Analysis of the DNA damage response in living cells - Elektronische

199 Pages·2008·7.85 MB·English

by  Oliver Mortusewicz

Analysis of the DNA damage
response in living cells

Oliver Mortusewicz

München 2007
Analysis of the DNA damage
response in living cells

Oliver Mortusewicz

Dissertation
an der Fakultät für Biologie
der Ludwig-Maximilians-Universität
München

vorgelegt von
Oliver Mortusewicz
aus Giessen

München, den 08. November 2007
Erstgutachter: Prof. Dr. Heinrich Leonhardt
Zweitgutachter: Prof. Dr. Manfred Schliwa

Tag der mündlichen Prüfung: 19.12.2007
CONTENT

CONTENT
CONTENT 1
SUMMARY 3
1. INTRODUCTION 7
1.1. DNA lesion detection and signalling 9
1.2. Checkpoint activation 12
1.3. Repair of genetic information 13
1.4. Repair of epigenetic information 16
1.5. A new assay to study protein-protein interactions in living cells 18
1.6. Technical Background 19
2. RESULTS 23
2.1. Feedback regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid
response to DNA damage in living cells. 23
2.2. Recruitment of RNA Polymerase II cofactor PC4 to DNA repair sites. 41
2.3. Spatiotemporal dynamics of p21CDKN1A protein recruitment to DNA damage
sites and interaction with proliferating cell nuclear antigen. 71
2.4. XRCC1 and PCNA are loading platforms with distinct kinetic properties and
different capacities to respond to multiple DNA lesions. 87
2.5. Differential recruitment of DNA Ligase I and III to DNA repair sites. 99
2.6. Recruitment of DNA methyltransferase 1 to DNA repair sites. 119
2.7. A fluorescent two-hybrid (F2H) assay for direct visualization of protein
interactions in living cells. 129
3. DISCUSSION 159
3.1. DNA lesion detection and signalling 160
3.2. The role of p21 in DNA repair 163
3.3. Coordination of DNA repair by central loading platforms 164
3.4. Maintenance of DNA methylation patterns in DNA repair 167
3.5. A new assay to visualize protein-protein interactions in living cells 169
4. ANNEX 171
4.1. Abbreviations 171
4.2. Contributions 173
4.3. Acknowledgements 177
4.4. References 179
5. CURRICULUM VITAE 191

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SUMMARY

SUMMARY
DNA lesions arising from environmental and endogenous sources induce various
cellular responses including cell cycle arrest, DNA repair and apoptosis. Although
detailed insights into the biochemical mechanisms and composition of DNA repair
pathways have been obtained from in vitro experiments, a better understanding of
the interplay and regulation of these pathways requires DNA repair studies in living
cells.
In this study we employed laser microirradiation and photobleaching techniques in
combination with specific mutants and inhibitors to analyze the real-time
accumulation of proteins at laser-induced DNA damage sites in vivo, thus unravelling
the mechanisms underlying the coordination of DNA repair in living cells.
The immediate and faithful recognition of DNA lesions is central to cellular survival,
but how these lesions are detected within the context of chromatin is still unclear. In
vitro data indicated that the DNA-damage dependent poly(ADP-ribose) polymerases,
PARP-1 and PARP-2, are involved in this crucial step of DNA repair. With specific
inhibitors, mutations and photobleaching analysis we could reveal a complex
feedback regulated mechanism for the recruitment of the DNA damage sensor
PARP-1 to microirradiated sites. Activation of PARP-1 results in localized poly(ADP-
ribosyl)ation and amplifies a signal for the subsequent rapid recruitment of the
loading platform XRCC1 which coordinates the assembly of the repair machinery.
Using similar techniques we could demonstrate the immediate and transient binding
of the RNA Polymerase II cofactor PC4 to DNA damage sites, which depended on its
single strand binding capacity. This establishes an interesting link between DNA
repair and transcription. We propose a role for PC4 in the early steps of the DNA
damage response, recognizing and stabilizing single stranded DNA (ssDNA) and
thereby facilitating DNA repair by enabling repair factors to access their substrates.
After DNA lesions have been successfully detected they have to be handed over to
the repair machinery which restores genome integrity. Efficient repair requires the
coordinated recruitment of multiple enzyme activities which is believed to be
controlled by central loading platforms. As laser microirradiation induces a variety of
different DNA lesions we could directly compare the recruitment kinetics of the two
loading platforms PCNA and XRCC1 which are involved in different repair pathways
side by side. We could demonstrate that PCNA and XRCC1 show distinct recruitment
and binding kinetics with the immediate and fast recruitment of XRCC1 preceding the
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SUMMARY

slow and continuous recruitment of PCNA. Introducing consecutively multiple DNA
lesions within a single cell, we further demonstrated that these different recruitment
and binding characteristics have functional consequences for the capacity of PCNA
and XRCC1 to respond to successive DNA damage events.
To further study the role of PCNA and XRCC1 as loading platforms in DNA repair, we
extended our analysis to their respective interaction partners DNA Ligase I and III.
Although these DNA Ligases are highly homologous and catalyze the same
enzymatic reaction, they are not interchangeable and fulfil unique functions in DNA
replication and repair. With deletion and mutational analysis we could identify
domains mediating the specific recruitment of DNA Ligase I and III to distinct repair
pathways through their interaction with PCNA and XRCC1. We conclude that this
specific targeting may have evolved to accommodate the particular requirements of
different repair pathways (single nucleotide replacement vs. synthesis of short
stretches of DNA) and thus enhances the efficiency of DNA repair.
Interestingly, we found that other PCNA-interacting proteins exhibit recruitment
kinetics similar to DNA Ligase I, indicating that PCNA not only serves as a central
loading platform during DNA replication, but also coordinates the recruitment of
multiple enzyme activities to DNA repair sites. Accordingly, we found that the
maintenance methyltransferase DNMT1, which is known to associate with replication
sites through binding to PCNA, is likewise recruited to DNA repair sites by PCNA. We
propose that DNMT1, like in DNA replication, preserves methylation patterns in the
newly synthesized DNA, thus contributing to the restoration of epigenetic information
in DNA repair.
In summary, we found immediate and transient binding of repair factors involved in
DNA damage detection and signalling, while repair factors involved in the later steps
of DNA repair, like damage processing, DNA ligation and restoration of epigenetic
information, showed a slow and persistent accumulation at DNA damage sites. We
conclude that DNA repair is not mediated by binding of a preassembled repair
machinery, but rather coordinated by the sequential recruitment of specific repair
factors to DNA damage sites.
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