Limits...
HSSB1 and hSSB2 form similar multiprotein complexes that participate in DNA damage response.

Li Y, Bolderson E, Kumar R, Muniandy PA, Xue Y, Richard DJ, Seidman M, Pandita TK, Khanna KK, Wang W - J. Biol. Chem. (2009)

Bottom Line: Here we present evidence that hSSB2, a homolog of hSSB1, plays a role similar to hSSB1 in DNA damage-response pathways.Notably, hSSB1 and hSSB2 form separate complexes with two identical proteins, INTS3 and hSSBIP1 (C9ORF80).Depletion of INTS3 decreased the stability of hSSB1 and hSSBIP1, suggesting that INTS3 may provide a scaffold to allow proper assembly of the hSSB complexes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetics, NIA, National Institutes of Health, NIH Biomedical Research Center, Baltimore, Maryland 21224, USA.

ABSTRACT
hSSB1 (human single strand DNA-binding protein 1) has been shown to participate in homologous recombination (HR)-dependent repair of DNA double strand breaks (DSBs) and ataxia telangiectasia-mutated (ATM)-mediated checkpoint pathways. Here we present evidence that hSSB2, a homolog of hSSB1, plays a role similar to hSSB1 in DNA damage-response pathways. This was evidenced by findings that hSSB2-depleted cells resemble hSSB1-depleted cells in hypersensitivity to DNA-damaging reagents, reduced efficiency in HR-dependent repair of DSBs, and defective ATM-dependent phosphorylation. Notably, hSSB1 and hSSB2 form separate complexes with two identical proteins, INTS3 and hSSBIP1 (C9ORF80). Cells depleted of INTS3 and hSSBIP1 also exhibited hypersensitivity to DNA damage reagents, chromosomal instability, and reduced ATM-dependent phosphorylation. hSSBIP1 was rapidly recruited to laser-induced DSBs, a feature also similar to that reported for hSSB1. Depletion of INTS3 decreased the stability of hSSB1 and hSSBIP1, suggesting that INTS3 may provide a scaffold to allow proper assembly of the hSSB complexes. Thus, our data demonstrate that hSSB1 and hSSB2 form two separate complexes with similar structures, and both are required for efficient HR-dependent repair of DSBs and ATM-dependent signaling pathways.

Show MeSH

Related in: MedlinePlus

Both hSSB1 and hSSB2 complexes are required for efficient ATM-dependent phosphorylation events in response to ionizing radiation. A, immunoblotting shows that phosphorylation of ATM itself and several of its substrates (indicated by circled P) in NFF cells was reduced by depletion of hSSB complex components. Cells were either unexposed or exposed to 6 Gy of ionizing radiation, and cell extracts were immunoblotted with anti-phosphorylation site-specific antibodies for ATM (Ser-1981), NBS1 (Ser-343), Chk1 (Ser-317), and Chk2 (Thr-68). ATM and actin were included as controls for equal protein loading. B–D, graphic representation shows quantification of the results in panel A. The result shown is a representative from two independent experiments that yielded reproducible data.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2749126&req=5

Figure 3: Both hSSB1 and hSSB2 complexes are required for efficient ATM-dependent phosphorylation events in response to ionizing radiation. A, immunoblotting shows that phosphorylation of ATM itself and several of its substrates (indicated by circled P) in NFF cells was reduced by depletion of hSSB complex components. Cells were either unexposed or exposed to 6 Gy of ionizing radiation, and cell extracts were immunoblotted with anti-phosphorylation site-specific antibodies for ATM (Ser-1981), NBS1 (Ser-343), Chk1 (Ser-317), and Chk2 (Thr-68). ATM and actin were included as controls for equal protein loading. B–D, graphic representation shows quantification of the results in panel A. The result shown is a representative from two independent experiments that yielded reproducible data.

Mentions: ATM-dependent phosphorylation of many checkpoint proteins is essential for IR-induced checkpoint activation (28). hSSB1-depleted cells have been shown to have impaired ATM-dependent phosphorylation of its substrates and itself (12) following IR. We found that hSSB2-depleted cells also had reduced ATM autophosphorylation (Ser-1981), as well as basal and IR-induced phosphorylation of substrates NBS1 (Ser-343) and Chk1 (Ser-317) (Fig. 3A, compare lanes 5 and 6 with lanes 1 and 2). The reduced phosphorylation in hSSB2-depleted cells was not as drastic as that in hSSB1-depleted cells in response to IR, with the former cells always displaying a higher level of the phosphorylated protein than the latter: ATM (45% versus 35%) (Fig. 3B), NBS1 (40% versus 26%) (Fig. 3C), Chk1 (66% versus 8%) (Fig. 3D), and Chk2 (109% versus 30%) (Fig. 3E). The results suggest that both hSSB1 and hSSB2 participate in ATM-dependent signaling pathways, but hSSB1 may play a relatively more important role.


