Limits...
TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

Harley ME, Murina O, Leitch A, Higgs MR, Bicknell LS, Yigit G, Blackford AN, Zlatanou A, Mackenzie KJ, Reddy K, Halachev M, McGlasson S, Reijns MA, Fluteau A, Martin CA, Sabbioneda S, Elcioglu NH, Altmüller J, Thiele H, Greenhalgh L, Chessa L, Maghnie M, Salim M, Bober MB, Nürnberg P, Jackson SP, Hurles ME, Wollnik B, Stewart GS, Jackson AP - Nat. Genet. (2015)

Bottom Line: Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism.TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes.Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

View Article: PubMed Central - PubMed

Affiliation: MRC Human Genetics Unit, IGMM, University of Edinburgh, Edinburgh, UK.

ABSTRACT
DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

Show MeSH

Related in: MedlinePlus

TRAIP localizes to sites of UV-induced DNA damage(a) TRAIP localizes to DNA damage sites induced by UV laser microirradiation both in the absence and presence of pre-treatment with BrdU as a damage sensitizer. Representative images, before and after UV laser microirradiation. Scale bar, 5 μm. (b) GFP-TRAIP colocalizes with γH2AX and with RFP-PCNA at sites of UV laser-induced damage. Representative images of UV laser-irradiated GFP-TRAIP expressing cells immunostained for γH2AX (pre-sensitized with BrdU) or co-expressing RFP-PCNA (no BrdU pre-treatment) as indicated. Scale bar, 5 μm. (c, d) GFP-TRAIP is detected by a Proximity Ligation Assay (PLA) in close proximity to PCNA, an association enhanced after UV-induced damage. (c) Representative images of PLA signals/nucleus in doxycycline-inducible GFP-TRAIP HeLa cells before and after damage with 25 J/m2 UV-C. Scale bar, 5 μm. (d) Quantification of PLA signals/nucleus. Box plots, center line denote mean values, box 25/75 %, whiskers 5/95 %, data pooled from n=2 independent experiments, n>65 data points per condition per experiment; Mann Whitney rank sum test: *** p<0.001. (e) TRAIP accumulates at sites of localized UV damage, colocalising with RPA and γH2AX. Representative immunofluorescence images of MRC5 cells transfected with GFP-TRAIP or GFP alone after UV-C irradiation through 3 μm microfilters. Scale bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4697364&req=5

Figure 3: TRAIP localizes to sites of UV-induced DNA damage(a) TRAIP localizes to DNA damage sites induced by UV laser microirradiation both in the absence and presence of pre-treatment with BrdU as a damage sensitizer. Representative images, before and after UV laser microirradiation. Scale bar, 5 μm. (b) GFP-TRAIP colocalizes with γH2AX and with RFP-PCNA at sites of UV laser-induced damage. Representative images of UV laser-irradiated GFP-TRAIP expressing cells immunostained for γH2AX (pre-sensitized with BrdU) or co-expressing RFP-PCNA (no BrdU pre-treatment) as indicated. Scale bar, 5 μm. (c, d) GFP-TRAIP is detected by a Proximity Ligation Assay (PLA) in close proximity to PCNA, an association enhanced after UV-induced damage. (c) Representative images of PLA signals/nucleus in doxycycline-inducible GFP-TRAIP HeLa cells before and after damage with 25 J/m2 UV-C. Scale bar, 5 μm. (d) Quantification of PLA signals/nucleus. Box plots, center line denote mean values, box 25/75 %, whiskers 5/95 %, data pooled from n=2 independent experiments, n>65 data points per condition per experiment; Mann Whitney rank sum test: *** p<0.001. (e) TRAIP accumulates at sites of localized UV damage, colocalising with RPA and γH2AX. Representative immunofluorescence images of MRC5 cells transfected with GFP-TRAIP or GFP alone after UV-C irradiation through 3 μm microfilters. Scale bar, 5 μm.

