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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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Mini-SLX4 Specifically Enhances XPF-ERCC1 Activity toward Y-Structured DNA(A–C) XE and SXE (5 nM) were reacted with different radiolabeled DNA substrates (∼1.5 pM), over a time course (A, long stem-loop; B, bubble; C, fork-structured DNA [Y11]). Substrates had identical primary sequence around the ss/ds bifurcation (depicted in red). The reaction products were separated by 12% denaturing PAGE gel (top panel), and the decay of the substrate band (S) was quantified and expressed as a percentage of initial substrate (middle panel). Data were fitted using single exponential decay in order to calculate reaction rates (bottom panel). XE data are plotted in blue; SXE data are plotted in red. SXE shows a modest stimulation of activity compared to XE toward stem-loop and bubble substrates and a pronounced induction of activity toward forked DNA (Y11). Error bars represent SEM.
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fig4: Mini-SLX4 Specifically Enhances XPF-ERCC1 Activity toward Y-Structured DNA(A–C) XE and SXE (5 nM) were reacted with different radiolabeled DNA substrates (∼1.5 pM), over a time course (A, long stem-loop; B, bubble; C, fork-structured DNA [Y11]). Substrates had identical primary sequence around the ss/ds bifurcation (depicted in red). The reaction products were separated by 12% denaturing PAGE gel (top panel), and the decay of the substrate band (S) was quantified and expressed as a percentage of initial substrate (middle panel). Data were fitted using single exponential decay in order to calculate reaction rates (bottom panel). XE data are plotted in blue; SXE data are plotted in red. SXE shows a modest stimulation of activity compared to XE toward stem-loop and bubble substrates and a pronounced induction of activity toward forked DNA (Y11). Error bars represent SEM.

Mentions: Our qualitative analysis had revealed an effect of mini-SLX4 on XPF-ERCC1 activity toward specific DNA structures. We next sought to test this definitively, assessing the reaction kinetics with an excess of enzyme and divalent metal (Mg2+) over substrate. We designed three substrates of identical length and sequence at the ss/ds junction (Figures 4 and S3B). The first two substrates (long stem loop and bubble) were similar to those used to characterize nuclease biochemistry in NER pathways (Enzlin and Schärer, 2002; Evans et al., 1997). Comparison of XE and SXE activities toward this stem loop revealed a modest rate enhancement of SXE (3.7-fold) (Figure 4A). These data differ from those for the short stem-loop substrate previously described, in which we observed marginally less activity of the SXE complex (Figures 3C and S4). The length of the duplex (and a possible contribution of DNA sequence; Bowles et al., 2012) could explain this difference. Next, we assessed the activity of the nuclease complexes toward bubble NER-like substrates (Figure 4B). There was a striking concordance for rates of catalysis of the bubble substrate with data for the stem loop. SXE displayed a similarly modest 3.5-fold induction in catalytic rate compared to XE. However, when we assayed fork-structured DNA, we observed a greater difference between the enzyme complexes (Figure 4C). XE processed the fork substrate (Y11) with similar efficiency to the loop and bubble substrates (half-life 16 min), indicating that in the absence of SLX4 it exhibited very little structural preference (on these substrates). In comparison, the Y11 fork was processed rapidly by SXE (half-life 1 min) compared to XE, a 16-fold increase in catalytic activity. Reaction rates are listed in Table S1. Thus, the structural DNA motif recognized by XE and SXE differs substantially; SLX4 effectively biases XPF-ERCC1 toward processing forked DNA structures.


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)

Mini-SLX4 Specifically Enhances XPF-ERCC1 Activity toward Y-Structured DNA(A–C) XE and SXE (5 nM) were reacted with different radiolabeled DNA substrates (∼1.5 pM), over a time course (A, long stem-loop; B, bubble; C, fork-structured DNA [Y11]). Substrates had identical primary sequence around the ss/ds bifurcation (depicted in red). The reaction products were separated by 12% denaturing PAGE gel (top panel), and the decay of the substrate band (S) was quantified and expressed as a percentage of initial substrate (middle panel). Data were fitted using single exponential decay in order to calculate reaction rates (bottom panel). XE data are plotted in blue; SXE data are plotted in red. SXE shows a modest stimulation of activity compared to XE toward stem-loop and bubble substrates and a pronounced induction of activity toward forked DNA (Y11). Error bars represent SEM.
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Related In: Results  -  Collection

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fig4: Mini-SLX4 Specifically Enhances XPF-ERCC1 Activity toward Y-Structured DNA(A–C) XE and SXE (5 nM) were reacted with different radiolabeled DNA substrates (∼1.5 pM), over a time course (A, long stem-loop; B, bubble; C, fork-structured DNA [Y11]). Substrates had identical primary sequence around the ss/ds bifurcation (depicted in red). The reaction products were separated by 12% denaturing PAGE gel (top panel), and the decay of the substrate band (S) was quantified and expressed as a percentage of initial substrate (middle panel). Data were fitted using single exponential decay in order to calculate reaction rates (bottom panel). XE data are plotted in blue; SXE data are plotted in red. SXE shows a modest stimulation of activity compared to XE toward stem-loop and bubble substrates and a pronounced induction of activity toward forked DNA (Y11). Error bars represent SEM.
Mentions: Our qualitative analysis had revealed an effect of mini-SLX4 on XPF-ERCC1 activity toward specific DNA structures. We next sought to test this definitively, assessing the reaction kinetics with an excess of enzyme and divalent metal (Mg2+) over substrate. We designed three substrates of identical length and sequence at the ss/ds junction (Figures 4 and S3B). The first two substrates (long stem loop and bubble) were similar to those used to characterize nuclease biochemistry in NER pathways (Enzlin and Schärer, 2002; Evans et al., 1997). Comparison of XE and SXE activities toward this stem loop revealed a modest rate enhancement of SXE (3.7-fold) (Figure 4A). These data differ from those for the short stem-loop substrate previously described, in which we observed marginally less activity of the SXE complex (Figures 3C and S4). The length of the duplex (and a possible contribution of DNA sequence; Bowles et al., 2012) could explain this difference. Next, we assessed the activity of the nuclease complexes toward bubble NER-like substrates (Figure 4B). There was a striking concordance for rates of catalysis of the bubble substrate with data for the stem loop. SXE displayed a similarly modest 3.5-fold induction in catalytic rate compared to XE. However, when we assayed fork-structured DNA, we observed a greater difference between the enzyme complexes (Figure 4C). XE processed the fork substrate (Y11) with similar efficiency to the loop and bubble substrates (half-life 16 min), indicating that in the absence of SLX4 it exhibited very little structural preference (on these substrates). In comparison, the Y11 fork was processed rapidly by SXE (half-life 1 min) compared to XE, a 16-fold increase in catalytic activity. Reaction rates are listed in Table S1. Thus, the structural DNA motif recognized by XE and SXE differs substantially; SLX4 effectively biases XPF-ERCC1 toward processing forked DNA structures.

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