Holliday junction resolution and its role in maintaining genome

Abstract –

Repair of DNA breaks by homologous recombination (HR) can lead to the formation of recombination intermediates, which often contain four-way structures known as Holliday junctions (HJs) that physically link sister chromatids. The efficient resolution of these joint molecules is essential for chromosome segregation. In human mitotic cells, HJs can be cleaved by resolvases, including MUS81-EME1, SLX1-SLX4 and GEN1. MUS81-EME1 associates with SLX1-SLX4 and a third nuclease, XPF-ERCC1, to form the SMX tri-nuclease complex. The SMX complex and GEN1 cleave recombination intermediates in two genetically distinct resolution pathways. To understand the molecular mechanism of HJ resolution in human cells, I have purified human GEN1 protein and showed that it mediates a pair of symmetrical nicks in the two opposite strands across the junction. I also discovered that GEN1 actions are restricted to the late stages of the cell cycle by nuclear exclusion to suppress the formation of sister chromatid exchanges. Furthermore, we have followed the fate of unresolved recombination intermediates that arise in GEN1^-/- knock-out cells depleted for MUS81. We found that the recombination intermediates persist until mitosis where they form a novel class of anaphase bridges, which we term homologous recombination ultra-fine bridges, or HR-UFBs. HR-UFBs are distinct from replication stress-associated UFBs that arise at common fragile sites, and also those that form between centromeres. Importantly, inhibiting the HR machinery by depleting RAD51 or BRCA2 leads to a reduction in the number of HR-UFBs. We found that the HR-UFBs are acted upon by BLM helicase to generate single-stranded RPA-coated bridges that are broken at cell division. In the next cell cycle, DNA breaks activate the DNA damage checkpoint and non-homologous end joining drives chromosome fusion events. Consequently, the cells undergo a cell cycle delay and massive cell death. Together, these results lead us to present a model detailing how unresolved recombination intermediates promote DNA damage and chromosomal instability.