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Human ISWI complexes are targeted by SMARCA5 ATPase and SLIDE domains to help resolve lesion-stalled transcription.

Aydin ÖZ, Marteijn JA, Ribeiro-Silva C, Rodríguez López A, Wijgers N, Smeenk G, van Attikum H, Poot RA, Vermeulen W, Lans H - Nucleic Acids Res. (2014)

Bottom Line: Using live cell imaging, we identify a novel function for two distinct mammalian ISWI adenosine triphosphate (ATP)-dependent chromatin remodeling complexes in resolving lesion-stalled transcription.After initial recruitment to UV damage, SMARCA5 re-localizes away from the center of DNA damage, requiring its HAND domain.Our studies support a model in which SMARCA5 targeting to DNA damage-stalled transcription sites is controlled by an ATP-hydrolysis-dependent scanning and proofreading mechanism, highlighting how SWI2/SNF2 chromatin remodelers identify and bind nucleosomes containing damaged DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Medical Genetics Cluster, Cancer Genomics Netherlands, Erasmus MC, Rotterdam, 3015 GE, The Netherlands.

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The ATPase, SLIDE and HAND domains of SMARCA5 regulate its damage recruitment. (A) Schematic representation of SMARCA5 domains (left). The invariant Lysine 211 in the ATPase domain is indicated by a ‘K’. Representative images (right) show the live cell accumulation pattern of wild type (WT), ATPase-dead, HAND, SANT and SLIDE deletions mutants at local UV-C damage in U2OS cells. Scale bar is 5 μm. (B) The SMARCA5 ATPase mutant shows impaired recruitment to DNA damage. Graph of the normalized fluorescence intensity of wild type and ATPase mutant (MT) at the damage center (left) and periphery (right; mean ± standard error of the mean; n > 10 cells). (C) Graphs of the normalized fluorescence intensity of wild type (WT), HAND, SANT and SLIDE deletion mutants (MT) at the damage center (left) and periphery (right; mean ± standard error of the mean; n > 10 cells). Recruitment of the SLIDE domain mutant is impaired, whereas the HAND domain mutant is only recruited to the center of damage. All results were confirmed using independent, duplicate experiments. RF denotes ‘relative fluorescence’.
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Figure 4: The ATPase, SLIDE and HAND domains of SMARCA5 regulate its damage recruitment. (A) Schematic representation of SMARCA5 domains (left). The invariant Lysine 211 in the ATPase domain is indicated by a ‘K’. Representative images (right) show the live cell accumulation pattern of wild type (WT), ATPase-dead, HAND, SANT and SLIDE deletions mutants at local UV-C damage in U2OS cells. Scale bar is 5 μm. (B) The SMARCA5 ATPase mutant shows impaired recruitment to DNA damage. Graph of the normalized fluorescence intensity of wild type and ATPase mutant (MT) at the damage center (left) and periphery (right; mean ± standard error of the mean; n > 10 cells). (C) Graphs of the normalized fluorescence intensity of wild type (WT), HAND, SANT and SLIDE deletion mutants (MT) at the damage center (left) and periphery (right; mean ± standard error of the mean; n > 10 cells). Recruitment of the SLIDE domain mutant is impaired, whereas the HAND domain mutant is only recruited to the center of damage. All results were confirmed using independent, duplicate experiments. RF denotes ‘relative fluorescence’.

Mentions: Next, we determined which SMARCA5 domains are responsible for its damage accumulation and subsequent re-localization. SMARCA5 harbors an ATPase domain at the N-terminus, for ATP hydrolysis, and HAND, SANT and SLIDE domains at the C-terminus (Figure 4A), which were suggested to associate with linker DNA to control nucleosome sliding (52,53). To test involvement of the ATPase domain, we introduced an inactivating mutation by replacing Lys211 in the nucleotide-binding motif with Arg (37). Intriguingly, ATPase dead SMARCA5-GFP did not localize to the center of UV damage and was even depleted from this area (Figure 4A and B). Furthermore, a reduced and delayed recruitment to the periphery of the damage was observed. This suggests that ATP hydrolysis directs SMARCA5 targeting to UV-C-induced DNA damage.


