Genome Instability as a Consequence of Defects in the Resolution of Recombination Intermediates.

Stephen C West,Ying Wai Chan

doi:10.1101/sqb.2017.82.034256

Stephen C West, Ying Wai Chan

Open Access

https://doi.org/10.1101/sqb.2017.82.034256

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Abstract

The efficient processing of homologous recombination (HR) intermediates, which often contain four-way structures known as Holliday junctions (HJs), is required for proper chromosome segregation at mitosis. Eukaryotic cells possess three distinct pathways of resolution: (i) HJ dissolution mediated by BLM-topoisomerase IIIα-RMI1-RMI2 (BTR) complex, and HJ resolution catalyzed by either (ii) SLX1-SLX4-MUS81-EME1-XPF-ERCC1 (SMX complex) or (iii) GEN1. The BTR pathway acts at all times throughout the cell cycle, whereas the actions of SMX and GEN1 are restrained in S phase and become elevated late in the cell cycle to ensure the resolution of persistent recombination intermediates before mitotic division. By developing a "resolvase-deficient" model system in which the activities of MUS81 and GEN1 are compromised, we have explored the fate of unresolved recombination intermediates. We find that covalently linked sister chromatids promote the formation of a new class of ultrafine bridges at anaphase that we term HR-UFBs. These bridges are broken at cell division, leading to activation of the DNA damage checkpoint and repair by nonhomologous end joining (NHEJ) in the next cell cycle. As a consequence, high levels of gross chromosomal rearrangements and aberrations are observed, together with frequent cell death. These results show that the HJ resolvases provide essential functions for the resolution of recombination intermediates, even in cells that remain proficient for BTR-mediated HJ dissolution.

Highlights

Homologous recombination provides an essential mechanism for the repair of DNA breaks that arise from the demise of stalled replication forks or are induced by genotoxic agents
Joint molecule intermediates of homologous recombination often take the form of a four-way junction, known as a Holliday junction (HJ), that needs to be resolved before chromosome segregation (Holliday 1964; West 2003; Wyatt and West 2014)
When the cells were treated with the DNA-PKcs inhibitor NU7026, which inhibits nonhomologous end joining (NHEJ), we observed suppression of the fusion phenotype (Fig. 5C). These results show that the chromosome fusions and rearrangements are generated through the repair of breaks produced at the first mitotic division. Our work with this resolvase-deficient model cell system provides a clear picture of the way that unresolved recombination intermediates lead to cell death

Summary

MECHANISMS AND REGULATION OF RECOMBINATION INTERMEDIATE

A four-subunit complex, containing BLM-topoisomerase IIIα-RMI1-RMI2, known as the BTR complex, promotes the dissolution of double HJs. Cells derived from individuals with Bloom syndrome (BS), which is caused by mutations in BLM, show the diagnostic feature of a high frequency of sister chromatid exchanges and increased genome instability. These individuals are predisposed to a broad spectrum of earlyonset cancers (Ray and German 1984; Hickson 2003). Persistent double HJs, and single HJs that cannot serve as a substrate for BLM, are resolved by structure-selective nucleases (resolvases) These enzymes cut HJs by introducing coordinated nicks across the junction and give rise to nicked duplex products that are crossovers (COs) or noncrossovers (NCOs).

WEST AND CHAN

CONCLUSION