The SMX DNA Repair Tri-nuclease.

The efficient removal of replication and recombination intermediates is essential for the maintenance of genome stability. Resolution of these potentially toxic structures requires the MUS81-EME1 endonuclease, which is activated at prometaphase by formation of the SMX tri-nuclease containing three DNA repair structure-selective endonucleases: SLX1-SLX4, MUS81-EME1, and XPF-ERCC1. Here we show that SMX tri-nuclease is more active than the three individual nucleases, efficiently cleaving replication forks and recombination intermediates. Within SMX, SLX4 co-ordinates the SLX1 and MUS81-EME1 nucleases for Holliday junction resolution, in a reaction stimulated by XPF-ERCC1. SMX formation activates MUS81-EME1 for replication fork and flap structure cleavage by relaxing substrate specificity. Activation involves MUS81's conserved N-terminal HhH domain, which mediates incision site selection and SLX4 binding. Cell cycle-dependent formation and activation of this tri-nuclease complex provides a unique mechanism by which cells ensure chromosome segregation and preserve genome integrity.

Roles of SLX1-SLX4, MUS81-EME1, and GEN1 in avoiding genome instability and mitotic catastrophe.

The resolution of recombination intermediates containing Holliday junctions (HJs) is critical for genome maintenance and proper chromosome segregation. Three pathways for HJ processing exist in human cells and involve the following enzymes/complexes: BLM-TopoIIIα-RMI1-RMI2 (BTR complex), SLX1-SLX4-MUS81-EME1 (SLX-MUS complex), and GEN1. Cycling cells preferentially use the BTR complex for the removal of double HJs in S phase, with SLX-MUS and GEN1 acting at temporally distinct phases of the cell cycle. Cells lacking SLX-MUS and GEN1 exhibit chromosome missegregation, micronucleus formation, and elevated levels of 53BP1-positive G1 nuclear bodies, suggesting that defects in chromosome segregation lead to the transmission of extensive DNA damage to daughter cells. In addition, however, we found that the effects of SLX4, MUS81, and GEN1 depletion extend beyond mitosis, since genome instability is observed throughout all phases of the cell cycle. This is exemplified in the form of impaired replication fork movement and S-phase progression, endogenous checkpoint activation, chromosome segmentation, and multinucleation. In contrast to SLX4, SLX1, the nuclease subunit of the SLX1-SLX4 structure-selective nuclease, plays no role in the replication-related phenotypes associated with SLX4/MUS81 and GEN1 depletion. These observations demonstrate that the SLX1-SLX4 nuclease and the SLX4 scaffold play divergent roles in the maintenance of genome integrity in human cells.

Phosphorylation of the DNA repair scaffold SLX4 drives folding of the SAP domain and activation of the MUS81-EME1 endonuclease.

The DNA repair scaffold SLX4 has multifaceted roles in genome stability, many of which depend on structure-selective endonucleases. SLX4 coordinates the cell cycle-regulated assembly of SLX1, MUS81-EME1, and XPF-ERCC1 into a tri-nuclease complex called SMX. Mechanistically, how the mitotic kinase CDK1 regulates the interaction between SLX4 and MUS81-EME1 remains unclear. Here, we show that CDK1-cyclin B phosphorylates SLX4 residues T1544, T1561, and T1571 in the MUS81-binding region (SLX4MBR). Phosphorylated SLX4MBR relaxes the substrate specificity of MUS81-EME1 and stimulates cleavage of replication and recombination structures, providing a biochemical explanation for the chromosome pulverization that occurs when SLX4 binds MUS81 in S-phase. Remarkably, phosphorylation of SLX4MBR drives folding of an SAP domain, which underpins the high-affinity interaction with MUS81. We also report the structure of phosphorylated SLX4MBR and identify the MUS81-binding interface. Our work provides mechanistic insights into how cell cycle-regulated phosphorylation of SLX4 drives the recruitment and activation of MUS81-EME1.

Exploring the Structures and Functions of Macromolecular SLX4-Nuclease Complexes in Genome Stability.

All organisms depend on the ability of cells to accurately duplicate and segregate DNA into progeny. However, DNA is frequently damaged by factors in the environment and from within cells. One of the most dangerous lesions is a DNA double-strand break. Unrepaired breaks are a major driving force for genome instability. Cells contain sophisticated DNA repair networks to counteract the harmful effects of genotoxic agents, thus safeguarding genome integrity. Homologous recombination is a high-fidelity, template-dependent DNA repair pathway essential for the accurate repair of DNA nicks, gaps and double-strand breaks. Accurate homologous recombination depends on the ability of cells to remove branched DNA structures that form during repair, which is achieved through the opposing actions of helicases and structure-selective endonucleases. This review focuses on a structure-selective endonuclease called SLX1-SLX4 and the macromolecular endonuclease complexes that assemble on the SLX4 scaffold. First, we discuss recent developments that illuminate the structure and biochemical properties of this somewhat atypical structure-selective endonuclease. We then summarize the multifaceted roles that are fulfilled by human SLX1-SLX4 and its associated endonucleases in homologous recombination and genome stability. Finally, we discuss recent work on SLX4-binding proteins that may represent integral components of these macromolecular nuclease complexes, emphasizing the structure and function of a protein called SLX4IP.

Homologous Recombination and the Formation of Complex Genomic Rearrangements.

The maintenance of genome integrity involves multiple independent DNA damage avoidance and repair mechanisms. However, the origin and pathways of the focal chromosomal reshuffling phenomena collectively referred to as chromothripsis remain mechanistically obscure. We discuss here the role, mechanisms, and regulation of homologous recombination (HR) in the formation of simple and complex chromosomal rearrangements. We emphasize features of the recently characterized multi-invasion (MI)-induced rearrangement (MIR) pathway which uniquely amplifies the initial DNA damage. HR intermediates and cellular contexts that endanger genomic stability are discussed as well as the emerging roles of various classes of nucleases in the formation of genome rearrangements. Long-read sequencing and improved mapping of repeats should enable better appreciation of the significance of recombination in generating genomic rearrangements.

Regulated Crossing-Over Requires Inactivation of Yen1/GEN1 Resolvase during Meiotic Prophase I.

During meiosis, crossover recombination promotes the establishment of physical connections between homologous chromosomes, enabling their bipolar segregation. To ensure that persistent recombination intermediates are disengaged prior to the completion of meiosis, the Yen1(GEN1) resolvase is strictly activated at the onset of anaphase II. Whether controlled activation of Yen1 is important for meiotic crossing-over is unknown. Here, we show that CDK-mediated phosphorylation of Yen1 averts its pervasive recruitment to recombination intermediates during prophase I. Yen1 mutants that are refractory to phosphorylation resolve DNA joint molecules prematurely and form crossovers independently of MutLγ, the central crossover resolvase during meiosis. Despite bypassing the requirement for MutLγ in joint molecule processing and promoting crossover-specific resolution, unrestrained Yen1 impairs the spatial distribution of crossover events, genome-wide. Thus, active suppression of Yen1 function, and by inference also of Mus81-Mms4(EME1) and Slx1-Slx4(BTBD12) resolvases, avoids precocious resolution of recombination intermediates to enable meiotic crossover patterning.