Limits...
RPA70 depletion induces hSSB1/2-INTS3 complex to initiate ATR signaling.

Kar A, Kaur M, Ghosh T, Khan MM, Sharma A, Shekhar R, Varshney A, Saxena S - Nucleic Acids Res. (2015)

Bottom Line: Depletion of homologs hSSB1/2 and INTS3 in RPA-deficient cells attenuates Chk1 phosphorylation, indicating that the cells are debilitated in responding to stress.We have identified that TopBP1 and the Rad9-Rad1-Hus1 complex are essential for the alternate mode of ATR activation.In summation, we report that the single-stranded DNA-binding protein complex, hSSB1/2-INTS3 can recruit the checkpoint complex to initiate ATR signaling.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India.

Show MeSH

Related in: MedlinePlus

The single-strand binding protein complex, hSSB1/2-INTS3 recruits ATRIP to single-stranded DNA. (A) Myc-hSSB1, Myc-INTS3, HA-ATRIP and HA-tagged non-specific control protein (HA-NS) were individually expressed in 293T cells, purified by immunoprecipitation with anti-Myc or anti-HA antibodies and were eluted with Myc or HA peptides. For the single-stranded DNA-binding assay, streptavidin-agarose was incubated with non-biotinylated (lane 2) or biotinylated (lane 3) single-stranded DNA followed by incubation with Myc-hSSB1 and Myc-INTS3. Next, HA-ATRIP or HA-NS purified from 293T cells were incubated with streptavidin-agarose bound biotinylated ssDNA either in the absence (lanes 4 and 6) or presence (lanes 5 and 7) of bound Myc-hSSB1 and Myc-INTS3. After washing, the bound proteins were identified by immunoblotting with anti-HA (top panel) and anti-Myc (bottom panel) antibodies. 10% of NS and ATRIP utilized for binding to streptavidin-agarose has been shown in lanes 1 and 8 respectively and specific proteins have been marked by arrowheads. The control protein (NS) did not bind to hSSB1-INTS3 complex, ruling out non-specific association. The numbers indicate relative binding of HA-ATRIP to ssDNA in the absence or presence of Myc-hSSB1 and Myc-INTS3. *P-value was calculated using two-tailed t-test which displays that the ATRIP binding observed in the absence or presence of hSSB1-INTS3 complex is significantly different (*P-value = 0.043). (B) RPA complex is absent in Myc-hSSB1 and Myc-INTS3 immunoprecipitates. Myc-hSSB1, Myc-INTS3, HA-ATRIP and HA-NS proteins expressed in 293T cells and purified by elution with Myc or HA peptides following immunoprecipitation were immunoblotted with anti-RPA70 (top panel) and anti-RPA32 (second panel) antibodies for detecting endogenous RPA70 and RPA32. As reported earlier, RPA complex physically associates with ATRIP but is absent from hSSB1-INTS3 complex. Note the high sensitivity of detection of endogenous RPA70 and RPA32 in 293T cell lysate. HA-ATRIP (hollow arrowhead), HA-NS (black arrow) Myc-INTS3 (black arrowhead) and Myc-hSSB1 (shaded arrowhead) have been marked.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4446429&req=5

Figure 6: The single-strand binding protein complex, hSSB1/2-INTS3 recruits ATRIP to single-stranded DNA. (A) Myc-hSSB1, Myc-INTS3, HA-ATRIP and HA-tagged non-specific control protein (HA-NS) were individually expressed in 293T cells, purified by immunoprecipitation with anti-Myc or anti-HA antibodies and were eluted with Myc or HA peptides. For the single-stranded DNA-binding assay, streptavidin-agarose was incubated with non-biotinylated (lane 2) or biotinylated (lane 3) single-stranded DNA followed by incubation with Myc-hSSB1 and Myc-INTS3. Next, HA-ATRIP or HA-NS purified from 293T cells were incubated with streptavidin-agarose bound biotinylated ssDNA either in the absence (lanes 4 and 6) or presence (lanes 5 and 7) of bound Myc-hSSB1 and Myc-INTS3. After washing, the bound proteins were identified by immunoblotting with anti-HA (top panel) and anti-Myc (bottom panel) antibodies. 10% of NS and ATRIP utilized for binding to streptavidin-agarose has been shown in lanes 1 and 8 respectively and specific proteins have been marked by arrowheads. The control protein (NS) did not bind to hSSB1-INTS3 complex, ruling out non-specific association. The numbers indicate relative binding of HA-ATRIP to ssDNA in the absence or presence of Myc-hSSB1 and Myc-INTS3. *P-value was calculated using two-tailed t-test which displays that the ATRIP binding observed in the absence or presence of hSSB1-INTS3 complex is significantly different (*P-value = 0.043). (B) RPA complex is absent in Myc-hSSB1 and Myc-INTS3 immunoprecipitates. Myc-hSSB1, Myc-INTS3, HA-ATRIP and HA-NS proteins expressed in 293T cells and purified by elution with Myc or HA peptides following immunoprecipitation were immunoblotted with anti-RPA70 (top panel) and anti-RPA32 (second panel) antibodies for detecting endogenous RPA70 and RPA32. As reported earlier, RPA complex physically associates with ATRIP but is absent from hSSB1-INTS3 complex. Note the high sensitivity of detection of endogenous RPA70 and RPA32 in 293T cell lysate. HA-ATRIP (hollow arrowhead), HA-NS (black arrow) Myc-INTS3 (black arrowhead) and Myc-hSSB1 (shaded arrowhead) have been marked.

