An open-source platform to quantify subnuclear foci and protein colocalization in response to replication stress

Panichnantakul, Pudchalaluck; Patel, Ayushi; Tse, Elizabeth Y.W.; Wyatt, Haley D.M.

doi:10.1016/j.dnarep.2021.103156

Cited by 8 publications

(9 citation statements)

References 63 publications

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“…Previous studies showed that the SLX1-SLX4 heterodimer is enriched in the nucleus, consistent with its role in DNA repair and recombination [59, 60]. Moreover, over-expressed SLX4 forms subnuclear foci in cells under basal growth conditions, and some of these foci correspond to telomeres [14, 24, 25, 61].…”

Section: Resultsmentioning

confidence: 60%

Comprehensive Interactome Mapping of the DNA Repair Scaffold SLX4 using Proximity Labeling and Affinity Purification

Aprosoff

Dyakov

Cheung

et al. 2022

Preprint

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The DNA repair scaffold SLX4 has pivotal roles in cellular processes that maintain genome stability, most notably homologous recombination. Germline mutations in SLX4 are associated with Fanconi anemia, a disease characterized by chromosome instability and cancer susceptibility. The role of mammalian SLX4 in homologous recombination depends critically on binding and activating structure-selective endonucleases, namely SLX1, MUS81-EME1, and XPF-ERCC1. Increasing evidence indicates that cells rely on distinct SLX4-dependent complexes to remove DNA lesions in specific regions of the genome. Despite our understanding of SLX4 as a scaffold for DNA repair proteins, a detailed repertoire of SLX4 interactors has never been reported. Here, we provide the first comprehensive map of the human SLX4 interactome using proximity-dependent biotin identification (BioID) and affinity purification coupled to mass spectrometry (AP-MS). We identified 237 high-confidence interactors, of which the vast majority represent novel SLX4 binding proteins. Network analysis of these hits revealed pathways with known involvement of SLX4, such as DNA repair, and novel or emerging pathways of interest, including RNA metabolism and chromatin remodeling. In summary, the comprehensive SLX4 interactome we report here provides a deeper understanding of how SLX4 functions in DNA repair while revealing new cellular processes that may involve SLX4.

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Section: Resultsmentioning

confidence: 60%

Comprehensive Interactome Mapping of the DNA Repair Scaffold SLX4 using Proximity Labeling and Affinity Purification

Aprosoff

Dyakov

Cheung

et al. 2022

Preprint

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“…Previous studies showed that the SLX1-SLX4 heterodimer is enriched in the nucleus, consistent with its role in DNA repair and recombination. 53,63 Moreover, overexpressed SLX4 forms subnuclear foci in cells under basal growth conditions, and some of these foci correspond to telomeres. 14,24,25,64 Likewise, BioID2-FLAG-SLX4 and SLX4-FLAG-BioID2 formed numerous discrete foci throughout the nucleus upon tetracycline addition (Figure 1D,E overlap between FLAG-and streptavidin-positive foci when cells were treated with tetracycline and biotin, indicative of live cell biotinylation of the bait and proteins in close proximity of the bait (Figure 1D,E).…”

Section: Expression and Validation Of Slx4 Bioid2 Fusion Proteinsmentioning

confidence: 99%

Comprehensive Interactome Mapping of the DNA Repair Scaffold SLX4 Using Proximity Labeling and Affinity Purification

et al. 2023

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The DNA repair scaffold SLX4 has pivotal roles in cellular processes that maintain genome stability, most notably homologous recombination. Germline mutations in SLX4 are associated with Fanconi anemia, a disease characterized by chromosome instability and cancer susceptibility. The role of mammalian SLX4 in homologous recombination depends critically on binding and activating structure-selective endonucleases, namely SLX1, MUS81-EME1, and XPF-ERCC1. Increasing evidence indicates that cells rely on distinct SLX4-dependent complexes to remove DNA lesions in specific regions of the genome. Despite our understanding of SLX4 as a scaffold for DNA repair proteins, a detailed repertoire of SLX4 interactors has never been reported. Here, we provide a comprehensive map of the human SLX4 interactome using proximity-dependent biotin identification (BioID) and affinity purification coupled to mass spectrometry (AP-MS). We identified 221 unique high-confidence interactors, of which the vast majority represent novel SLX4-binding proteins. Network analysis of these hits revealed pathways with known involvement of SLX4, such as DNA repair, and several emerging pathways of interest, including RNA metabolism and chromatin remodeling. In summary, the comprehensive SLX4 interactome we report here provides a deeper understanding of how SLX4 functions in DNA repair while revealing new cellular processes that may involve SLX4.

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“…Together, these data suggest that monoubiquitylated FANCD2 is not the primary ligand for SLX4 UBZ-1. Nevertheless, there are reports that SLX4 and FANCD2 colocalize in subnuclear foci under certain cellular conditions ( Minocherhomji et al, 2015 ; Panichnantakul et al, 2021 ). Further studies are needed to determine the ubiquitylated ligand(s) that recruit SLX4 to DNA interstrand crosslinks.…”

Section: Slx1-slx4 Has Pivotal Roles In Dna Repair and Genome Stabilitymentioning

confidence: 99%

“…Biochemical fractionation and coimmunoprecipitation experiments revealed that human SLX1-SLX4 is constitutively associated with XPF-ERCC1 throughout the cell cycle, forming the SX complex ( Figure 6 ) ( Wyatt et al, 2017 ; Panichnantakul et al, 2021 ). The SX complex is held together through direct contacts between the SLX4 MLR domain and the XPF N-terminal helicase-like domain ( Fekairi et al, 2009 ; Svendsen et al, 2009 ; Kim et al, 2013 ; Guervilly et al, 2015 ; Hashimoto et al, 2015 ; Zhang et al, 2019 ).…”

Section: Human Slx4 Is the Scaffold For Macromolecular Structure-selective Endonuclease Complexesmentioning

confidence: 99%

“…The majority of interstrand crosslink repair occurs during S-phase, which is the phase of the cell cycle when the SX complex predominates ( Figure 6 ) ( Wyatt et al, 2017 ; Panichnantakul et al, 2021 ). In a cell-free system using Xenopus egg extracts, the interstrand crosslink is recognized when two replication forks converge ( Figure 7 ) ( Raschle et al, 2008 ; Zhang et al, 2015 ).…”

Section: Human Slx4 Is the Scaffold For Macromolecular Structure-selective Endonuclease Complexesmentioning

confidence: 99%

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

et al. 2021

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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.

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An open-source platform to quantify subnuclear foci and protein colocalization in response to replication stress

Cited by 8 publications

References 63 publications

Comprehensive Interactome Mapping of the DNA Repair Scaffold SLX4 using Proximity Labeling and Affinity Purification

Comprehensive Interactome Mapping of the DNA Repair Scaffold SLX4 using Proximity Labeling and Affinity Purification

Comprehensive Interactome Mapping of the DNA Repair Scaffold SLX4 Using Proximity Labeling and Affinity Purification

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

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