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

ATR-ATRIP complex associates with hSSB1/2-INTS3. (A and B) 293T cells were co-transfected with plasmids expressing HA-ATR (1–400 aa), HA-ATRIP or a non-specific protein (HA-NS) in combination with Myc-INTS3 or Myc-hSSB1 as indicated followed by immunoprecipitation (IP) with HA antibody. The lysates were normalized for the expression of HA-tagged and Myc-tagged proteins, shown by immunoblotting (IB) in the first and second panels, respectively. The bottom panel displays the co-immunoprecipitated Myc-tagged protein. HA-ATR (hollow arrowhead), HA-ATRIP (black arrowhead) and HA-NS (double arrowhead) have been marked while (*) displays multiple expression products of the non-specific protein (HA-NS). The expression of an endogenous non-specific protein (LC) in different transfected samples as visualized by Ponceau S staining has been shown. Note that lanes 2 and 3 of the bottom panel in part B are separated by an intervening lane to prevent any spill over artifacts. (C and D) 293T cells were co-transfected with plasmids expressing HA-ATR (1–400 aa) or HA-ATRIP in combination with Myc-INTS3 or a non-specific protein (Myc-NS) as indicated followed by immunoprecipitation with Myc antibody. The expression of Myc-tagged and HA-tagged proteins have been shown in the first and second panels respectively while the bottom panel displays the co-immunoprecipitated HA-tagged protein (black arrowhead). Myc-INTS3 and Myc-NS have similar mobility. Note that lanes 1 and 2 of the bottom panel in part D are separated by an intervening lane to prevent any spill over artifacts and other details are same as parts A and B.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: ATR-ATRIP complex associates with hSSB1/2-INTS3. (A and B) 293T cells were co-transfected with plasmids expressing HA-ATR (1–400 aa), HA-ATRIP or a non-specific protein (HA-NS) in combination with Myc-INTS3 or Myc-hSSB1 as indicated followed by immunoprecipitation (IP) with HA antibody. The lysates were normalized for the expression of HA-tagged and Myc-tagged proteins, shown by immunoblotting (IB) in the first and second panels, respectively. The bottom panel displays the co-immunoprecipitated Myc-tagged protein. HA-ATR (hollow arrowhead), HA-ATRIP (black arrowhead) and HA-NS (double arrowhead) have been marked while (*) displays multiple expression products of the non-specific protein (HA-NS). The expression of an endogenous non-specific protein (LC) in different transfected samples as visualized by Ponceau S staining has been shown. Note that lanes 2 and 3 of the bottom panel in part B are separated by an intervening lane to prevent any spill over artifacts. (C and D) 293T cells were co-transfected with plasmids expressing HA-ATR (1–400 aa) or HA-ATRIP in combination with Myc-INTS3 or a non-specific protein (Myc-NS) as indicated followed by immunoprecipitation with Myc antibody. The expression of Myc-tagged and HA-tagged proteins have been shown in the first and second panels respectively while the bottom panel displays the co-immunoprecipitated HA-tagged protein (black arrowhead). Myc-INTS3 and Myc-NS have similar mobility. Note that lanes 1 and 2 of the bottom panel in part D are separated by an intervening lane to prevent any spill over artifacts and other details are same as parts A and B.

Mentions: It is known that RPA physically associates with ATRIP and since we propose that hSSB1/2-INTS3 complex substitutes the function of RPA, we evaluated the physical interaction between hSSB1/2-INTS3 and ATRIP. Since the antibodies against endogenous proteins immunoprecipitated poorly, we expressed the proteins using affinity tags and assayed the physical interactions. It has been previously shown that the C-terminus of ATRIP (aa 508–776) binds to the N- terminus of ATR (aa 1–388) and this interaction is essential not only for ATR recruitment but also for ATRIP localization to the sites of DNA damage (16). Since the expression of full-length ATR was poor, we tested the interaction of hSSB1-INTS3 with this domain of ATR which is known to be vital for binding to ATRIP. We co-expressed HA-ATR or HA-ATRIP with Myc-INTS3 in 293T cells and observed that HA-ATR as well as HA-ATRIP co-immunoprecipitated Myc-INTS3 indicating in vivo interaction between ATR/ATRIP and INTS3 (Figure 5A). We also observed that HA-ATR and HA-ATRIP co-immunoprecipitated Myc-hSSB1 (Figure 5B). To validate the interactions, we carried out reverse immunoprecipitation with anti-Myc antibody: we co-expressed Myc-INTS3 along with HA-ATR or HA-ATRIP and observed that Myc-INTS3 co-immunoprecipitated HA-ATR and HA-ATRIP, authenticating their physical association (Figure 5C and D).