HSSB1 and hSSB2 form similar multiprotein complexes that participate in DNA damage response.

Li Y, Bolderson E, Kumar R, Muniandy PA, Xue Y, Richard DJ, Seidman M, Pandita TK, Khanna KK, Wang W - J. Biol. Chem. (2009)

Both hSSB1 and hSSB2 complexes are required for efficient ATM-dependent phosphorylation events in response to ionizing radiation. A, immunoblotting shows that phosphorylation of ATM itself and several of its substrates (indicated by circled P) in NFF cells was reduced by depletion of hSSB complex components. Cells were either unexposed or exposed to 6 Gy of ionizing radiation, and cell extracts were immunoblotted with anti-phosphorylation site-specific antibodies for ATM (Ser-1981), NBS1 (Ser-343), Chk1 (Ser-317), and Chk2 (Thr-68). ATM and actin were included as controls for equal protein loading. B–D, graphic representation shows quantification of the results in panel A. The result shown is a representative from two independent experiments that yielded reproducible data.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2749126&req=5

Figure 3: Both hSSB1 and hSSB2 complexes are required for efficient ATM-dependent phosphorylation events in response to ionizing radiation. A, immunoblotting shows that phosphorylation of ATM itself and several of its substrates (indicated by circled P) in NFF cells was reduced by depletion of hSSB complex components. Cells were either unexposed or exposed to 6 Gy of ionizing radiation, and cell extracts were immunoblotted with anti-phosphorylation site-specific antibodies for ATM (Ser-1981), NBS1 (Ser-343), Chk1 (Ser-317), and Chk2 (Thr-68). ATM and actin were included as controls for equal protein loading. B–D, graphic representation shows quantification of the results in panel A. The result shown is a representative from two independent experiments that yielded reproducible data.
Mentions: ATM-dependent phosphorylation of many checkpoint proteins is essential for IR-induced checkpoint activation (28). hSSB1-depleted cells have been shown to have impaired ATM-dependent phosphorylation of its substrates and itself (12) following IR. We found that hSSB2-depleted cells also had reduced ATM autophosphorylation (Ser-1981), as well as basal and IR-induced phosphorylation of substrates NBS1 (Ser-343) and Chk1 (Ser-317) (Fig. 3A, compare lanes 5 and 6 with lanes 1 and 2). The reduced phosphorylation in hSSB2-depleted cells was not as drastic as that in hSSB1-depleted cells in response to IR, with the former cells always displaying a higher level of the phosphorylated protein than the latter: ATM (45% versus 35%) (Fig. 3B), NBS1 (40% versus 26%) (Fig. 3C), Chk1 (66% versus 8%) (Fig. 3D), and Chk2 (109% versus 30%) (Fig. 3E). The results suggest that both hSSB1 and hSSB2 participate in ATM-dependent signaling pathways, but hSSB1 may play a relatively more important role.

Bottom Line: Here we present evidence that hSSB2, a homolog of hSSB1, plays a role similar to hSSB1 in DNA damage-response pathways.Notably, hSSB1 and hSSB2 form separate complexes with two identical proteins, INTS3 and hSSBIP1 (C9ORF80).Depletion of INTS3 decreased the stability of hSSB1 and hSSBIP1, suggesting that INTS3 may provide a scaffold to allow proper assembly of the hSSB complexes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetics, NIA, National Institutes of Health, NIH Biomedical Research Center, Baltimore, Maryland 21224, USA.

ABSTRACT
hSSB1 (human single strand DNA-binding protein 1) has been shown to participate in homologous recombination (HR)-dependent repair of DNA double strand breaks (DSBs) and ataxia telangiectasia-mutated (ATM)-mediated checkpoint pathways. Here we present evidence that hSSB2, a homolog of hSSB1, plays a role similar to hSSB1 in DNA damage-response pathways. This was evidenced by findings that hSSB2-depleted cells resemble hSSB1-depleted cells in hypersensitivity to DNA-damaging reagents, reduced efficiency in HR-dependent repair of DSBs, and defective ATM-dependent phosphorylation. Notably, hSSB1 and hSSB2 form separate complexes with two identical proteins, INTS3 and hSSBIP1 (C9ORF80). Cells depleted of INTS3 and hSSBIP1 also exhibited hypersensitivity to DNA damage reagents, chromosomal instability, and reduced ATM-dependent phosphorylation. hSSBIP1 was rapidly recruited to laser-induced DSBs, a feature also similar to that reported for hSSB1. Depletion of INTS3 decreased the stability of hSSB1 and hSSBIP1, suggesting that INTS3 may provide a scaffold to allow proper assembly of the hSSB complexes. Thus, our data demonstrate that hSSB1 and hSSB2 form two separate complexes with similar structures, and both are required for efficient HR-dependent repair of DSBs and ATM-dependent signaling pathways.

Show MeSH
Related in: MedlinePlus