumulating GCRs in a wild-type strain. A survey of 48 different mutations identified those that either increased or decreased the rate of Ty-mediated GCRs and demonstrated that suppression of Ty-mediated GCRs differs from that of both low copy repeat sequence- and single copy sequence-mediated GCRs. The majority of the Ty912-mediated GCRs observed were monocentric nonreciprocal translocations mediated by RAD52-dependent homologous recombination (HR) between Ty912 and a Ty element on another chromosome arm. The remaining Ty912-mediated GCRs appeared to involve Ty912-mediated formation of unstable dicentric translocation chromosomes that were resolved by one or more Ty-mediated breakage-fusion-bridge cycles. Overall, the results demonstrate that the Ty912-mediated GCR assay is an excellent model for understanding mechanisms and pathways that suppress genome rearrangements mediated by high copy number repeat sequences, as well as the mechanisms by which such rearrangements occur

ETUDE DU FACTEUR DE REPLICATION C (CARACTERISATION DE NOUVELLES FONCTIONS DE LA LARGE SOUS-UNITE RF-CP145)

LE FACTEUR DE REPLICATION C (RF-C), UN COMPLEXE DE CINQ POLYPEPTIDES SE LIE AUX AMORCES D'ARN ET FIXE LE PCNA SUR L'ADN, ACTIVANT AINSI L'ACTIVITE PROCESSIVE DES ADN POLYMERASES. NOUS AVONS MIS A JOUR, DANS DES CELLULES DE MAMMIFERES, UNE NOUVELLE FONCTION DE RF-C INFLUENCANT LA SURVIE CELLULAIRE APRES L'ENDOMMAGEMENT DE L'ADN. NOUS AVONS DETERMINE QUE LA LARGE SOUS-UNITE DE RF-C, RF-CP145, FAVORISE LA SURVIE CELLULAIRE APRES ENDOMMAGEMENT DE L'ADN PAR RADIATION UV PAR UNE VOIE DE SIGNALISATION DEPENDANTE DE LA PROTEINE DU RETINOBLASTOME (RB). LA COOPERATION ENTRE RF-CP145 ET RB FAVORISANT LA SURVIE CELLULAIRE EST ABROGEE PAR LA MUTATION DU MOTIF LXCXE DE RF-CP145 OU MUTATION DANS RB DU SITE D'INTERACTION AVEC LE MOTIF LXCXE. NOUS SUGGERONS QUE CETTE NOUVELLE FONCTION DE RF-CP145 REQUIERT LA LIAISON AVEC RB VIA LE MOTIF LXCXE. NOUS AVONS PU EGALEMENT ETABLIR QUE LE DOMAINE B DE RF-CP145 PEUT PROMOUVOIR LA SURVIE CELLULAIRE APRES INDUCTION DE L'APOPTOSE PAR TNF- (TUMOR NECROSIS FACTOR-ALPHA) ET BAX. RF-CP145 INHIBE LA CONDENSATION DE LA CHROMATINE, LA FRAGMENTATION DE L'ADN ET L'ACTIVATION DE LA CASPASE-3 INDUITE PAR BAX. NOUS AVONS EGALEMENT MONTRE QUE IN VITRO, RF-CP145 DOMAINE B INHIBE L'ACTIVATION PAR PROTEOLYSE DE LA PRO-CASPASE-3. NOS RESULTATS SUGGERENT QUE L'ACTIVITE ANTI-APOPTOTIQUE DU DOMAINE B EST DIRIGE VERS LA REGULATION DE LA PHASE EXECUTRICE DE LA MORT CELLULAIRE. D'AUTRE PART, NOUS AVONS MONTRE QUE LE DOMAINE DE LIAISON A L'ADN DE RF-C, DOMAINE A, A LA PROPRIETE D'INTERAGIR AVEC DES PROTEINES. L'UNE DES PROTEINES INTERAGISSANT AVEC LE DOMAINE A A ETE CARACTERISE COMME ETANT PARP (POLY(ADP-RIBOSE) POLYMERASE). NOUS AVONS DETERMINE QUE DIFFERENTS ACIDES AMINES DU DOMAINE A SONT IMPLIQUES DANS LA LIAISON AVEC L'ADN ET AVEC PARP. DE PLUS LES DOMAINES BRCT DE XRCC1 ET DE L'ADN LIGASE III INTERAGISSENT AVEC LE DOMAINE A IN VITRO. NOS RESULTATS SUGGERENT QUE NON SEULEMENT RF-CP145 JOUE UN ROLE CENTRAL LORS DE LA REPLICATION DE L'ADN, MAIS POURRAIT EGALEMENT INTERVENIR DANS LA REGULATION DES VOIES DE SIGNALISATION ACTIVEES LORS DE STRESS GENOTOXIQUES. L'ETUDE DE LA REGULATION DE CES VOIES DE SIGNALISATION PAR RF-C EST D'UNE GRANDE IMPORTANCE POUR LA COMPREHENSION DES MECANISMES CONTROLANT LE DEVENIR CELLULAIRE PAR LES ACTEURS DE LA PROGRESSION NORMALE DU CYCLE CELLULAIRE.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Saccharomyces cerevisiae as a Model System To Define the Chromosomal Instability Phenotype

Translocations, deletions, and chromosome fusions are frequent events seen in cancers with genome instability. Here we analyzed 358 genome rearrangements generated in Saccharomyces cerevisiae selected by the loss of the nonessential terminal segment of chromosome V. The rearrangements appeared to be generated by both nonhomologous end joining and homologous recombination and targeted all chromosomes. Fifteen percent of the rearrangements occurred independently more than once. High levels of specific classes of rearrangements were isolated from strains with specific mutations: translocations to Ty elements were increased in telomerase-defective mutants, potential dicentric translocations and dicentric isochromosomes were associated with cell cycle checkpoint defects, chromosome fusions were frequent in strains with both telomerase and cell cycle checkpoint defects, and translocations to homolog genes were seen in strains with defects allowing homoeologous recombination. An analysis of human cancer-associated rearrangements revealed parallels to the effects that strain genotypes have on classes of rearrangement in S. cerevisiae

Saccharomyces cerevisiae chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability

Some spontaneous gross chromosomal rearrangements (GCRs) seem to result from DNA-replication errors. The chromatin-assembly factor I (CAF-I) and replication-coupling assembly factor (RCAF) complexes function in chromatin assembly during DNA replication and repair and could play a role in maintaining genome stability. Inactivation of CAF-I or RCAF increased the rate of accumulating different types of GCRs including translocations and deletion of chromosome arms with associated de novo telomere addition. Inactivation of CAF-I seems to cause damage that activates the DNA-damage checkpoints, whereas inactivation of RCAF seems to cause damage that activates the DNA-damage and replication checkpoints. Both defects result in increased genome instability that is normally suppressed by these checkpoints, RAD52-dependent recombination, and PIF1-dependent inhibition of de novo telomere addition. Treatment of CAF-I- or RCAF-defective cells with methyl methanesulfonate increased the induction of GCRs compared with that seen for a wild-type strain. These results indicate that coupling of chromatin assembly to DNA replication and DNA repair is critical to maintaining genome stability

De novo telomere addition at chromosome breaks: Dangerous Liaisons

International audienceTelomerase counteracts the loss of terminal DNA sequences from chromosome ends; however, it may erroneously add telomeric repeats to DNA double-strand breaks. In this issue, Ouenzar et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201610071) uncover cell cycle–dependent sequestration of the telomerase RNA in nucleoli, a process that excludes telomerase from DNA repair sites