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SLX4 contributes to telomere preservation and regulated processing of telomeric joint molecule intermediates.

Sarkar J, Wan B, Yin J, Vallabhaneni H, Horvath K, Kulikowicz T, Bohr VA, Zhang Y, Lei M, Liu Y - Nucleic Acids Res. (2015)

Bottom Line: The nucleolytic activity of SLX1-SLX4 is negatively regulated by telomeric DNA-binding proteins TRF1 and TRF2 and is suppressed by the RecQ helicase BLM in vitro.In vivo, in the presence of functional BLM, telomeric circle formation and telomere sister chromatid exchange, both arising out of nucleolytic processing of telomeric homologous recombination intermediates, are suppressed.We propose that the SLX4-toolkit is a telomere accessory complex that, in conjunction with other telomere maintenance proteins, ensures unhindered, but regulated telomere maintenance.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Gerontology, National Institute on Aging/National Institute of Health, 251 Bayview Blvd, Baltimore, MD 21224, USA.

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Telomeric DNA-binding proteins limit SLX1–SLX4-sponsored processing of telomeric branched DNA forms in vitro. (A–D) Telomeric D-loop and (E–H) telomeric HJ are protected from nucleolytic cleavage by the purified SLX1WT − SLX4SBR complex in vitro. Representative native gel images (A, E) and quantification of cleaved products (B, C and F, G) and uncleaved substrate (D, H) in the presence of increasing concentrations (0–60 nM) of TRF2 or TRF1. The substrate (0.5 nM) was pre-bound to TRF2 or TRF1 on ice for 5 min, followed by addition of 0.5-nM purified SLX1WT − SLX4SBR complex. S, substrate (D-loop or HJ); P1 and P2, products of HJ cleavage; P3, P4 and P5, products of D-loop cleavage; Total, total cleaved products formed due to substrate (D-loop or HJ) cleavage.
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Figure 4: Telomeric DNA-binding proteins limit SLX1–SLX4-sponsored processing of telomeric branched DNA forms in vitro. (A–D) Telomeric D-loop and (E–H) telomeric HJ are protected from nucleolytic cleavage by the purified SLX1WT − SLX4SBR complex in vitro. Representative native gel images (A, E) and quantification of cleaved products (B, C and F, G) and uncleaved substrate (D, H) in the presence of increasing concentrations (0–60 nM) of TRF2 or TRF1. The substrate (0.5 nM) was pre-bound to TRF2 or TRF1 on ice for 5 min, followed by addition of 0.5-nM purified SLX1WT − SLX4SBR complex. S, substrate (D-loop or HJ); P1 and P2, products of HJ cleavage; P3, P4 and P5, products of D-loop cleavage; Total, total cleaved products formed due to substrate (D-loop or HJ) cleavage.

Mentions: We then pre-bound the telomeric substrates (D-loop or HJ) with increasing concentrations (0–60 nM) of either TRF1 or TRF2, followed by nuclease cleavage reactions with catalytic amounts (0.5 nM) of purified SLX1WT − SLX4SBR. We analyzed the extent of TRF2 or TRF1 protection by separately quantifying amount of all product bands formed and also the amount of uncleaved substrate on a native gel (Figure 4A and E). The fraction of cleaved products or uncleaved substrate did not show significant changes at low concentrations of TRF2 or TRF1 [1:1 or 1:2 molar ratios of (SLX1WT − SLX4SBR:TRF2 or TRF1)], both for the telomeric D-loop (Figure 4A–D) and the HJ (Figure 4E–H). However, at higher concentrations of TRF2 or TRF1 [1:10 or higher molar ratios of (SLX1WT − SLX4SBR:TRF2 or TRF1)], protection of telomeric substrates by TRF2 or TRF1 (both in terms of cleaved products and uncleaved substrate) reached as high as 60–90% (Figure 4A–D for D-loop and Figure 4E–H for HJ). Thus, at higher concentrations, both TRF2 and TRF1 limit the extent of SLX1WT − SLX4SBR nuclease activity on telomeric substrates in vitro, implying that the extent of substrate protection offered by TRF2/TRF1 may depend on the amount of these proteins bound to telomeres at any given time. Because the purified SLX1WT − SLX4SBR complex lacked the TRF2-binding domain (TBM) of SLX4 (Figure 3B) (12), we wondered if the SLX4–TRF2 interaction would impact the observed trend of TRF2/TRF1 protection on nucleolytic processing of telomeric substrates. To address this possibility, we exogenously expressed both wild-type and full-length SLX1 and SLX4, co-immunoprecipitated the complex (SLX1WT + SLX4WT) and used it to perform similar TRF2/TRF1 protection experiments as described above. Higher concentrations of pre-bound TRF2 (or TRF1, data not shown) resulted in substrate protection and inhibition of cleavage of both telomeric HJ and D-loop by the immunoprecipitated (SLX1WT + SLX4WT) complex (Supplementary Figure S5B, HJ, lanes 1–6; D-loop, lanes 7–12), consistent with results from experiments with purified SLX1WT − SLX4SBR. Thus, telomeric substrate protection rendered by pre-bound TRF2/TRF1 is unlikely to be affected by the SLX4–TRF2 interaction.


