Cell unable to complete the recombinational repair process show abnormalities at cell division.

Stephen West : Genome instability disorders linked to defects in the resolution of recombination intermediates

Introduction

Recombinational repair requires a reciprocal exchange of DNA strands between sister chromatids or homologous chromosomes, leading to the formation of DNA intermediates, such as Holliday junctions (HJs), in which the two interacting DNAs become covalently interlinked.

Recombination intermediates are resolved by three distinct mechanisms that ensure chromosome segregation.

Figure 1: Recombination intermediates are resolved by three distinct mechanisms that ensure chromosome segregation.

Recombinational repair requires a reciprocal exchange of DNA strands between sister chromatids or homologous chromosomes, leading to the formation of DNA intermediates, such as Holliday junctions (HJs), in which the two interacting DNAs become covalently interlinked.
The efficient processing of these joint molecules is essential for chromosome segregation at cell division, and is also important for determining the outcome of recombination: for example, crossovers (COs) between homologous chromosomes are required for meiotic division, whereas non-crossovers (NCOs) are favoured in mitotic cells in order to avoid 'loss of heterozygosity' (LOH), a known driver of tumourigenesis.

This image shows defects in chromosome segregation, indicated by the presence of ultrafine bridges.

Figure 2: Loss of MUS-SLX and/or GEN1 resolvase activity allows recombination intermediates to persist to mitosis where they cause defects in chromosome segregation that leads to severe genome instability. This image shows defects in chromosome segregation, indicated by the presence of ultrafine bridges.

A four-protein complex, made up from the BLM helicase, topoisomerase IIIα, RMI1 and RMI2 (the BTR complex) promotes the dissolution of HJs to form non-crossovers. Individuals with mutations in the BLM helicase suffer from a genetic disorder called Bloom's Syndrome (BS) that leads to dwarfism, immunodeficiency and reduced fertility. BS patients also develop various types of cancers, often at a young age. Cells derived from individuals with BS exhibit an extreme form of genome instability, the hallmark feature of which is an elevated frequency of sister chromatid exchanges (SCEs).

The high frequency of SCEs observed in cells derived from individuals with BS arise from the use of different pathways for HJ processing that involve the MUS81-EME1, SLX1-SLX4 and GEN1 endonucleases. Recent work has shown that the nucleolytic resolution of recombination intermediates is coordinated with cell cycle progression, and is restricted to the late stages of the cell cycle.

Using yeast as a model system, we discovered that the HJ-resolving activities of Mus81-Mms4 (the ortholog of MUS81-EME1) and Yen1 (the equivalent of GEN1) are controlled by phosphorylation events that modulate their activities throughout the cell cycle. In mitotic cells, Mus81-Mms4 is hyper-activated by Cdc5-mediated phosphorylation at G2/M, whereas Yen1 is activated at anaphase, where it catalyses the resolution of persistent Holliday junctions that would otherwise block chromosome segregation.

Similar regulatory systems exist in human cells, where MUS81-EME1 is activated by CDK/PLK1 phosphorylation at prometaphase. In this case, however, the phosphorylation of MUS81-EME1 stimulates its interaction with SLX1-SLX4, to form a tri-nuclease complex that we have named SMX (SLX1-SLX4-MUS81-EME1-XPF-ERCC1). Within this supernuclease complex, the MUS81-EME1 nuclease is activated to cut recombination and replication intermediates late in the cell cycle. Like, SMX, the activity of GEN1 is also tightly controlled, in this case by nuclear extrusion so that the nuclease only gains access to the DNA when the nuclear membrane breaks down. These two waves of Holliday junction resolution, which occur at prometaphase and mitosis, respectively, process persistent HJs and ensure chromosome segregation (Figure 1).

These three pathways for HJ resolution are essential for cell viability. Inactivation of MUS81 and GEN1 from cells derived from individuals with Bloom's syndrome leads to an unusual aberrant chromosome morphology and cell death. It appears that the BTR complex normally resolves joint molecules in a manner that specifically avoids SCEs (and loss of heterozygosity when recombination occurs between homologous chromosomes rather than sister chromatids), and that, without BTR, joint molecule resolution is mediated by MUS81-EME1-SLX1-SLX4 and GEN1. However, the importance of these nucleases is demonstrates by observations showing that cells suffer severe chromosome abnormalities and show signs of mitotic catastrophe, such as ultrafine anaphase bridges (Figure 2), lagging chromosomes, and a high frequency of multi-nucleated cells.