The Role of Adaptor Proteins in rDNA Double-Strand Break Repair by Homologous Recombination

Genome stability and integrity in eukaryotic cells are continuously threatened by a number of endogenous as well as exogenous DNA damaging agents. Among the various lesions, DNA double strand breaks (DSBs) represent a particularly deleterious threat for a cell. In order to maintain genome stability and integrity, cells have evolved a highly organized network of cellular pathways that sense, signal and ultimately repair DNA lesions. This network is called the DNA damage response (DDR) and its activity is crucial for a cellular health and survival. Depending on the cell cycle, two major pathways were described to repair genotoxic DSBs in mammalian cells. The non-homologous end joining (NHEJ) is mainly active in G1 and early-S phase and is an error-prone pathway, occasionally leading to small insertions or deletions at the break site. In contrast, the homologous recombination (HR) occurs mainly in G2 and S phase when a sister chromatid is present, which serves as a homologous dsDNA template for an effective and accurate repair of DSBs. Although the DNA damage response (DDR) is a conserved mechanism, a few variations have been observed. One example is the DSBs repair in repetitive DNA sequences, which are highly abundant in mammalian genomes. DNA breaks within repetitive regions of the genome pose a potential threat to chromosomal stability because, due to their repetitive nature, they are prone to non- allelic (ectopic) recombination. During this process, cells use identical DNA repeats located on another chromosome as the repair template, which may lead to chromosome rearrangements and widespread genome instability. Previous work, carried out mostly in yeasts and Drosophila cells, showed that cells are well equipped to suppress ectopic recombination of repetitive DNA sequences. The key element in this process is the temporary pausing of HR progression followed by the movement of the DNA break 6 sites to a “safe location”, where DSBs are finally repaired by HR. A similar mechanism was observed in mammalian cells, namely in mouse heterochromatin regions and human ribosomal DNA (rDNA) repeats, which reside in the nucleoli. It is well established that upon break induction in the rDNA repeats, cells shut down ribosomal RNA transcription, which triggers the movement of broken rDNA repeats from inside of the nucleoli in the nuclear periphery, where they accumulate in so-called nucleolar caps. Within this thesis, I contribute to the discovery of the molecular mechanism which underlies the rDNA breaks repair by HR. I describe that the adaptor proteins Treacle and MDC1 play a central role in coordinating the sequence of events that ensue in response to rDNA DSBs. Firstly, the phosphorylation of the histone variant H2AX by the ATM kinase is dependent on Treacle and its two interaction partners TOPBP1 and NBS1, which together mediate mobilization of damaged rDNA repeats to the nucleolar periphery. The subsequent recruitment of the BRCA1-PALB2-BRCA2 complex occurs downstream of the MDC1-RNF8-RNF168 axis, which depends on the phosphorylation of H2AX, and thus on the Treacle-TOPBP1-NBS1 complex and on rDNA break mobilization. This ensures that rDNA break repair by HR (RAD51 loading) only occurs after rDNA break mobilization, hence preventing ectopic recombination. The mechanism may ensure the maintenance of rDNA repeats stability and integrity, which is essential for cellular health and survival.

Author dc:creator

• Hänel, Andrea

• 610 Medicine & health

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

eng