DNA Repair

Research project

Our lab studies the DNA repair mechanisms that operate in eukaryotic cells, particularly during replication, and the regulatory pathways that coordinate their usage.

DNA synthesis is frequently associated with nucleotide misincorporation, slippage at repetitive sequences, and aberrant transitions at replication forks. These events often result in the formation of single or double strand breaks, which can trigger recombination and endanger the stability of the chromosomes if not appropriately repaired.

When replication occurs in the presence of a damaged template, repriming downstream of the lesion generates gaps that can be filled in by using different strategies. One mechanism (referred to as template switch) is essentially error-free and uses the undamaged information of the sister duplex to bypass the DNA lesion. This resembles a homologous recombination reaction and leads to the formation of transient X-shaped chromosome structures. If left unresolved, however, these molecules can be potentially cleaved by nucleases and trigger recombination. We can detect these DNA molecules by using a combination of techniques, mostly based on the analysis of replication intermediates. The RecQ helicase Sgs1/BLM plays an important role in the resolution of these structures, and we have previously shown that Ubc9 and Mms21 dependent sumoylation controls this process. Sgs1/BLM is sumoylated but in an Mms21-independent fashion indicating that there may be other factors that act in concert with Sgs1/BLM to promote the dissolution of these X-shaped intermediates.

1. We are interested in uncovering the other factors implicated in the template switch process and in understanding the molecular mechanism through which they promote DNA repair. We have already identified two additional factors, Smc5-6 and Esc2, that prevent X-shaped molecule accumulation at damaged replication forks, and their present characterization suggests that they act in concert with Sgs1, likely by integrating sumoylation and ubiquitin ligase activities to regulate the repair pathway in response to damaged DNA (J. Sollier et. al, MBC, 2009).
   
   
2. We have previously found that sumoylation is important in regulating the transitions that occur at damaged replication forks. However, the molecular mechanism that regulates sumoylation events in response to DNA damage remains unknown. We have obtained evidence that one enzyme of the SUMO pathway is phosphorylated in response to DNA damage and prolonged replication fork stalling and that this event may play an important role in regulating the repair events that occur in response to intra-S damage.
   
   
3. We have recently found that two molecular mechanisms can lead to the formation of X-shaped sister chromatid junctions template switching. Their choice is regulated by SUMO-modification of a key replication and repair factor but it is independent of the Mms21-dependent sumoylation (Branzei et. al, Nature, 2008). We are addressing the other factors that contribute to these two pathways and the consequence of their usage for genome stability.
   
   
4. We are interested in understanding the repair pathways that operate in the cell to promote the resolution/repair of these structures when they fail to be dissolved in S-phase by Sgs1 and sumoylation, and the implications for replication termination.
   
   
5. The replication checkpoint is required for the formation of template switch intermediates during replication of damaged templates, but the targets and the mechanisms controlling the appropriate formation and resolution of the X molecules formed in the proximity of replication forks following DNA damage are not known. We plan to characterize the factors and the DNA transaction pathways promoting replication bypass of chromosomal lesions and filling in of gaps via the formation of X-shaped intermediates and the molecular mechanisms through which SUMO and the damage/replication checkpoint control this process.

The DNA replication machinery as well as most of the DNA repair factors and the DNA damage response pathways are conserved from yeast to humans. In the lab, we are using the yeast Saccharomyces cerevisiae, chicken B-cell lines, DT-40, and human cells. The budding yeast is a good model system that helps us identify and characterize in molecular detail the mechanisms promoting DNA repair and replication fork transactions. Some of our findings are presently extended in chicken DT40 cells, which are also genetically amenable. DT40 cells spend more than 60% of the cell cycle time in S phase, and are particularly suitable for studies in post replication repair. Furthermore, chromosome instability can be directly assessed by performing mitotic chromosome analysis. Well-conserved factors that appear to impact on genome integrity are tested in human cells for their role in replication, recombination, and the DNA damage response.