SLX4 contributes to telomere preservation and regulated processing of telomeric joint molecule intermediates.

Sarkar J, Wan B, Yin J, Vallabhaneni H, Horvath K, Kulikowicz T, Bohr VA, Zhang Y, Lei M, Liu Y - Nucleic Acids Res. (2015)

Telomeric DNA-binding proteins limit SLX1–SLX4-sponsored processing of telomeric branched DNA forms in vitro. (A–D) Telomeric D-loop and (E–H) telomeric HJ are protected from nucleolytic cleavage by the purified SLX1WT − SLX4SBR complex in vitro. Representative native gel images (A, E) and quantification of cleaved products (B, C and F, G) and uncleaved substrate (D, H) in the presence of increasing concentrations (0–60 nM) of TRF2 or TRF1. The substrate (0.5 nM) was pre-bound to TRF2 or TRF1 on ice for 5 min, followed by addition of 0.5-nM purified SLX1WT − SLX4SBR complex. S, substrate (D-loop or HJ); P1 and P2, products of HJ cleavage; P3, P4 and P5, products of D-loop cleavage; Total, total cleaved products formed due to substrate (D-loop or HJ) cleavage.
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Related In: Results  -  Collection

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Figure 4: Telomeric DNA-binding proteins limit SLX1–SLX4-sponsored processing of telomeric branched DNA forms in vitro. (A–D) Telomeric D-loop and (E–H) telomeric HJ are protected from nucleolytic cleavage by the purified SLX1WT − SLX4SBR complex in vitro. Representative native gel images (A, E) and quantification of cleaved products (B, C and F, G) and uncleaved substrate (D, H) in the presence of increasing concentrations (0–60 nM) of TRF2 or TRF1. The substrate (0.5 nM) was pre-bound to TRF2 or TRF1 on ice for 5 min, followed by addition of 0.5-nM purified SLX1WT − SLX4SBR complex. S, substrate (D-loop or HJ); P1 and P2, products of HJ cleavage; P3, P4 and P5, products of D-loop cleavage; Total, total cleaved products formed due to substrate (D-loop or HJ) cleavage.
Mentions: We then pre-bound the telomeric substrates (D-loop or HJ) with increasing concentrations (0–60 nM) of either TRF1 or TRF2, followed by nuclease cleavage reactions with catalytic amounts (0.5 nM) of purified SLX1WT − SLX4SBR. We analyzed the extent of TRF2 or TRF1 protection by separately quantifying amount of all product bands formed and also the amount of uncleaved substrate on a native gel (Figure 4A and E). The fraction of cleaved products or uncleaved substrate did not show significant changes at low concentrations of TRF2 or TRF1 [1:1 or 1:2 molar ratios of (SLX1WT − SLX4SBR:TRF2 or TRF1)], both for the telomeric D-loop (Figure 4A–D) and the HJ (Figure 4E–H). However, at higher concentrations of TRF2 or TRF1 [1:10 or higher molar ratios of (SLX1WT − SLX4SBR:TRF2 or TRF1)], protection of telomeric substrates by TRF2 or TRF1 (both in terms of cleaved products and uncleaved substrate) reached as high as 60–90% (Figure 4A–D for D-loop and Figure 4E–H for HJ). Thus, at higher concentrations, both TRF2 and TRF1 limit the extent of SLX1WT − SLX4SBR nuclease activity on telomeric substrates in vitro, implying that the extent of substrate protection offered by TRF2/TRF1 may depend on the amount of these proteins bound to telomeres at any given time. Because the purified SLX1WT − SLX4SBR complex lacked the TRF2-binding domain (TBM) of SLX4 (Figure 3B) (12), we wondered if the SLX4–TRF2 interaction would impact the observed trend of TRF2/TRF1 protection on nucleolytic processing of telomeric substrates. To address this possibility, we exogenously expressed both wild-type and full-length SLX1 and SLX4, co-immunoprecipitated the complex (SLX1WT + SLX4WT) and used it to perform similar TRF2/TRF1 protection experiments as described above. Higher concentrations of pre-bound TRF2 (or TRF1, data not shown) resulted in substrate protection and inhibition of cleavage of both telomeric HJ and D-loop by the immunoprecipitated (SLX1WT + SLX4WT) complex (Supplementary Figure S5B, HJ, lanes 1–6; D-loop, lanes 7–12), consistent with results from experiments with purified SLX1WT − SLX4SBR. Thus, telomeric substrate protection rendered by pre-bound TRF2/TRF1 is unlikely to be affected by the SLX4–TRF2 interaction.

Bottom Line: The nucleolytic activity of SLX1-SLX4 is negatively regulated by telomeric DNA-binding proteins TRF1 and TRF2 and is suppressed by the RecQ helicase BLM in vitro.In vivo, in the presence of functional BLM, telomeric circle formation and telomere sister chromatid exchange, both arising out of nucleolytic processing of telomeric homologous recombination intermediates, are suppressed.We propose that the SLX4-toolkit is a telomere accessory complex that, in conjunction with other telomere maintenance proteins, ensures unhindered, but regulated telomere maintenance.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Gerontology, National Institute on Aging/National Institute of Health, 251 Bayview Blvd, Baltimore, MD 21224, USA.

Show MeSH