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Mouse SLX4 is a tumor suppressor that stimulates the activity of the nuclease XPF-ERCC1 in DNA crosslink repair.

Hodskinson MR, Silhan J, Crossan GP, Garaycoechea JI, Mukherjee S, Johnson CM, Schärer OD, Patel KJ - Mol. Cell (2014)

Bottom Line: Slx4-deficient mice develop epithelial cancers and have a contracted hematopoietic stem cell pool.The N-terminal domain of SLX4 (mini-SLX4) that only binds to XPF-ERCC1 is sufficient to confer resistance to DNA crosslinking agents.Recombinant mini-SLX4 enhances XPF-ERCC1 nuclease activity up to 100-fold, directing specificity toward DNA forks.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.

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Related in: MedlinePlus

SLX4 1-758 Partially Complements Crosslinker Sensitivity and Can Be Purified in a Complex with XPF-ERCC1(A) Cartoon depicts the SLX4 polypeptide (1–1565), domains, and interactions with the three nucleases: XPF-ERCC1, MUS81-EME1, and SLX1. A truncated SLX4 1-758 (mini-SLX4) contains the region that interacts with XPF-ERCC1.(B) Full-length FLAG-tagged SLX4 or FLAG-tagged mini-SLX4 was expressed in Slx4f3/f3 MEFs. Anti-FLAG immunoprecipitation shows that the ectopically expressed SLX4 polypeptides can be copurified with XPF-ERCC1. Note: ectopically expressed full-length SLX4 is prone to degradation/aggregation, accounting for the three bands seen by western blot (WB).(C) MTS viability of Slx4f3/f3 MEFs stably expressing full-length FLAG-SLX4 or FLAG-mini-SLX4, exposed to varying doses of Mitomycin C (MMC) for 4 days.(D) Expression and purification of a recombinant mini-SLX4 (1–758) in complex with XPF-ERCC1 (SXE) from insect cells. The purification scheme is shown next to a Coomassie gel depicting the various stages of purification.(E) Analytical gel filtration and Coomassie gels of purified mini-SLX4, XPF-ERCC1 (XE), and SXE complexes. Italicized letters correspond to the proteins shown on the Commassie gel. The shaded box represents those fractions loaded on SDS gels (below). A red arrow denotes the column void volume (∼2 MDa). Error bars represent SEM.
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fig2: SLX4 1-758 Partially Complements Crosslinker Sensitivity and Can Be Purified in a Complex with XPF-ERCC1(A) Cartoon depicts the SLX4 polypeptide (1–1565), domains, and interactions with the three nucleases: XPF-ERCC1, MUS81-EME1, and SLX1. A truncated SLX4 1-758 (mini-SLX4) contains the region that interacts with XPF-ERCC1.(B) Full-length FLAG-tagged SLX4 or FLAG-tagged mini-SLX4 was expressed in Slx4f3/f3 MEFs. Anti-FLAG immunoprecipitation shows that the ectopically expressed SLX4 polypeptides can be copurified with XPF-ERCC1. Note: ectopically expressed full-length SLX4 is prone to degradation/aggregation, accounting for the three bands seen by western blot (WB).(C) MTS viability of Slx4f3/f3 MEFs stably expressing full-length FLAG-SLX4 or FLAG-mini-SLX4, exposed to varying doses of Mitomycin C (MMC) for 4 days.(D) Expression and purification of a recombinant mini-SLX4 (1–758) in complex with XPF-ERCC1 (SXE) from insect cells. The purification scheme is shown next to a Coomassie gel depicting the various stages of purification.(E) Analytical gel filtration and Coomassie gels of purified mini-SLX4, XPF-ERCC1 (XE), and SXE complexes. Italicized letters correspond to the proteins shown on the Commassie gel. The shaded box represents those fractions loaded on SDS gels (below). A red arrow denotes the column void volume (∼2 MDa). Error bars represent SEM.

Mentions: As already mentioned, transformed MEF cells lines obtained from Slx4f3/f3 embryos were hypersensitive to ICL agents, such as Mitomycin C (MMC). The introduction of a full-length Slx4 transgene into these cells can complement this key phenotypic feature. This simple, cell-intrinsic DNA repair defect provides a system for functional dissection of the SLX4 polypeptide. SLX4 is a large 1565 amino acid polypeptide that serves as a binding platform for three nucleases (Figure 2A). An N-terminal MLR domain mediates the interaction with XPF-ERCC1, whereas MUS81-EME1 and SLX1 bind through regions mapping near the C terminus of SLX4 (Fekairi et al., 2009; Kim et al., 2013; Svendsen et al., 2009). Additionally, SLX4 possess two N-terminal UBZ domains and a central BTB/POZ protein dimerization/interaction domain. We created a truncation of mouse SLX4 (SLX4 1-758: mini-SLX4) that includes the XPF-ERCC1 binding region (MLR) and ectopically expressed this in Slx4f3/f3-deficient MEFs. Mini-SLX4 binds to endogenous XPF-ERCC1 as efficiently as the full-length SLX4 polypeptide (Figure 2B). This mini-SLX4 also significantly complements resistance to MMC (Figure 2C) (LD50 values: Slx4f3/f3 4 ng/ml, Mini-SLX4 23 ng/ml, and full-length SLX4 80 ng/ml).


