Regulation of recombination-mediated DSB repair and its importance for genome stability


Year:

Session type:

Stephen West1
1London Research Institute, South Mimms, UK

Abstract

The repair of DNA double strand breaks in mitotic cells can lead to the formation of Holliday junctions (HJs) that link sister chromatids. There are several mechanisms for the removal of these junctions from DNA, and these are essential for proper chromosome segregation. Additionally, the formation and resolution of Holliday junctions during meiosis is an essential cellular event because crossovers are needed to provide the tension required for the segregation of homologous chromosomes.

In human mitotic cells, the BLM protein, inactivated in individuals with Bloom’s Syndrome (BS), acts in combination with Topoisomerase IIIα, RMI1 and RMI2 (BTR complex) to promote HJ ‘dissolution’, a reaction that is important for the avoidance of sister chromatid exchanges (SCEs). We find that elevated SCE formation, a characteristic feature of BS cell lines, results from the actions of MUS81-EME1, SLX1-SLX4 and GEN1. These enzymes process HJs but, in contrast to the BTR complex, do so by endonucleolytic cleavage. Cells defective for BLM develop a broad spectrum of early onset cancers caused by chromosome instability, and it is likely that the aberrant use of these nucleolytic HJ resolution pathways contributes to tumourigenesis.

We show that the BTR complex provides the primary pathway for the processing of recombination intermediates in S-phase human mitotic cells, with the MUS81-EME1 and GEN1 junction resolvases becoming activated in G2/M in order to resolve any junctions that have escaped the attentions of BTR. Using siRNA to down-regulate MUS81 and GEN1 in the BLM-defective background, effectively producing a cell lacking all pathways of HJ resolution, we observed severe chromosome abnormalities due to problems with chromosome condensation leading to cell death. How normal cells regulate these pathways for the processing of recombination intermediates will be described.