hSnm1B Is a Novel Telomere-associated Protein

Abstract

Artemis, a member of the ␤-CASP family, has been implicated in the regulation of both telomere stability and length. Prompted by this, we examined whether the other two putative DNA-binding members of this family, hSnm1A and hSnm1B, may associate with telomeres. hSnm1A was found to not interact with the telomere. Conversely, hSnm1B was found to associate with telomeres in vivo by both immunofluorescence and chromatin immunoprecipitation. Furthermore, the C terminus of hSnm1B was shown to interact with the TRF homology domain of TRF2 indicating that hSnm1B is likely recruited to the telomere via interaction with the doublestranded telomere-binding protein TRF2.The ends of human chromosomes are capped and protected by a DNAprotein structure termed the telomere. The DNA portion of human telomeres is composed of a G-rich repeat (TTAGGG) that extends past the complementary C-rich strand, forming a 3Ј extension. This extension has been found by electron microscopy to loop back and invade the double-stranded region, forming a large loop structure termed the t-loop (1). The protein portion of telomeres is composed of a core of six proteins termed the telosome or shelterin (2-4), three of which directly bind telomeric DNA, TRF1 (5), TRF2 (6, 7), and hPot1 (8), and three that associate with the DNA-binding proteins, Tin2 (9), Tpp1 (10 -12), and Rap1 (13). TRF1 and TRF2 bind double-stranded telomeric DNA (7), whereas hPot1 binds the single-stranded region of the telomere (8). In addition to this core complex, many other proteins are known to associate and function at the telomere, albeit at a smaller concentration and often indirectly through binding to TRF1 or TRF2 (4).One protein that influences the stability of the telomere but is not a member of the described core telomeric complex is Artemis. This protein is a member of the ␤-CASP family of proteins that contain a unique metallo-␤-lactamase domain that hydrolyzes nucleic acids (14). Artemis is well established to play a role in non-homologous recombination where it forms a complex with DNA-PK CS , which cleaves the intermediate hairpin loop structure formed during V(D)J recombination and non-homologous end joining (15). However, Artemis-deficient mice have increased telomeric fusions, suggesting that this family of proteins plays a role in telomere structure or function (16). Indeed, Artemis deficient cell lines have increased rates of telomeric shortening as well as rapid accumulation of anaphase bridges (17).Five mammalian proteins belonging to the ␤-CASP family have been identified, two of which hydrolyze RNA (18 -21), whereas the remaining three, Artemis, Snm1A, and Snm1B, are believed to function solely on DNA substrates (22). Snm1A localizes to DNA double-strand breaks after ionizing radiation; although cells lacking Snm1A are sensitive only to mitomycin C and not ionizing radiation (23,24). Snm1A co-localizes with the DNA damage response protein 53BP before and after exposure to ionizing radiation (23). Snm1A is also highly up-regulated during mitosis and is be...

Endogenous hSNM1B/Apollo interacts with TRF2 and stimulates ATM in response to ionizing radiation

“…Further IF studies revealed that the majority of hSNM1B-foci co-localized with the telomere core protein, TRF1, and are therefore localized at telomeres. These findings substantiate previous reports on the localization of ectopic expressed hSNM1B at telomeres [12,14,16]. The observation that only a fraction of cells contained hSNM1B foci suggests a transient, cell cycle dependent function for hSNM1B at telomeres consistent with reports that hSNM1B functions in repressing the "DNA-damage" signal at telomeres during or after their replication [13].…”

“…As shown above, the anti-hSNM1B antibodies were able to detect hSNM1B in IF experiments which allowed us to determine whether endogenous hSNM1B localizes to telomeres, as suggested by the yeast-two-hybrid and co-IP results and previously published results on ectopic overexpressed hSNM1B [12][13][14][15]. Double staining of hSNM1B and either of the telomere markers, TRF1 or TRF2, demonstrated a high degree of co-localization of these proteins ( Figure 2C) and showing, for the first time, that the majority of endogenous hSNM1B foci are localized at telomers.…”

“…Freibaum and Counter found transiently expressed EGFP-hSNM1B colocalized and coimmunoprecipitated with TRF2 [12]. Another group identified this interaction by employing a combination of co-immunoprecipitation and mass spectrometry [13].…”

Snm1B/Apollo mediates replication fork collapse and S Phase checkpoint activation in response to DNA interstrand cross-links

“…Together our findings demonstrate that Snm1B is specifically involved in mediating resistance to ICLinducing compounds by its role in promoting replication fork collapse, which leads to activation of ATMmediated cell cycle arrest and likely initiation of removal of the lesion. As Snm1B/Apollo has also been implicated in telomere protection during S phase (Freibaum and Counter, 2006;Lenain et al, 2006;van Overbeek and de Lange, 2006), it is clear that it has a multifunctional role in maintenance of genomic stability, and experiments designed to assess its role as a tumor suppressor merit further investigation.…”

“…In human cells siRNA-mediated knockdown of SNM1B has been shown to result in moderate hypersensitivity to cisplatin, MMC and IR (Demuth et al, 2004). Also, several recent papers have demonstrated a novel function for Snm1B/Apollo namely that it interacts with the telomere protein TRF2 and protects telomeres from the DNA repair machinery during S phase (Freibaum and Counter, 2006;Lenain et al, 2006;van Overbeek and de Lange, 2006).…”