Mouse SLX4 is a tumor suppressor that stimulates the activity of the nuclease XPF-ERCC1 in DNA crosslink repair.

Hodskinson MR, Silhan J, Crossan GP, Garaycoechea JI, Mukherjee S, Johnson CM, Schärer OD, Patel KJ - Mol. Cell (2014)

SLX4 1-758 Partially Complements Crosslinker Sensitivity and Can Be Purified in a Complex with XPF-ERCC1(A) Cartoon depicts the SLX4 polypeptide (1–1565), domains, and interactions with the three nucleases: XPF-ERCC1, MUS81-EME1, and SLX1. A truncated SLX4 1-758 (mini-SLX4) contains the region that interacts with XPF-ERCC1.(B) Full-length FLAG-tagged SLX4 or FLAG-tagged mini-SLX4 was expressed in Slx4f3/f3 MEFs. Anti-FLAG immunoprecipitation shows that the ectopically expressed SLX4 polypeptides can be copurified with XPF-ERCC1. Note: ectopically expressed full-length SLX4 is prone to degradation/aggregation, accounting for the three bands seen by western blot (WB).(C) MTS viability of Slx4f3/f3 MEFs stably expressing full-length FLAG-SLX4 or FLAG-mini-SLX4, exposed to varying doses of Mitomycin C (MMC) for 4 days.(D) Expression and purification of a recombinant mini-SLX4 (1–758) in complex with XPF-ERCC1 (SXE) from insect cells. The purification scheme is shown next to a Coomassie gel depicting the various stages of purification.(E) Analytical gel filtration and Coomassie gels of purified mini-SLX4, XPF-ERCC1 (XE), and SXE complexes. Italicized letters correspond to the proteins shown on the Commassie gel. The shaded box represents those fractions loaded on SDS gels (below). A red arrow denotes the column void volume (∼2 MDa). Error bars represent SEM.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4017094&req=5

fig2: SLX4 1-758 Partially Complements Crosslinker Sensitivity and Can Be Purified in a Complex with XPF-ERCC1(A) Cartoon depicts the SLX4 polypeptide (1–1565), domains, and interactions with the three nucleases: XPF-ERCC1, MUS81-EME1, and SLX1. A truncated SLX4 1-758 (mini-SLX4) contains the region that interacts with XPF-ERCC1.(B) Full-length FLAG-tagged SLX4 or FLAG-tagged mini-SLX4 was expressed in Slx4f3/f3 MEFs. Anti-FLAG immunoprecipitation shows that the ectopically expressed SLX4 polypeptides can be copurified with XPF-ERCC1. Note: ectopically expressed full-length SLX4 is prone to degradation/aggregation, accounting for the three bands seen by western blot (WB).(C) MTS viability of Slx4f3/f3 MEFs stably expressing full-length FLAG-SLX4 or FLAG-mini-SLX4, exposed to varying doses of Mitomycin C (MMC) for 4 days.(D) Expression and purification of a recombinant mini-SLX4 (1–758) in complex with XPF-ERCC1 (SXE) from insect cells. The purification scheme is shown next to a Coomassie gel depicting the various stages of purification.(E) Analytical gel filtration and Coomassie gels of purified mini-SLX4, XPF-ERCC1 (XE), and SXE complexes. Italicized letters correspond to the proteins shown on the Commassie gel. The shaded box represents those fractions loaded on SDS gels (below). A red arrow denotes the column void volume (∼2 MDa). Error bars represent SEM.
Mentions: As already mentioned, transformed MEF cells lines obtained from Slx4f3/f3 embryos were hypersensitive to ICL agents, such as Mitomycin C (MMC). The introduction of a full-length Slx4 transgene into these cells can complement this key phenotypic feature. This simple, cell-intrinsic DNA repair defect provides a system for functional dissection of the SLX4 polypeptide. SLX4 is a large 1565 amino acid polypeptide that serves as a binding platform for three nucleases (Figure 2A). An N-terminal MLR domain mediates the interaction with XPF-ERCC1, whereas MUS81-EME1 and SLX1 bind through regions mapping near the C terminus of SLX4 (Fekairi et al., 2009; Kim et al., 2013; Svendsen et al., 2009). Additionally, SLX4 possess two N-terminal UBZ domains and a central BTB/POZ protein dimerization/interaction domain. We created a truncation of mouse SLX4 (SLX4 1-758: mini-SLX4) that includes the XPF-ERCC1 binding region (MLR) and ectopically expressed this in Slx4f3/f3-deficient MEFs. Mini-SLX4 binds to endogenous XPF-ERCC1 as efficiently as the full-length SLX4 polypeptide (Figure 2B). This mini-SLX4 also significantly complements resistance to MMC (Figure 2C) (LD50 values: Slx4f3/f3 4 ng/ml, Mini-SLX4 23 ng/ml, and full-length SLX4 80 ng/ml).

Bottom Line: Slx4-deficient mice develop epithelial cancers and have a contracted hematopoietic stem cell pool.The N-terminal domain of SLX4 (mini-SLX4) that only binds to XPF-ERCC1 is sufficient to confer resistance to DNA crosslinking agents.Recombinant mini-SLX4 enhances XPF-ERCC1 nuclease activity up to 100-fold, directing specificity toward DNA forks.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.

Show MeSH
Related in: MedlinePlus