Header

UZH-Logo

Maintenance Infos

Molecular and Cellular Aspects of CtIP Function in DNA Double-Strand Break Processing


Ferretti, Lorenza P. Molecular and Cellular Aspects of CtIP Function in DNA Double-Strand Break Processing. 2015, University of Zurich, Faculty of Science.

Abstract

The causes and consequences of DNA damage are of major relevance to cancer biology. DNA double strand breaks (DSBs) are one of the most cytotoxic lesions induced by ionizing radiation and chemotherapy drugs, with the potential to cause chromosomal rearrangements. Thus, faithful repair of DSBs is essential to preserve genomic integrity. Cells have evolved two major DSB repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). Originally discovered as a transcriptional regulator, human CtIP has emerged as a multivalent adaptor protein involved various biological processes. Importantly, CtIP plays an essential role in DNA-end resection, the initial step HR. However, still very little is known about its exact role in this process and the cellular pathways regulating its function.
In the first part of my PhD thesis, we have investigated the role of CtIP ubiquitnation in the DNA damage response. Using a mass spectrometry-based proteomic screen, we have identified human Kelch-like protein 15 (KLHL15), a substrate-specific adaptor for cullin 3-based (CUL3) E3 ubiquitin ligases, as a novel CtIP interaction partner. We could show that the KLHL15-CUL3 complex mediates CtIP ubiquitination, leading to its proteasome-dependent degradation. We have found that KLHL15 specifically recognizes an evolutionary conserved 'FRY' motif in the C-terminal domain of CtIP. Remarkably, U2OS cells expressing a CtIP-Y842A mutant deficient in KLHL15 interaction displayed deregulated DNA-end resection. Taken together, our findings established a new role for KLHL15-mediated ubiquitination of CtIP in repair of DSBs.
My second PhD thesis project was aimed at getting further insight into the biochemical and structural features of CtIP. As human CtIP is a rather large polypeptide (897 amino acids), we decided to biochemically characterize Paramecium tetraurelia (PtCtIP), that with a length of only 198 amino acids constitutes the shortest CtIP homolog known. Following successful expression and purification of recombinant PtCtIP from insect cells, we have found that PtCtIP harbors a DNA-binding domain in its C-terminus. Collectively, work presented in my thesis highlights the importance of CtIP functions in DNA-end resection and in the maintenance of genomic stability.

Abstract

The causes and consequences of DNA damage are of major relevance to cancer biology. DNA double strand breaks (DSBs) are one of the most cytotoxic lesions induced by ionizing radiation and chemotherapy drugs, with the potential to cause chromosomal rearrangements. Thus, faithful repair of DSBs is essential to preserve genomic integrity. Cells have evolved two major DSB repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). Originally discovered as a transcriptional regulator, human CtIP has emerged as a multivalent adaptor protein involved various biological processes. Importantly, CtIP plays an essential role in DNA-end resection, the initial step HR. However, still very little is known about its exact role in this process and the cellular pathways regulating its function.
In the first part of my PhD thesis, we have investigated the role of CtIP ubiquitnation in the DNA damage response. Using a mass spectrometry-based proteomic screen, we have identified human Kelch-like protein 15 (KLHL15), a substrate-specific adaptor for cullin 3-based (CUL3) E3 ubiquitin ligases, as a novel CtIP interaction partner. We could show that the KLHL15-CUL3 complex mediates CtIP ubiquitination, leading to its proteasome-dependent degradation. We have found that KLHL15 specifically recognizes an evolutionary conserved 'FRY' motif in the C-terminal domain of CtIP. Remarkably, U2OS cells expressing a CtIP-Y842A mutant deficient in KLHL15 interaction displayed deregulated DNA-end resection. Taken together, our findings established a new role for KLHL15-mediated ubiquitination of CtIP in repair of DSBs.
My second PhD thesis project was aimed at getting further insight into the biochemical and structural features of CtIP. As human CtIP is a rather large polypeptide (897 amino acids), we decided to biochemically characterize Paramecium tetraurelia (PtCtIP), that with a length of only 198 amino acids constitutes the shortest CtIP homolog known. Following successful expression and purification of recombinant PtCtIP from insect cells, we have found that PtCtIP harbors a DNA-binding domain in its C-terminus. Collectively, work presented in my thesis highlights the importance of CtIP functions in DNA-end resection and in the maintenance of genomic stability.

Statistics

Additional indexing

Item Type:Dissertation
Referees:Sartori Alessandro A, B├ętermier Mireille, Cejka Petr, Peter Matthias
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2015
Deposited On:28 Jan 2016 11:11
Last Modified:05 Apr 2016 20:01

Download

Full text not available from this repository.