NADRCT

Nuclear architecture in DNA repair and formation of chromosomal translocatons

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 Obiettivo del progetto (Objective)

'Double-strand breaks (DSBs) occur frequently in the genome by DNA damaging agents or during genome replication. DSBs are hazardous because interaction between DNA ends from different double strand breaks can produce tumorigenic chromosome translocations. Little is known about how repair factors function in the context of chromatin and how translocations form in vivo. I developed a cell system in which a DSB can specifically be induced at a defined genomic site and follow the fate of damaged DNA in living cells. Using this system, I showed but that the broken ends are positionally stable and unable to roam the cell nucleus and I identified that the repair factor Ku80 is required for maintaining the alignment of broken ends. I extended the use of this system to probe how DSBs are recognized in vivo and how DDR pathways are triggered in the context of chromatin. I found that tethering a single component of a DSB repair complex to chromatin is sufficient to elicit the DDR in the absence of DNA damage and that stable association of the mediator of DDR MDC1 induces high local chromatin decondensation. I will use a combination of advanced live cell imaging and biochemistry techniques to further test the importance of nuclear architecture in maintaining genomic integrity and I will identify novel factors involved in DSB repair and alignment of broken chromosomes. More specifically I plan to: 1. Visualize the formation of chromosome translocations in vivo and determine the role of chromatin structure and nuclear organization in the process using time-lapse imaging. 2. Investigate the role of the repair factor MDC1 in chromatin decondensation using yeast two hybrid and advanced biochemistry in mammalian cells. 3. Identify novel proteins that accumulate in DSBs using biochemical fractionation and purification methods. 4. Identify novel proteins that align broken chromosome ends by high-through-put siRNA screening.'

Introduzione (Teaser)

Chromosome translocation is an abnormality caused by rearrangement of parts between different chromosomes. Several cell pathways participate in the detection of DNA damage and mediation of its repair.

Descrizione progetto (Article)

Cells continuously experience stress and damage from number of sources, such as UV light, irradiation, or oxidative by-products of metabolism. This endangers genome stability and could cause DNA breaks such as double strand breaks (DSBs). DSBs are the most harmful because their inaccurate repair can lead to chromosomal translocations.

The EU-funded 'Nuclear architecture in DNA repair and formation of chromosomal translocations' (NADRCT) project investigates the role of nuclear architecture in the sensing and repair of DSBs. Preliminary findings suggest that nuclear compartmentalisation may contribute to the mechanism linking DNA damage response (DDR) and DNA repair.

Researchers developed an experimental system to induce DSBs at specific locations and follow its repair. During first project period, they identified new repair factors involved in chromatin de-condensation. For instance, the polymerases TNKS 1 and 2 were found to be recruited to DNA lesions by the check-point protein MDC1 to promote reparative recombination. The same TNKS proteins counteract de-condensation and facilitate bridging of distal broken DNA ends.

Using this system, researchers' visualised how breaks are recognised and repaired in the two different sub-compartments: the nuclear lamina and the nuclear pores. They showed that the DDR induced by a break inflicted at the nuclear lamina is delayed and the nuclear pores appear to be an activating microenvironment for DDR and repair.

Finally, siRNA screening helped identify novel chromatin related proteins whose down regulation led to persistent and unrepaired DSBs. This approach revealed several novel proteins that are currently being investigated.

Project outcomes revealed the role of chromatin structure in the differential regulation of DDR and repair at the two distinct compartments of the nuclear periphery. They reveal a new level of regulation of DSB repair through spatial organisation of DNA in the nucleus. This has important implications for the development of gene-based therapies.