Mentions: We assayed if hSSB1/2-INTS3 complex can promote the binding of ATRIP to ssDNA in an in vitro assay, which permits for controlled manipulation of proteins and is impervious to the toxicity that may occur due to in vivo depletions. Previous studies have addressed the effect of RPA on ATRIP binding to ssDNA: while one study reported that ATRIP efficiently bound to ssDNA only in the presence of RPA, another study reported that there was no significant difference in ATRIP binding to both naked and RPA-coated ssDNA and it has been proposed that a distinct ATRIP-ssDNA binding mode exists, that does not require RPA (11,35,36). Myc-hSSB1, Myc-INTS3, HA-ATRIP and a non-specific control protein (HA-NS) were purified from 293T cells. Streptavidin-agarose was incubated with biotinylated or non-biotinylated single-stranded DNA followed by incubation with Myc-hSSB1 and Myc-INTS3. Purified hSSB1 and INTS3 bound to streptavidin-agarose if coated with biotinylated ssDNA indicating that hSSB1-INTS3 complex specifically binds to ssDNA (FigureĀ 6A, lane 3, bottom panel). Next, we incubated HA-ATRIP with biotinylated ssDNA either in the absence or presence of the bound hSSB1-INTS3 complex. As reported by other groups, we also observed low affinity ATRIP binding to ssDNA in vitro (lane 6, top panel) (35,36). Binding of ATRIP to ssDNA was enhanced by 2.6 fold if it was coated with hSSB1-INTS3 complex, demonstrating that ATRIP can be recruited to ssDNA by hSSB1-INTS3 complex (compare lanes 6 and 7, top panel). To rule out the possibility that enhanced ATRIP binding to hSSB1-INTS3 coated ssDNA is due to contaminating RPA, the purified proteins were immunoblotted for detecting endogenous RPA70 and RPA32 (Figure 6B). As reported earlier, RPA complex physically associates with ATRIP but is absent in hSSB1 and INTS3 eluates, ruling out the possibility that enhanced ATRIP binding is due to contaminating RPA (35,36).


RPA70 depletion induces hSSB1/2-INTS3 complex to initiate ATR signaling.

Kar A, Kaur M, Ghosh T, Khan MM, Sharma A, Shekhar R, Varshney A, Saxena S - Nucleic Acids Res. (2015)

The single-strand binding protein complex, hSSB1/2-INTS3 recruits ATRIP to single-stranded DNA. (A) Myc-hSSB1, Myc-INTS3, HA-ATRIP and HA-tagged non-specific control protein (HA-NS) were individually expressed in 293T cells, purified by immunoprecipitation with anti-Myc or anti-HA antibodies and were eluted with Myc or HA peptides. For the single-stranded DNA-binding assay, streptavidin-agarose was incubated with non-biotinylated (lane 2) or biotinylated (lane 3) single-stranded DNA followed by incubation with Myc-hSSB1 and Myc-INTS3. Next, HA-ATRIP or HA-NS purified from 293T cells were incubated with streptavidin-agarose bound biotinylated ssDNA either in the absence (lanes 4 and 6) or presence (lanes 5 and 7) of bound Myc-hSSB1 and Myc-INTS3. After washing, the bound proteins were identified by immunoblotting with anti-HA (top panel) and anti-Myc (bottom panel) antibodies. 10% of NS and ATRIP utilized for binding to streptavidin-agarose has been shown in lanes 1 and 8 respectively and specific proteins have been marked by arrowheads. The control protein (NS) did not bind to hSSB1-INTS3 complex, ruling out non-specific association. The numbers indicate relative binding of HA-ATRIP to ssDNA in the absence or presence of Myc-hSSB1 and Myc-INTS3. *P-value was calculated using two-tailed t-test which displays that the ATRIP binding observed in the absence or presence of hSSB1-INTS3 complex is significantly different (*P-value = 0.043). (B) RPA complex is absent in Myc-hSSB1 and Myc-INTS3 immunoprecipitates. Myc-hSSB1, Myc-INTS3, HA-ATRIP and HA-NS proteins expressed in 293T cells and purified by elution with Myc or HA peptides following immunoprecipitation were immunoblotted with anti-RPA70 (top panel) and anti-RPA32 (second panel) antibodies for detecting endogenous RPA70 and RPA32. As reported earlier, RPA complex physically associates with ATRIP but is absent from hSSB1-INTS3 complex. Note the high sensitivity of detection of endogenous RPA70 and RPA32 in 293T cell lysate. HA-ATRIP (hollow arrowhead), HA-NS (black arrow) Myc-INTS3 (black arrowhead) and Myc-hSSB1 (shaded arrowhead) have been marked.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446429&req=5

