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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.

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Related in: MedlinePlus

RPA70 depletion-induced phosphorylation of Chk1. (A) HeLa cells were transfected on three consecutive days with control GL2 or siRNA targeting two different regions of RPA70 (RPA70(1), RPA70(2)) in combination with CHK1 siRNA as indicated. Level of RPA70 was evaluated by immunoblotting with two different antibodies (Ab1 and Ab2). Phosphorylation of Chk1 at Ser345 and Ser317 was assessed by specific antibodies. LC refers to the loading control, a non-specific band that displays equal protein load in different lanes. The numbers indicate levels of phosphorylated-Chk1 (P-Chk1) relative to RPA70(1) siRNA transfected cells and normalized with the protein loading control. (B) HeLa cells were transfected with control GL2 or RPA70 siRNA and the level of RPA70 mRNA was quantified. The numbers indicate the RPA70 mRNA levels following RPA70 siRNA depletion relative to control GL2 transfected cells. β-2 microglobulin (BMG) serves as the internal RNA loading control. (C) RPA70 deficiency-induced Chk1 activation is dependent on active DNA replication. GL2 or RPA70 siRNA was transfected either in asynchronous cells (Asn) or cells blocked in G1-phase with mevastatin treatment. After transfection on two consecutive days, the cells were either harvested in the presence of mevastatin (Mev-0 h) or 8 h after removal of mevastatin (Mev-8 h) and the levels of RPA70 and phosphorylated-Chk1 were assayed. The numbers indicate phosphorylated-Chk1 levels after different treatments relative to asynchronous RPA70 siRNA transfected cells. (D) Flow cytometry of propidium iodide stained DNA from HeLa cells demonstrates that RPA70 depletion induces S-phase accumulation, which is prevented in the presence of mevastatin.
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Figure 1: RPA70 depletion-induced phosphorylation of Chk1. (A) HeLa cells were transfected on three consecutive days with control GL2 or siRNA targeting two different regions of RPA70 (RPA70(1), RPA70(2)) in combination with CHK1 siRNA as indicated. Level of RPA70 was evaluated by immunoblotting with two different antibodies (Ab1 and Ab2). Phosphorylation of Chk1 at Ser345 and Ser317 was assessed by specific antibodies. LC refers to the loading control, a non-specific band that displays equal protein load in different lanes. The numbers indicate levels of phosphorylated-Chk1 (P-Chk1) relative to RPA70(1) siRNA transfected cells and normalized with the protein loading control. (B) HeLa cells were transfected with control GL2 or RPA70 siRNA and the level of RPA70 mRNA was quantified. The numbers indicate the RPA70 mRNA levels following RPA70 siRNA depletion relative to control GL2 transfected cells. β-2 microglobulin (BMG) serves as the internal RNA loading control. (C) RPA70 deficiency-induced Chk1 activation is dependent on active DNA replication. GL2 or RPA70 siRNA was transfected either in asynchronous cells (Asn) or cells blocked in G1-phase with mevastatin treatment. After transfection on two consecutive days, the cells were either harvested in the presence of mevastatin (Mev-0 h) or 8 h after removal of mevastatin (Mev-8 h) and the levels of RPA70 and phosphorylated-Chk1 were assayed. The numbers indicate phosphorylated-Chk1 levels after different treatments relative to asynchronous RPA70 siRNA transfected cells. (D) Flow cytometry of propidium iodide stained DNA from HeLa cells demonstrates that RPA70 depletion induces S-phase accumulation, which is prevented in the presence of mevastatin.

