The integrity of the genome is continuously jeopardized by endogenous reactive byproducts of cellular metabolism and genotoxic insults by environmental agents, as well as by the DNA transactions (replication, transcription and recombination) required for cell survival and proliferation. Failure of the mechanisms deputed to the maintenance of genome integrity leads to genome instability, which is a hallmark of cancer and a driving force of tumorigenesis. To fully understand the mechanisms leading to genome instability and the cellular pathways counteracting them, three basic tasks must be achieved: i) identify all the genes implicated in the control of genome integrity; ii) unravel their biological role; iii) define the mechanistic molecular details of the processes in which they are implicated. This thesis describes work performed in the budding yeast Saccharomyces cerevisiae to explore the genome stability landscape at all these three levels. This model system is extremely useful for two main reasons: a) its high genetic tractability allows the application of genome-wide genetic screenings; b) the large conservation of the genome integrity pathways allows to extend the findings obtained in yeast to other eukaryotic organisms. We performed a genome-wide screen, based on the overexpression of the DDC2 DNA damage checkpoint gene in the yeast deletion collection, to identify genome stability genes on the basis of spontaneous accumulation of endogenous DNA damage in the corresponding mutant strains. Our screen identified several genes implicated in the control of genome integrity, highlighting, in particular, a key role for pathways protecting against oxidative stress. We present here the preliminary characterization of a new genome integrity gene, VID22. We also investigated the mechanisms counteracting a newly discovered source of genome instability, namely ribonucleotides (rNTPs) incorporated in genomic DNA during replication. We uncovered a role for RNase H enzymes, template switch pathways and Pol ζ translesion polymerase in protecting from misincorporated rNTPs. Given that mutations in any of the three human RNase H2 subunits were proven to cause Aicardi-Goutiéres Syndrome, these results might contribute to shed light on the complex and largely unknown pathogenetic mechanism of this rare genetic disease. Finally, we studied the molecular details underlying the role of Rad9 mediator protein in DNA damage checkpoint activation, exploring the dynamics of Rad9 dimerization, chromatin binding, CDK-dependent phosphorylation and checkpoint activation in G1 and M phases of the cell cycle; in particular, we characterized an M-phase specific pathway for checkpoint activation which is relies on Rad9-Dpb11 interaction.

MECHANISMS PRESERVING GENOME INTEGRITY IN SACCHAROMYCES CEREVISIAE / D. Novarina ; tutor: P. Plevani ; supervisore: M. Muzi Falconi. - : . UNIVERSITA' DEGLI STUDI DI MILANO, 2013 Jan 25. ((24. ciclo, Anno Accademico 2011. [10.13130/novarina-daniele_phd2013-01-25].

MECHANISMS PRESERVING GENOME INTEGRITY IN SACCHAROMYCES CEREVISIAE

D. Novarina
2013-01-25

Abstract

The integrity of the genome is continuously jeopardized by endogenous reactive byproducts of cellular metabolism and genotoxic insults by environmental agents, as well as by the DNA transactions (replication, transcription and recombination) required for cell survival and proliferation. Failure of the mechanisms deputed to the maintenance of genome integrity leads to genome instability, which is a hallmark of cancer and a driving force of tumorigenesis. To fully understand the mechanisms leading to genome instability and the cellular pathways counteracting them, three basic tasks must be achieved: i) identify all the genes implicated in the control of genome integrity; ii) unravel their biological role; iii) define the mechanistic molecular details of the processes in which they are implicated. This thesis describes work performed in the budding yeast Saccharomyces cerevisiae to explore the genome stability landscape at all these three levels. This model system is extremely useful for two main reasons: a) its high genetic tractability allows the application of genome-wide genetic screenings; b) the large conservation of the genome integrity pathways allows to extend the findings obtained in yeast to other eukaryotic organisms. We performed a genome-wide screen, based on the overexpression of the DDC2 DNA damage checkpoint gene in the yeast deletion collection, to identify genome stability genes on the basis of spontaneous accumulation of endogenous DNA damage in the corresponding mutant strains. Our screen identified several genes implicated in the control of genome integrity, highlighting, in particular, a key role for pathways protecting against oxidative stress. We present here the preliminary characterization of a new genome integrity gene, VID22. We also investigated the mechanisms counteracting a newly discovered source of genome instability, namely ribonucleotides (rNTPs) incorporated in genomic DNA during replication. We uncovered a role for RNase H enzymes, template switch pathways and Pol ζ translesion polymerase in protecting from misincorporated rNTPs. Given that mutations in any of the three human RNase H2 subunits were proven to cause Aicardi-Goutiéres Syndrome, these results might contribute to shed light on the complex and largely unknown pathogenetic mechanism of this rare genetic disease. Finally, we studied the molecular details underlying the role of Rad9 mediator protein in DNA damage checkpoint activation, exploring the dynamics of Rad9 dimerization, chromatin binding, CDK-dependent phosphorylation and checkpoint activation in G1 and M phases of the cell cycle; in particular, we characterized an M-phase specific pathway for checkpoint activation which is relies on Rad9-Dpb11 interaction.
PLEVANI, PAOLO
MUZI FALCONI, MARCO
DNA damage ; genome stability ; Rad9 ; RNase H ; screen ; Saccharomyces cerevisiae
Settore BIO/11 - Biologia Molecolare
MECHANISMS PRESERVING GENOME INTEGRITY IN SACCHAROMYCES CEREVISIAE / D. Novarina ; tutor: P. Plevani ; supervisore: M. Muzi Falconi. - : . UNIVERSITA' DEGLI STUDI DI MILANO, 2013 Jan 25. ((24. ciclo, Anno Accademico 2011. [10.13130/novarina-daniele_phd2013-01-25].
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/215589
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