DNA repair and chromatin.
Technische Universität, Darmstadt
[Ph.D. Thesis], (2014)
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|Item Type:||Ph.D. Thesis|
|Title:||DNA repair and chromatin|
Laser microirradiation can be used to induce DNA damage with high spatial and temporal resolution, representing a powerful tool to analyze DNA repair in living cells. However, most lasers induce a mixture of DNA damage leading to the activation of multiple DNA repair pathways, making it impossible to study the kinetics of individual repair processes or the interaction of repair factors. To overcome this, we established conditions to discriminate specific repair pathways using lasers commonly available in confocal microscopes. We used cells expressing fluorescently tagged proteins specific for different repair processes and monitored their accumulation after microirradiation with lasers of different wavelengths (405, 488, 561, 633 nm). In addition, we validated DNA damage using a series of DNA damage markers (CPDs, TUNEL,gH2AX-foci). Our data showed that irradiation with a 405 nm laser led to activation of all tested repair pathways. Irradiation with 488 or 561 nm lasers led to selective activation of short-patch base excision repair. These conditions discriminated against processive long-patch base excision repair, nucleotide excision repair as well as double strand break repair pathways.
We applied this microirradiation strategy to study differences between eu- and heterochromatin upon DNA damage. Highly compacted heterochromatic regions in human and mouse cells were fluorescently marked and microirradiated. We demonstrated recruitment of repair factors inside these dense heterochromatic regions and showed that conditions leading to processive DNA synthesis resulted in local decondensation with about two fold increase in heterochromatin area. Using pulse labeling with thymidine analogs added immediately before microirradiation, we measured processive DNA synthesis repair directly inside heterochromatic regions. These data are not in agreement with the proposal that repair of heterochromatic DNA damage takes place at the periphery of the domain but show that processive DNA synthesis repair can take place inside heterochromatic regions.
We furthermore took advantage of the discriminating microirradiation strategy to study the newly described post-replicative DNA repair pathway ribonucleotide excision repair. This pathway was proposed to degrade millions of ribonucleotides misincorporated by DNA polymerases during replication. The excision of ribonucleotide is carried out by the nuclease RNase H2. We showed that RNase H2 is not only recruited to sites of DNA replication, but also to sites of processive DNA repair, suggesting that pathway activation takes place not only after replication but also after repair. Mutations in RNase H2 are linked to the autoimmune disease Aicardi-Goutieres syndrome. To shed more light into the pathology of these mutations, we demonstrated that recruitment efficiency and kinetics of exchange at repair and replication sites were diminished by mutations affecting complex formation, PCNA binding and catalysis.
|Place of Publication:||Darmstadt|
|Classification DDC:||500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie|
|Divisions:||10 Department of Biology
10 Department of Biology > Cell Biology and Epigenetics
|Date Deposited:||20 Jun 2014 11:10|
|Last Modified:||20 Jun 2014 11:10|
|Referees:||Cardoso, Prof. Dr. M. Cristina and Layer, Prof. Dr Paul|
|Refereed:||23 May 2014|
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