Coordinated actions of SLX1-SLX4 and MUS81-EME1 for Holliday junction resolution in human cells.
Holliday junctions (HJs) are four-way DNA intermediates that form during homologous recombination, and their efficient resolution is essential for chromosome segregation. Here, we show that three structure-selective endonucleases, namely SLX1-SLX4, MUS81-EME1, and GEN1, define two pathways of HJ resolution in human cells. One pathway is mediated by GEN1, whereas SLX1-SLX4 and MUS81-EME1 provide a second and genetically distinct pathway (SLX-MUS). Cells depleted for SLX-MUS or GEN1 pathway proteins exhibit severe defects in chromosome segregation and reduced survival. In response to CDK-mediated phosphorylation, SLX1-SLX4 and MUS81-EME1 associate at the G2/M transition to form a stable SLX-MUS holoenzyme, which can be reconstituted in vitro. Biochemical studies show that SLX-MUS is a HJ resolvase that coordinates the active sites of two distinct endonucleases during HJ resolution. This cleavage reaction is more efficient and orchestrated than that mediated by SLX1-SLX4 alone, which exhibits a potent nickase activity that acts promiscuously upon DNA secondary structures.
London Research Institute, Cancer Research UK, Clare Hall Laboratories, South Mimms, Herts EN6 3LD, UK.
Haley D M
HD
Wyatt
Shriparna
S
Sarbajna
Joao
J
Matos
Stephen C
SC
West
D001483:Base Sequence; D002461:Cell Line, Transformed; D004260:DNA Repair; D045566:DNA, Cruciform; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D059565:G2 Phase Cell Cycle Checkpoints / Q000235:genetics; D006367:HeLa Cells; D045565:Holliday Junction Resolvases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008957:Models, Genetic; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D034622:RNA Interference; D045522:Recombinases / Q000235:genetics / Q000378:metabolism*; D012854:Sister Chromatid Exchange; D013379:Substrate Specificity
D045566:DNA, Cruciform; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D045522:Recombinases; C507947:Eme1 protein, human; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C498772:MUS81 protein, human; C542320:SLX1 protein, human; C542319:SLX4 protein, human; C533748:GEN1 protein, human; D045565:Holliday Junction Resolvases