Spatio-temporal properties of the mammalian DNA damage response

Simon Bekker-Jensen

Research output: Book/ReportPh.D. thesis

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Abstract

Organizing the mammalian DNA damage response in time and space is a major challenge to the cell. Of the many roles of this response, the ability to control cell cycle progression and various aspects of DNA metabolism poses the greatest challenge, as such interventions require a damage alert signal to be spread from focal lesions to distant replication origins, gene promoters etc. In the present report, I explore some of the spatio-temporal features of the DNA damage response that enable detection of DNA double strand breaks (DSBs), organization of the surrounding nuclear space and the communication with distant and immobile nuclear structures.

To strengthen our existing capabilities in experimental live cell imaging techniques, I have developed a mathematical framework based on linear differential equations to describe the accumulation of proteins into foci at sites of DNA damage. This proved to be a useful tool in analyzing data from DSB-generating laser microirradiation experiments. In this report, I also devise mathematical models for the analysis of photobleaching experiments, the non-invasive microscopy technique for studying protein dynamics.

Mdc1 is the physiological binding partner of γ-H2AX, and in two publications my co-workers and I showed how this interaction at sites of DNA damage attracts other factors by distinct mechanisms. First, by a physical interaction with Nbs1, Mdc1 recruits the Mre11-Nbs1-Rad50 (MRN) complex to γ-H2AX modified chromatin. Second, Mdc1 is required for productive assembly of 53BP1 at sites of DNA damage, as 53BP1 recruitment was strongly diminished in Mdc1-depleted cells. This requirement is evident at early as well as late stages of checkpoint signalling and points to an important role for Mdc1 in structuring DSB-flanking chromatin. Kinetic studies of micro-laser induced protein redistribution revealed that accumulation of Mdc1 and Nbs1 at sites of DNA damage occurred simultaneously and almost instantly after generation of DSBs. Conversely, accumulation of 53BP1 lagged significantly behind. These kinetic discrepancies reflect the direct recruiting versus chromatin organizing functions of Mdc1.

In this thesis I show that the DSB-containing nuclear space is organized in two non-overlapping compartments. One of these is modified chromatin which provides a binding platform for chromatin interacting proteins like Mdc1 and 53BP1 and from this compartment ATM signalling emanates. The other and significantly smaller compartment, dubbed “micro foci”, is cell cycle regulated and consists of single stranded DNA (ssDNA) which attracts proteins involved in ATR signalling, including ATR itself and the homologous recombination machinery. Thus, despite that they share many substrates, ATM and ATR markedly differ in their sub-nuclear localization following a DSB-generating insult. The MRN complex and BRCA1 are unique in the sense that they can be detected within both compartments. I speculate that the Mdc1-independent accumulation of MRN in micro foci precedes ssDNA formation and reflects the important sensory role of this complex. A large and heterogeneous group of proteins involved in the DNA damage response do not accumulate in either of the spatially restricted compartments. Chk1 and Chk2 belong to this category, and this behaviour was previously shown to reflect the pan-nuclear messenger roles of these kinases. In the present study, I add the novel ATM substrate Kap1 to this category and show that this protein transiently interacts with, and is activated at, the site of DNA damage, but does so with very slow kinetics compared to Chk1 and Chk2. This spatio-temporal profile may be important for the role of Kap1 in mediating global chromatin relaxation following activation by ATM.

TopBP1 and Claspin are two mediators that operate in the ATR pathway to phosphorylate and activate Chk1. Together with my co-workers, I show that TopBP1, which tightly co-localizes with ATR in micro foci, impacts on all tested phosphorylation events carried out by this kinase. This points to a role for TopBP1 in directly activating ATR, a view that is supported by the existing literature. Claspin, on the other hand, only impacts on Chk1 phosphorylation and these two proteins also display spatio-temporal properties that are virtually identical. Thus, this study provides an example of how the sub-nuclear localization of a protein acting in the DNA damage response can provide a clue to its function. I speculate that such an approach can be used as a diagnostic tool to assign novel DSB regulators to distinct signalling pathways.

In an effort to improve our understanding of Chk1 regulation, we discovered that the mediator protein Claspin is a target for SCF-mediated proteolysis via the F box protein β-Trcp. We found Claspin to be eliminated by this mechanism at the onset of mitosis and during recovery from genotoxic stress. In both cases, removal of Claspin strongly hampered the cells’ ability to activate or sustain activity of Chk1.

In summary, I have successfully applied mathematical and analytical methods to extract relevant biological information from live cell imaging data. Together with my co-workers I have discovered several new spatio-temporally important mechanisms operating during all stages of the DNA damage response, from its inception to its termination.
Original languageEnglish
Place of PublicationKgs. Lyngby
PublisherTechnical University of Denmark
Number of pages160
Publication statusPublished - 2006
Externally publishedYes

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