Mentions: Depletion of RPA70 in asynchronous cells resulted in phosphorylation of Chk1 at Ser345 as well as Ser317, both of which are known to be mediated by ATR (Figure 1A and B) (3). Different siRNA duplexes against RPA70 ((RPA70(1) and RPA70(2)) led to phosphorylation of Chk1 at Ser345 and Ser317, ruling out off-target effects and co-depletion of Chk1 authenticated the phosphorylated-Chk1 band on immunoblots. Moreover, co-expression of RNAi resistant RPA70 suppressed the phosphorylation of Chk1 (Supplementary Figure S1C). Apart from HeLa cells, RPA70 depletion in cell lines of different lineages resulted in Chk1 phosphorylation, which was also confirmed using multiple antibodies (Supplementary Figure S1A and B). RPA70 depletion leads to S-phase accumulation and in order to verify whether RPA70 deficiency-induced Chk1 phosphorylation occurs during the S-phase, we utilized mevastatin to block the cells in G1-phase and then carried out RPA70 siRNA transfection (Figure 1C) (31). We observed that Chk1 phosphorylation was significantly decreased in G1 blocked RPA70-depleted cells (Figure 1D and Supplementary Figure S5). It is known that cells released from G1 block caused by statins progress slowly into the cell cycle and we observed that as a subpopulation of RPA70-depleted cells entered S-phase 8 h after the removal of mevastatin, Chk1 was phosphorylated (32). Therefore, the RPA depletion-induced Chk1 phosphorylation occurs during DNA replication, presumably because of replication fork stalling (Supplementary Figure S1D) (15,32). It has been reported that depletion of a second human SSB, hSSB1, abrogates the DSB response, so we assayed if it mediates ATR activation (23). We observed that hSSB1 formed punctate foci in RPA70-depleted cells similar to what has been reported after gamma-irradiation, indicating the localization of hSSB1 at the sites of genomic stress (Figure 2A and C and Supplementary Figure S2A). The foci were absent after co-depletion of hSSB1 along with RPA70, confirming that the observed immunofluorescence signal was from hSSB1. HSSB1 is part of the single-stranded DNA-binding complex, wherein its partner protein, INTS3 serves as a central adaptor required for assembly of the complex and recruitment of hSSB1 to the sites of DNA damage (26,27). We observed that after RPA70 depletion INTS3 also formed punctate foci, similar to hSSB1 (Figure 2B and C and Supplementary Figure S2A and B). Therefore, we demonstrate that alternate single-strand binding protein complex, hSSB1-INTS3, forms punctate foci after RPA depletion.


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)