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)

ATR-ATRIP complex associates with hSSB1/2-INTS3. (A and B) 293T cells were co-transfected with plasmids expressing HA-ATR (1–400 aa), HA-ATRIP or a non-specific protein (HA-NS) in combination with Myc-INTS3 or Myc-hSSB1 as indicated followed by immunoprecipitation (IP) with HA antibody. The lysates were normalized for the expression of HA-tagged and Myc-tagged proteins, shown by immunoblotting (IB) in the first and second panels, respectively. The bottom panel displays the co-immunoprecipitated Myc-tagged protein. HA-ATR (hollow arrowhead), HA-ATRIP (black arrowhead) and HA-NS (double arrowhead) have been marked while (*) displays multiple expression products of the non-specific protein (HA-NS). The expression of an endogenous non-specific protein (LC) in different transfected samples as visualized by Ponceau S staining has been shown. Note that lanes 2 and 3 of the bottom panel in part B are separated by an intervening lane to prevent any spill over artifacts. (C and D) 293T cells were co-transfected with plasmids expressing HA-ATR (1–400 aa) or HA-ATRIP in combination with Myc-INTS3 or a non-specific protein (Myc-NS) as indicated followed by immunoprecipitation with Myc antibody. The expression of Myc-tagged and HA-tagged proteins have been shown in the first and second panels respectively while the bottom panel displays the co-immunoprecipitated HA-tagged protein (black arrowhead). Myc-INTS3 and Myc-NS have similar mobility. Note that lanes 1 and 2 of the bottom panel in part D are separated by an intervening lane to prevent any spill over artifacts and other details are same as parts A and B.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: ATR-ATRIP complex associates with hSSB1/2-INTS3. (A and B) 293T cells were co-transfected with plasmids expressing HA-ATR (1–400 aa), HA-ATRIP or a non-specific protein (HA-NS) in combination with Myc-INTS3 or Myc-hSSB1 as indicated followed by immunoprecipitation (IP) with HA antibody. The lysates were normalized for the expression of HA-tagged and Myc-tagged proteins, shown by immunoblotting (IB) in the first and second panels, respectively. The bottom panel displays the co-immunoprecipitated Myc-tagged protein. HA-ATR (hollow arrowhead), HA-ATRIP (black arrowhead) and HA-NS (double arrowhead) have been marked while (*) displays multiple expression products of the non-specific protein (HA-NS). The expression of an endogenous non-specific protein (LC) in different transfected samples as visualized by Ponceau S staining has been shown. Note that lanes 2 and 3 of the bottom panel in part B are separated by an intervening lane to prevent any spill over artifacts. (C and D) 293T cells were co-transfected with plasmids expressing HA-ATR (1–400 aa) or HA-ATRIP in combination with Myc-INTS3 or a non-specific protein (Myc-NS) as indicated followed by immunoprecipitation with Myc antibody. The expression of Myc-tagged and HA-tagged proteins have been shown in the first and second panels respectively while the bottom panel displays the co-immunoprecipitated HA-tagged protein (black arrowhead). Myc-INTS3 and Myc-NS have similar mobility. Note that lanes 1 and 2 of the bottom panel in part D are separated by an intervening lane to prevent any spill over artifacts and other details are same as parts A and B.
Mentions: It is known that RPA physically associates with ATRIP and since we propose that hSSB1/2-INTS3 complex substitutes the function of RPA, we evaluated the physical interaction between hSSB1/2-INTS3 and ATRIP. Since the antibodies against endogenous proteins immunoprecipitated poorly, we expressed the proteins using affinity tags and assayed the physical interactions. It has been previously shown that the C-terminus of ATRIP (aa 508–776) binds to the N- terminus of ATR (aa 1–388) and this interaction is essential not only for ATR recruitment but also for ATRIP localization to the sites of DNA damage (16). Since the expression of full-length ATR was poor, we tested the interaction of hSSB1-INTS3 with this domain of ATR which is known to be vital for binding to ATRIP. We co-expressed HA-ATR or HA-ATRIP with Myc-INTS3 in 293T cells and observed that HA-ATR as well as HA-ATRIP co-immunoprecipitated Myc-INTS3 indicating in vivo interaction between ATR/ATRIP and INTS3 (Figure 5A). We also observed that HA-ATR and HA-ATRIP co-immunoprecipitated Myc-hSSB1 (Figure 5B). To validate the interactions, we carried out reverse immunoprecipitation with anti-Myc antibody: we co-expressed Myc-INTS3 along with HA-ATR or HA-ATRIP and observed that Myc-INTS3 co-immunoprecipitated HA-ATR and HA-ATRIP, authenticating their physical association (Figure 5C and D).

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