hSSB1 and hSSB2 Form Similar Multiprotein Complexes That Participate in DNA Damage Response

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.

Single strand DNA binding proteins 1 and 2 protect newly replicated telomeres

“…In mSSB1 ∆/∆ MEFs, mSSB1 protein was undetectable, confirming that we successfully generated a mSSB1-null allele ( Figure 1B and 1C). The localization of mSSB2 to IR-induced DNA damage foci was not affected upon mSSB1 deletion, in agreement with previous reports [17,18,20] (Figure 1C). Interestingly, mSSB2 protein level was upregulated in the absence of mSSB1 ( Figure 1B).…”

“…Previous studies suggest that hSSB1 and hSSB2 both form complexes with INTS3 and possess overlapping functions in the DNA damage response (DDR) [17,18]. The elevated expression of mSSB2 observed when mSSB1 is deleted ( Figures 1C, 2B, 2C and 3F) suggests that upregulation of mSSB2 likely compensates for mSSB1 loss, thereby complicating the analysis of mSSB1 function.…”

“…siRNA knockdown experiments suggest that INTS3 links hSSB1/2 and C9orf80 into heterotrimeric complexes [17,18,20]. As INTS3 controls the levels of hSSB1 through transcriptional regulation [20], it is unclear whether hSSB1 is able to localize to damaged DNA independent of INTS3.…”

“…Notably, long trains of end-to-end fused chromosomes, a hallmark of TRF2 loss, were rarely observed in the setting of mSSB1 deficiency. As activation of the ATM kinase is negatively impacted when hSSB1 is depleted [17,18,20], we monitored the Chk2 phosphorylation status in mSSB1 ∆/∆ MEFs treated with shTRF2. Chk2 phosphorylation in mSSB1 ∆/∆ MEFs appears similar to that observed in WT MEFs, although this analysis is complicated by the fact that mSSB2 levels increased following mSSB1 deletion and therefore any decline in Chk2 phosphorylation due to mSSB1 loss will not be evident ( Figure 2B).…”

“…Telomeres consist of TTAGGG repetitive sequences that terminate in a 3′ ss G-rich overhang, and invasion of the 3′ end into duplex regions of the telomere is postulated to be important to protect DNA ends from being recognized by RPA, which would INTS3 and C9orf80, termed sensor of ssDNA complex 1 and 2 (SOSS1/2) [17][18][19][20]. Although RPA recruitment to damaged DNA requires both CtIP and the MRN complex, localization of SOSS1 to damaged DNA in S/ G2 phase requires only MRN [17].…”