The integrity of genomic DNA is continuously jeopardized through of environmental stresses such as UV light, ionizing radiations and various chemicals in addition to cellular byproducts such as reactive oxygen species. Furthermore, structural or chemical hindrances also affect the basic cellular processes (replication, transcription and translation) compromising genome stability. All the eukaryotic cells have thus evolved mechanisms to detect such genomic lesions and activate a surveillance mechanisms termed as checkpoint activation to arrest cell cycle, which in term provide time to repair the lesion using a suitable pathway to maintain genome stability. The resumption of cell cycle after the repair is also an important and finely regulated mechanisms. Indeed, resumption of cell cycle in case of faulty/un-repaired damage compromises genome integrity and may lead to cancer. In this thesis, I studied the role of Polo-kinase Cdc5 and DNA repair scaffold complex-Slx-Rtt107, specifically in response to one of the most deleterious lesion, DNA double strand break (DSB) in budding yeast Saccharomyces cerevisiae. The human counterpart Polo-like kinase 1 is overexpressed in many cancers, while Slx4/FANCP is one of the proteins involved in Fanconi anemia repair pathway. In first part, we characterized the role of phosphorylation of Threonine 238 in the activation loop of the Cdc5 kinase domain in unperturbed cell cycle and in response to repairable and unrepairable DSB. Using alanine/ aspartic acid mutagenesis and genetic approaches we delineated the requirement of T238 phosphorylation of Cdc5. Interestingly, we discovered that absence of T238 phosphorylation of Cdc5, even though doesn’t affect the normal cell cycle, affects kinase activity and leads to defect in checkpoint adaptation and recovery after one DSB. Importantly, we also found that cdc5-T238A cells also have altered genome stability, assessed by using multiple genetic approaches. In second part, we characterized the role of Slx4-Rtt107 complex in modulating the level of checkpoint signalling and initial processing of DSB. Indeed in the absence of functional Slx4-Rtt107 complex, we found slower processing of DSB and hyper-activated checkpoint signalling which is due to increased binding of checkpoint adaptor protein Rad9 at the lesion. Importantly, this hyper-activated checkpoint has consequent effect on cell cycle resumption and proliferation in response to various DNA damaging agents.

ROLE OF POLO KINASE CDC5 AND SLX4-RTT107 COMPLEX IN CHECKPOINT SIGNALING DURING DNA DAMAGE IN S. CEREVISIAE / C. Rawal ; scientific tutor: A. Pellicioli. DIPARTIMENTO DI BIOSCIENZE, 2015 Dec 09. 28. ciclo, Anno Accademico 2015. [10.13130/c-rawal_phd2015-12-09].

ROLE OF POLO KINASE CDC5 AND SLX4-RTT107 COMPLEX IN CHECKPOINT SIGNALING DURING DNA DAMAGE IN S. CEREVISIAE

C. Rawal
2015

Abstract

The integrity of genomic DNA is continuously jeopardized through of environmental stresses such as UV light, ionizing radiations and various chemicals in addition to cellular byproducts such as reactive oxygen species. Furthermore, structural or chemical hindrances also affect the basic cellular processes (replication, transcription and translation) compromising genome stability. All the eukaryotic cells have thus evolved mechanisms to detect such genomic lesions and activate a surveillance mechanisms termed as checkpoint activation to arrest cell cycle, which in term provide time to repair the lesion using a suitable pathway to maintain genome stability. The resumption of cell cycle after the repair is also an important and finely regulated mechanisms. Indeed, resumption of cell cycle in case of faulty/un-repaired damage compromises genome integrity and may lead to cancer. In this thesis, I studied the role of Polo-kinase Cdc5 and DNA repair scaffold complex-Slx-Rtt107, specifically in response to one of the most deleterious lesion, DNA double strand break (DSB) in budding yeast Saccharomyces cerevisiae. The human counterpart Polo-like kinase 1 is overexpressed in many cancers, while Slx4/FANCP is one of the proteins involved in Fanconi anemia repair pathway. In first part, we characterized the role of phosphorylation of Threonine 238 in the activation loop of the Cdc5 kinase domain in unperturbed cell cycle and in response to repairable and unrepairable DSB. Using alanine/ aspartic acid mutagenesis and genetic approaches we delineated the requirement of T238 phosphorylation of Cdc5. Interestingly, we discovered that absence of T238 phosphorylation of Cdc5, even though doesn’t affect the normal cell cycle, affects kinase activity and leads to defect in checkpoint adaptation and recovery after one DSB. Importantly, we also found that cdc5-T238A cells also have altered genome stability, assessed by using multiple genetic approaches. In second part, we characterized the role of Slx4-Rtt107 complex in modulating the level of checkpoint signalling and initial processing of DSB. Indeed in the absence of functional Slx4-Rtt107 complex, we found slower processing of DSB and hyper-activated checkpoint signalling which is due to increased binding of checkpoint adaptor protein Rad9 at the lesion. Importantly, this hyper-activated checkpoint has consequent effect on cell cycle resumption and proliferation in response to various DNA damaging agents.
9-dic-2015
scientific tutor: A. Pellicioli
DIPARTIMENTO DI BIOSCIENZE
English
28
2015
SCIENZE BIOLOGICHE E MOLECOLARI
Settore BIO/11 - Biologia Molecolare
Ricerca di base
Pubblicazione scientifica
DNA damage checkpoint; DNA double strand breaks; Polo kinases/Cdc5; Slx4-Rtt107 complex
PELLICIOLI, ACHILLE
PELLICIOLI, ACHILLE
Doctoral Thesis
Prodotti della ricerca::Tesi di dottorato
-2.0
open
Università degli Studi di Milano
info:eu-repo/semantics/doctoralThesis
1
C. Rawal
ROLE OF POLO KINASE CDC5 AND SLX4-RTT107 COMPLEX IN CHECKPOINT SIGNALING DURING DNA DAMAGE IN S. CEREVISIAE / C. Rawal ; scientific tutor: A. Pellicioli. DIPARTIMENTO DI BIOSCIENZE, 2015 Dec 09. 28. ciclo, Anno Accademico 2015. [10.13130/c-rawal_phd2015-12-09].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/335192
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