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Genome fideltiy and DNA repair in plants: the characterization of the MBD4 DNA glycosylase homolog in Arabidopsis thaliana

Joke Baute UGent (2008)
abstract
The maintenance of genome stability is a continuous challenge for all organisms that is encountered by DNA repair mechanisms. In the human genome, the most important individual source of point mutations is spontaneous hydrolytic deamination of 5- methylcytosine (Sved and Bird, 1990; Krawczak et al., 1998; Fryxell and Moon, 2005). This results in the formation of G:T mismatches and, if not properly repaired, in C-to-T transitions after replication (Salser, 1977). To reduce the number of mutations generated by deamination of 5-methylcytosine, vertebrates have developed a pathway to specifically repair G:T mismatched base pairs to G:C (Neddermann and Jiricny, 1993; Hendrich et al., 1999). The first step in this pathway is the recognition of the mismatched base pair by a DNA glycosylase, MBD4 or TDG, followed by removal of the thymine and restoration of the abasic site by the BER pathway. In contrast to vertebrates, little is known about the impact of spontaneous hydrolytic deamination on genome stability in plants and about the capacity to repair the damage generated in this way in plants. To contribute to answering these questions, we aimed to establish the role of the homolog of the DNA glycosylase MBD4 in Arabidopsis thaliana, both in vitro and in vivo. Completion of the genome sequence of Arabidopsis thaliana allowed us to identify this homolog based on amino acid similarities with human, mouse and chicken MBD4 homologs (chapter 2). Also in poplar and rice, MBD4 homologs were identified, implying that this repair pathway is conserved in plants. In chapter 3, we describe our attempts to determine the biochemical activity of AtMBD4 in vitro, using different approaches, while chapter 4 focuses on the identification of interactors, to gain further insight into the biological function of AtMBD4. The temporal and spatial expression pattern of AtMBD4 is unravelled in chapter 5. Also, the effects of various environmental stimuli on AtMBD4 expression are analyzed. Chapter 6 reports on the development of a scorable system to monitor C-to-T transition mutations in Arabidopsis and the validation of the system. This system was then used to evaluate if C:G-to-T:A reversion frequencies were altered in AtMBD4 overexpressing and silenced plants. In addition, the response of these mutants to various genotoxic treatments was compared to wild type (chapter 7). G:T mismatches not only arise through deamination of 5-methylcytosine, but can also originate from replication errors. MSH2 is a protein involved in amongst others the repair of these replication errors. In chapter 8, the in vivo role of the Arabidopsis homolog AtMSH2 in the repair of G:T mismatches was determined using the scorable system described in chapter 6. Additionally, we analyzed the sensitivity of an AtMSH2::T-DNA insertion mutant to different genotoxic agents, aiming to further unravel the biological functions of AtMSH2.
Please use this url to cite or link to this publication:
author
promoter
UGent
organization
year
type
dissertation (monograph)
subject
pages
244 pages
publisher
Ghent University. Faculty of Sciences
place of publication
Ghent, Belgium
defense location
Zwijnaarde : Technologiepark (FSVM building)
defense date
2008-02-19 16:30
language
English
UGent publication?
yes
classification
D1
additional info
dissertation consists of copyrighted material
copyright statement
I have transferred the copyright for this publication to the publisher
id
3007662
handle
http://hdl.handle.net/1854/LU-3007662
date created
2012-10-05 15:00:34
date last changed
2012-10-08 15:15:34
@phdthesis{3007662,
  abstract     = {The maintenance of genome stability is a continuous challenge for all organisms that is encountered by DNA repair mechanisms. In the human genome, the most important individual source of point mutations is spontaneous hydrolytic deamination of 5- methylcytosine (Sved and Bird, 1990; Krawczak et al., 1998; Fryxell and Moon, 2005). This results in the formation of G:T mismatches and, if not properly repaired, in C-to-T transitions after replication (Salser, 1977). To reduce the number of mutations generated by deamination of 5-methylcytosine, vertebrates have developed a pathway to specifically repair G:T mismatched base pairs to G:C (Neddermann and Jiricny, 1993; Hendrich et al., 1999). The first step in this pathway is the recognition of the mismatched base pair by a DNA glycosylase, MBD4 or TDG, followed by removal of the thymine and restoration of the abasic site by the BER pathway. In contrast to vertebrates, little is known about the impact of spontaneous hydrolytic deamination on genome stability in plants and about the capacity to repair the damage generated in this way in plants. To contribute to answering these questions, we aimed to establish the role of the homolog of the DNA glycosylase MBD4 in Arabidopsis thaliana, both in vitro and in vivo. Completion of the genome sequence of Arabidopsis thaliana allowed us to identify this homolog based on amino acid similarities with human, mouse and chicken MBD4 homologs (chapter 2). Also in poplar and rice, MBD4 homologs were identified, implying that this repair pathway is conserved in plants. In chapter 3, we describe our attempts to determine the biochemical activity of AtMBD4 in vitro, using different approaches, while chapter 4 focuses on the identification of interactors, to gain further insight into the biological function of AtMBD4. The temporal and spatial expression pattern of AtMBD4 is unravelled in chapter 5. Also, the effects of various environmental stimuli on AtMBD4 expression are analyzed. Chapter 6 reports on the development of a scorable system to monitor C-to-T transition mutations in Arabidopsis and the validation of the system. This system was then used to evaluate if C:G-to-T:A reversion frequencies were altered in AtMBD4 overexpressing and silenced plants. In addition, the response of these mutants to various genotoxic treatments was compared to wild type (chapter 7). G:T mismatches not only arise through deamination of 5-methylcytosine, but can also originate from replication errors. MSH2 is a protein involved in amongst others the repair of these replication errors. In chapter 8, the in vivo role of the Arabidopsis homolog AtMSH2 in the repair of G:T mismatches was determined using the scorable system described in chapter 6. Additionally, we analyzed the sensitivity of an AtMSH2::T-DNA insertion mutant to different genotoxic agents, aiming to further unravel the biological functions of AtMSH2.},
  author       = {Baute, Joke},
  language     = {eng},
  pages        = {244},
  publisher    = {Ghent University. Faculty of Sciences},
  school       = {Ghent University},
  title        = {Genome fideltiy and DNA repair in plants: the characterization of the MBD4 DNA glycosylase homolog in Arabidopsis thaliana},
  year         = {2008},
}

Chicago
Baute, Joke. 2008. “Genome Fideltiy and DNA Repair in Plants: The Characterization of the MBD4 DNA Glycosylase Homolog in Arabidopsis Thaliana”. Ghent, Belgium: Ghent University. Faculty of Sciences.
APA
Baute, J. (2008). Genome fideltiy and DNA repair in plants: the characterization of the MBD4 DNA glycosylase homolog in Arabidopsis thaliana. Ghent University. Faculty of Sciences, Ghent, Belgium.
Vancouver
1.
Baute J. Genome fideltiy and DNA repair in plants: the characterization of the MBD4 DNA glycosylase homolog in Arabidopsis thaliana. [Ghent, Belgium]: Ghent University. Faculty of Sciences; 2008.
MLA
Baute, Joke. “Genome Fideltiy and DNA Repair in Plants: The Characterization of the MBD4 DNA Glycosylase Homolog in Arabidopsis Thaliana.” 2008 : n. pag. Print.