Human ISWI complexes are targeted by SMARCA5 ATPase and SLIDE domains to help resolve lesion-stalled transcription.

Aydin ÖZ, Marteijn JA, Ribeiro-Silva C, Rodríguez López A, Wijgers N, Smeenk G, van Attikum H, Poot RA, Vermeulen W, Lans H - Nucleic Acids Res. (2014)

The ATPase, SLIDE and HAND domains of SMARCA5 regulate its damage recruitment. (A) Schematic representation of SMARCA5 domains (left). The invariant Lysine 211 in the ATPase domain is indicated by a ‘K’. Representative images (right) show the live cell accumulation pattern of wild type (WT), ATPase-dead, HAND, SANT and SLIDE deletions mutants at local UV-C damage in U2OS cells. Scale bar is 5 μm. (B) The SMARCA5 ATPase mutant shows impaired recruitment to DNA damage. Graph of the normalized fluorescence intensity of wild type and ATPase mutant (MT) at the damage center (left) and periphery (right; mean ± standard error of the mean; n > 10 cells). (C) Graphs of the normalized fluorescence intensity of wild type (WT), HAND, SANT and SLIDE deletion mutants (MT) at the damage center (left) and periphery (right; mean ± standard error of the mean; n > 10 cells). Recruitment of the SLIDE domain mutant is impaired, whereas the HAND domain mutant is only recruited to the center of damage. All results were confirmed using independent, duplicate experiments. RF denotes ‘relative fluorescence’.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4117783&req=5

Figure 4: The ATPase, SLIDE and HAND domains of SMARCA5 regulate its damage recruitment. (A) Schematic representation of SMARCA5 domains (left). The invariant Lysine 211 in the ATPase domain is indicated by a ‘K’. Representative images (right) show the live cell accumulation pattern of wild type (WT), ATPase-dead, HAND, SANT and SLIDE deletions mutants at local UV-C damage in U2OS cells. Scale bar is 5 μm. (B) The SMARCA5 ATPase mutant shows impaired recruitment to DNA damage. Graph of the normalized fluorescence intensity of wild type and ATPase mutant (MT) at the damage center (left) and periphery (right; mean ± standard error of the mean; n > 10 cells). (C) Graphs of the normalized fluorescence intensity of wild type (WT), HAND, SANT and SLIDE deletion mutants (MT) at the damage center (left) and periphery (right; mean ± standard error of the mean; n > 10 cells). Recruitment of the SLIDE domain mutant is impaired, whereas the HAND domain mutant is only recruited to the center of damage. All results were confirmed using independent, duplicate experiments. RF denotes ‘relative fluorescence’.
Mentions: Next, we determined which SMARCA5 domains are responsible for its damage accumulation and subsequent re-localization. SMARCA5 harbors an ATPase domain at the N-terminus, for ATP hydrolysis, and HAND, SANT and SLIDE domains at the C-terminus (Figure 4A), which were suggested to associate with linker DNA to control nucleosome sliding (52,53). To test involvement of the ATPase domain, we introduced an inactivating mutation by replacing Lys211 in the nucleotide-binding motif with Arg (37). Intriguingly, ATPase dead SMARCA5-GFP did not localize to the center of UV damage and was even depleted from this area (Figure 4A and B). Furthermore, a reduced and delayed recruitment to the periphery of the damage was observed. This suggests that ATP hydrolysis directs SMARCA5 targeting to UV-C-induced DNA damage.

Bottom Line: Using live cell imaging, we identify a novel function for two distinct mammalian ISWI adenosine triphosphate (ATP)-dependent chromatin remodeling complexes in resolving lesion-stalled transcription.After initial recruitment to UV damage, SMARCA5 re-localizes away from the center of DNA damage, requiring its HAND domain.Our studies support a model in which SMARCA5 targeting to DNA damage-stalled transcription sites is controlled by an ATP-hydrolysis-dependent scanning and proofreading mechanism, highlighting how SWI2/SNF2 chromatin remodelers identify and bind nucleosomes containing damaged DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Medical Genetics Cluster, Cancer Genomics Netherlands, Erasmus MC, Rotterdam, 3015 GE, The Netherlands.

Show MeSH
Related in: MedlinePlus