Genome instability, defined as the occurrence and amplification of mutations and chromosomal rearrangements, is a hallmark of neoplasic transformation and cancer onset. Cells have evolved DNA damage checkpoint processes that act in response to replicative stress induced by genotoxic agents. In Saccharomyces cerevisiae the DNA damage checkpoint requires the essential- Rad53 kinase. Checkpoint-defective cells accumulate gross chromosomal rearrangements and loose viability following exposure to hydroxyurea (HU), mainly due to their failure to stabilize stalled replication forks. In this work I show that mutations in THO/TREX and TREX2 genes, that encode factors required for mRNA transcription, export and association of transcribed genes to the nuclear envelope (Gene gating), rescue the viability of rad53 checkpoint deficient mutants treated with low concentrations of HU. This suppression is not affected by the RNAse H overexpression, suggesting that is not related to R-loop formation, transcription defects or hyper-recombination. By 2D gels and BrdU-ChIP analyses, I found that SAC3 (TREX2) deletion suppresses replication fork progression defects and collapse in rad53 cells treated with HU. Ablation of genes encoding Nuclear Pore Complex inner basket factors, that are required for gene gating, also suppresses the HU-sensitivity ofcheckpoint mutants. Moreover I found that upon replication stress, transcribed gene dissociate from the nuclear envelope in a Rad53-dependent manner. Based on these results, I propose that in cells experiencing replication stress the DNA damage checkpoint stabilizes replication forks by releasing the topological constraints imposed by NPC-associated genes.

THE DNA DAMAGE CHECKPOINT PRESERVES REPLICATION FORK INTEGRITY BY REGULATING TRANSCRIBED GENES ASSOCIATION TO THE NUCLEAR PORE COMPLEX / T. Capra ; tutor: Marco Fojani ; correlatore: Rodrigo Bermejo Moreno ; coordinatore del dottorato: Roberto Mantovani. - : . Universita' degli Studi di Milano, 2010 Dec 10. ((23. ciclo, Anno Accademico 2010.

THE DNA DAMAGE CHECKPOINT PRESERVES REPLICATION FORK INTEGRITY BY REGULATING TRANSCRIBED GENES ASSOCIATION TO THE NUCLEAR PORE COMPLEX.

T. Capra
2010

Abstract

Genome instability, defined as the occurrence and amplification of mutations and chromosomal rearrangements, is a hallmark of neoplasic transformation and cancer onset. Cells have evolved DNA damage checkpoint processes that act in response to replicative stress induced by genotoxic agents. In Saccharomyces cerevisiae the DNA damage checkpoint requires the essential- Rad53 kinase. Checkpoint-defective cells accumulate gross chromosomal rearrangements and loose viability following exposure to hydroxyurea (HU), mainly due to their failure to stabilize stalled replication forks. In this work I show that mutations in THO/TREX and TREX2 genes, that encode factors required for mRNA transcription, export and association of transcribed genes to the nuclear envelope (Gene gating), rescue the viability of rad53 checkpoint deficient mutants treated with low concentrations of HU. This suppression is not affected by the RNAse H overexpression, suggesting that is not related to R-loop formation, transcription defects or hyper-recombination. By 2D gels and BrdU-ChIP analyses, I found that SAC3 (TREX2) deletion suppresses replication fork progression defects and collapse in rad53 cells treated with HU. Ablation of genes encoding Nuclear Pore Complex inner basket factors, that are required for gene gating, also suppresses the HU-sensitivity ofcheckpoint mutants. Moreover I found that upon replication stress, transcribed gene dissociate from the nuclear envelope in a Rad53-dependent manner. Based on these results, I propose that in cells experiencing replication stress the DNA damage checkpoint stabilizes replication forks by releasing the topological constraints imposed by NPC-associated genes.
FOIANI, MARCO
MANTOVANI, ROBERTO
Gene Gating ; Nuclear Pore Complex ; DNA Damage Checkpoint ; Genome instability ; THO/TREX complex ; TREX2 complex ; Replication stress
Settore BIO/11 - Biologia Molecolare
THE DNA DAMAGE CHECKPOINT PRESERVES REPLICATION FORK INTEGRITY BY REGULATING TRANSCRIBED GENES ASSOCIATION TO THE NUCLEAR PORE COMPLEX / T. Capra ; tutor: Marco Fojani ; correlatore: Rodrigo Bermejo Moreno ; coordinatore del dottorato: Roberto Mantovani. - : . Universita' degli Studi di Milano, 2010 Dec 10. ((23. ciclo, Anno Accademico 2010.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/150161
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