Figure 6: The single-strand binding protein complex, hSSB1/2-INTS3 recruits ATRIP to single-stranded DNA. (A) Myc-hSSB1, Myc-INTS3, HA-ATRIP and HA-tagged non-specific control protein (HA-NS) were individually expressed in 293T cells, purified by immunoprecipitation with anti-Myc or anti-HA antibodies and were eluted with Myc or HA peptides. For the single-stranded DNA-binding assay, streptavidin-agarose was incubated with non-biotinylated (lane 2) or biotinylated (lane 3) single-stranded DNA followed by incubation with Myc-hSSB1 and Myc-INTS3. Next, HA-ATRIP or HA-NS purified from 293T cells were incubated with streptavidin-agarose bound biotinylated ssDNA either in the absence (lanes 4 and 6) or presence (lanes 5 and 7) of bound Myc-hSSB1 and Myc-INTS3. After washing, the bound proteins were identified by immunoblotting with anti-HA (top panel) and anti-Myc (bottom panel) antibodies. 10% of NS and ATRIP utilized for binding to streptavidin-agarose has been shown in lanes 1 and 8 respectively and specific proteins have been marked by arrowheads. The control protein (NS) did not bind to hSSB1-INTS3 complex, ruling out non-specific association. The numbers indicate relative binding of HA-ATRIP to ssDNA in the absence or presence of Myc-hSSB1 and Myc-INTS3. *P-value was calculated using two-tailed t-test which displays that the ATRIP binding observed in the absence or presence of hSSB1-INTS3 complex is significantly different (*P-value = 0.043). (B) RPA complex is absent in Myc-hSSB1 and Myc-INTS3 immunoprecipitates. Myc-hSSB1, Myc-INTS3, HA-ATRIP and HA-NS proteins expressed in 293T cells and purified by elution with Myc or HA peptides following immunoprecipitation were immunoblotted with anti-RPA70 (top panel) and anti-RPA32 (second panel) antibodies for detecting endogenous RPA70 and RPA32. As reported earlier, RPA complex physically associates with ATRIP but is absent from hSSB1-INTS3 complex. Note the high sensitivity of detection of endogenous RPA70 and RPA32 in 293T cell lysate. HA-ATRIP (hollow arrowhead), HA-NS (black arrow) Myc-INTS3 (black arrowhead) and Myc-hSSB1 (shaded arrowhead) have been marked.
Mentions: We assayed if hSSB1/2-INTS3 complex can promote the binding of ATRIP to ssDNA in an in vitro assay, which permits for controlled manipulation of proteins and is impervious to the toxicity that may occur due to in vivo depletions. Previous studies have addressed the effect of RPA on ATRIP binding to ssDNA: while one study reported that ATRIP efficiently bound to ssDNA only in the presence of RPA, another study reported that there was no significant difference in ATRIP binding to both naked and RPA-coated ssDNA and it has been proposed that a distinct ATRIP-ssDNA binding mode exists, that does not require RPA (11,35,36). Myc-hSSB1, Myc-INTS3, HA-ATRIP and a non-specific control protein (HA-NS) were purified from 293T cells. Streptavidin-agarose was incubated with biotinylated or non-biotinylated single-stranded DNA followed by incubation with Myc-hSSB1 and Myc-INTS3. Purified hSSB1 and INTS3 bound to streptavidin-agarose if coated with biotinylated ssDNA indicating that hSSB1-INTS3 complex specifically binds to ssDNA (FigureĀ 6A, lane 3, bottom panel). Next, we incubated HA-ATRIP with biotinylated ssDNA either in the absence or presence of the bound hSSB1-INTS3 complex. As reported by other groups, we also observed low affinity ATRIP binding to ssDNA in vitro (lane 6, top panel) (35,36). Binding of ATRIP to ssDNA was enhanced by 2.6 fold if it was coated with hSSB1-INTS3 complex, demonstrating that ATRIP can be recruited to ssDNA by hSSB1-INTS3 complex (compare lanes 6 and 7, top panel). To rule out the possibility that enhanced ATRIP binding to hSSB1-INTS3 coated ssDNA is due to contaminating RPA, the purified proteins were immunoblotted for detecting endogenous RPA70 and RPA32 (Figure 6B). As reported earlier, RPA complex physically associates with ATRIP but is absent in hSSB1 and INTS3 eluates, ruling out the possibility that enhanced ATRIP binding is due to contaminating RPA (35,36).

Bottom Line: Depletion of homologs hSSB1/2 and INTS3 in RPA-deficient cells attenuates Chk1 phosphorylation, indicating that the cells are debilitated in responding to stress.We have identified that TopBP1 and the Rad9-Rad1-Hus1 complex are essential for the alternate mode of ATR activation.In summation, we report that the single-stranded DNA-binding protein complex, hSSB1/2-INTS3 can recruit the checkpoint complex to initiate ATR signaling.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India.

Show MeSH
Related in: MedlinePlus