RPA70 depletion-induced phosphorylation of Chk1. (A) HeLa cells were transfected on three consecutive days with control GL2 or siRNA targeting two different regions of RPA70 (RPA70(1), RPA70(2)) in combination with CHK1 siRNA as indicated. Level of RPA70 was evaluated by immunoblotting with two different antibodies (Ab1 and Ab2). Phosphorylation of Chk1 at Ser345 and Ser317 was assessed by specific antibodies. LC refers to the loading control, a non-specific band that displays equal protein load in different lanes. The numbers indicate levels of phosphorylated-Chk1 (P-Chk1) relative to RPA70(1) siRNA transfected cells and normalized with the protein loading control. (B) HeLa cells were transfected with control GL2 or RPA70 siRNA and the level of RPA70 mRNA was quantified. The numbers indicate the RPA70 mRNA levels following RPA70 siRNA depletion relative to control GL2 transfected cells. β-2 microglobulin (BMG) serves as the internal RNA loading control. (C) RPA70 deficiency-induced Chk1 activation is dependent on active DNA replication. GL2 or RPA70 siRNA was transfected either in asynchronous cells (Asn) or cells blocked in G1-phase with mevastatin treatment. After transfection on two consecutive days, the cells were either harvested in the presence of mevastatin (Mev-0 h) or 8 h after removal of mevastatin (Mev-8 h) and the levels of RPA70 and phosphorylated-Chk1 were assayed. The numbers indicate phosphorylated-Chk1 levels after different treatments relative to asynchronous RPA70 siRNA transfected cells. (D) Flow cytometry of propidium iodide stained DNA from HeLa cells demonstrates that RPA70 depletion induces S-phase accumulation, which is prevented in the presence of mevastatin.
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Figure 1: RPA70 depletion-induced phosphorylation of Chk1. (A) HeLa cells were transfected on three consecutive days with control GL2 or siRNA targeting two different regions of RPA70 (RPA70(1), RPA70(2)) in combination with CHK1 siRNA as indicated. Level of RPA70 was evaluated by immunoblotting with two different antibodies (Ab1 and Ab2). Phosphorylation of Chk1 at Ser345 and Ser317 was assessed by specific antibodies. LC refers to the loading control, a non-specific band that displays equal protein load in different lanes. The numbers indicate levels of phosphorylated-Chk1 (P-Chk1) relative to RPA70(1) siRNA transfected cells and normalized with the protein loading control. (B) HeLa cells were transfected with control GL2 or RPA70 siRNA and the level of RPA70 mRNA was quantified. The numbers indicate the RPA70 mRNA levels following RPA70 siRNA depletion relative to control GL2 transfected cells. β-2 microglobulin (BMG) serves as the internal RNA loading control. (C) RPA70 deficiency-induced Chk1 activation is dependent on active DNA replication. GL2 or RPA70 siRNA was transfected either in asynchronous cells (Asn) or cells blocked in G1-phase with mevastatin treatment. After transfection on two consecutive days, the cells were either harvested in the presence of mevastatin (Mev-0 h) or 8 h after removal of mevastatin (Mev-8 h) and the levels of RPA70 and phosphorylated-Chk1 were assayed. The numbers indicate phosphorylated-Chk1 levels after different treatments relative to asynchronous RPA70 siRNA transfected cells. (D) Flow cytometry of propidium iodide stained DNA from HeLa cells demonstrates that RPA70 depletion induces S-phase accumulation, which is prevented in the presence of mevastatin.
Mentions: Depletion of RPA70 in asynchronous cells resulted in phosphorylation of Chk1 at Ser345 as well as Ser317, both of which are known to be mediated by ATR (Figure 1A and B) (3). Different siRNA duplexes against RPA70 ((RPA70(1) and RPA70(2)) led to phosphorylation of Chk1 at Ser345 and Ser317, ruling out off-target effects and co-depletion of Chk1 authenticated the phosphorylated-Chk1 band on immunoblots. Moreover, co-expression of RNAi resistant RPA70 suppressed the phosphorylation of Chk1 (Supplementary Figure S1C). Apart from HeLa cells, RPA70 depletion in cell lines of different lineages resulted in Chk1 phosphorylation, which was also confirmed using multiple antibodies (Supplementary Figure S1A and B). RPA70 depletion leads to S-phase accumulation and in order to verify whether RPA70 deficiency-induced Chk1 phosphorylation occurs during the S-phase, we utilized mevastatin to block the cells in G1-phase and then carried out RPA70 siRNA transfection (Figure 1C) (31). We observed that Chk1 phosphorylation was significantly decreased in G1 blocked RPA70-depleted cells (Figure 1D and Supplementary Figure S5). It is known that cells released from G1 block caused by statins progress slowly into the cell cycle and we observed that as a subpopulation of RPA70-depleted cells entered S-phase 8 h after the removal of mevastatin, Chk1 was phosphorylated (32). Therefore, the RPA depletion-induced Chk1 phosphorylation occurs during DNA replication, presumably because of replication fork stalling (Supplementary Figure S1D) (15,32). It has been reported that depletion of a second human SSB, hSSB1, abrogates the DSB response, so we assayed if it mediates ATR activation (23). We observed that hSSB1 formed punctate foci in RPA70-depleted cells similar to what has been reported after gamma-irradiation, indicating the localization of hSSB1 at the sites of genomic stress (Figure 2A and C and Supplementary Figure S2A). The foci were absent after co-depletion of hSSB1 along with RPA70, confirming that the observed immunofluorescence signal was from hSSB1. HSSB1 is part of the single-stranded DNA-binding complex, wherein its partner protein, INTS3 serves as a central adaptor required for assembly of the complex and recruitment of hSSB1 to the sites of DNA damage (26,27). We observed that after RPA70 depletion INTS3 also formed punctate foci, similar to hSSB1 (Figure 2B and C and Supplementary Figure S2A and B). Therefore, we demonstrate that alternate single-strand binding protein complex, hSSB1-INTS3, forms punctate foci after RPA depletion.

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