Mentions: Phenotypically, TRAIP patients were reminiscent of individuals with Seckel syndrome that have defects in ATR pathway signaling7,8, particularly with their disproportionately reduced head size. We therefore postulated that TRAIP could be a DDR protein acting in this pathway. Consistent with this, live-imaging of GFP-TRAIP protein demonstrated rapid re-localization from a pan-nuclear distribution to sites of DNA damage after UV laser microirradiation, irrespective of addition of BrdU as a DNA damage sensitizer (Fig. 3a). Colocalization was observed both with γH2AX and RFP-PCNA (Fig. 3b). Proximity ligation assays (PLA) detected GFP-TRAIP in close proximity (<40 nm) to PCNA in undamaged cells, suggesting it can be present at replication foci in undamaged cells. This association was substantially enriched after UV-C irradiation (Fig. 3c, d, p< 0.001), consistent with TRAIP localizing with PCNA at sites of DNA damage. Relocalisation of TRAIP to sites of UV-induced DNA damage was confirmed using localized UV-C irradiation through 3 μm isopore membrane filters, after which GFP-TRAIP was detected in irradiated regions, along with multiple DNA damage repair markers, including γH2AX and RPA2 (Fig. 3e). Notably, TRAIP was a highly significant hit in a recently reported high-throughput mass spectroscopy screen for proteins recruited to replication blocking DNA lesions27. As well as re-localizing to DNA damage sites, we also noted that TRAIP protein levels were regulated by DNA damage, with proteasome-mediated degradation observed at later time points after UV treatment (Supplementary Fig. 5), similar to certain DDR proteins involved in repair of UV lesions28.


TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

Harley ME, Murina O, Leitch A, Higgs MR, Bicknell LS, Yigit G, Blackford AN, Zlatanou A, Mackenzie KJ, Reddy K, Halachev M, McGlasson S, Reijns MA, Fluteau A, Martin CA, Sabbioneda S, Elcioglu NH, Altmüller J, Thiele H, Greenhalgh L, Chessa L, Maghnie M, Salim M, Bober MB, Nürnberg P, Jackson SP, Hurles ME, Wollnik B, Stewart GS, Jackson AP - Nat. Genet. (2015)

TRAIP localizes to sites of UV-induced DNA damage(a) TRAIP localizes to DNA damage sites induced by UV laser microirradiation both in the absence and presence of pre-treatment with BrdU as a damage sensitizer. Representative images, before and after UV laser microirradiation. Scale bar, 5 μm. (b) GFP-TRAIP colocalizes with γH2AX and with RFP-PCNA at sites of UV laser-induced damage. Representative images of UV laser-irradiated GFP-TRAIP expressing cells immunostained for γH2AX (pre-sensitized with BrdU) or co-expressing RFP-PCNA (no BrdU pre-treatment) as indicated. Scale bar, 5 μm. (c, d) GFP-TRAIP is detected by a Proximity Ligation Assay (PLA) in close proximity to PCNA, an association enhanced after UV-induced damage. (c) Representative images of PLA signals/nucleus in doxycycline-inducible GFP-TRAIP HeLa cells before and after damage with 25 J/m2 UV-C. Scale bar, 5 μm. (d) Quantification of PLA signals/nucleus. Box plots, center line denote mean values, box 25/75 %, whiskers 5/95 %, data pooled from n=2 independent experiments, n>65 data points per condition per experiment; Mann Whitney rank sum test: *** p<0.001. (e) TRAIP accumulates at sites of localized UV damage, colocalising with RPA and γH2AX. Representative immunofluorescence images of MRC5 cells transfected with GFP-TRAIP or GFP alone after UV-C irradiation through 3 μm microfilters. Scale bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4697364&req=5

Figure 3: TRAIP localizes to sites of UV-induced DNA damage(a) TRAIP localizes to DNA damage sites induced by UV laser microirradiation both in the absence and presence of pre-treatment with BrdU as a damage sensitizer. Representative images, before and after UV laser microirradiation. Scale bar, 5 μm. (b) GFP-TRAIP colocalizes with γH2AX and with RFP-PCNA at sites of UV laser-induced damage. Representative images of UV laser-irradiated GFP-TRAIP expressing cells immunostained for γH2AX (pre-sensitized with BrdU) or co-expressing RFP-PCNA (no BrdU pre-treatment) as indicated. Scale bar, 5 μm. (c, d) GFP-TRAIP is detected by a Proximity Ligation Assay (PLA) in close proximity to PCNA, an association enhanced after UV-induced damage. (c) Representative images of PLA signals/nucleus in doxycycline-inducible GFP-TRAIP HeLa cells before and after damage with 25 J/m2 UV-C. Scale bar, 5 μm. (d) Quantification of PLA signals/nucleus. Box plots, center line denote mean values, box 25/75 %, whiskers 5/95 %, data pooled from n=2 independent experiments, n>65 data points per condition per experiment; Mann Whitney rank sum test: *** p<0.001. (e) TRAIP accumulates at sites of localized UV damage, colocalising with RPA and γH2AX. Representative immunofluorescence images of MRC5 cells transfected with GFP-TRAIP or GFP alone after UV-C irradiation through 3 μm microfilters. Scale bar, 5 μm.
Mentions: Phenotypically, TRAIP patients were reminiscent of individuals with Seckel syndrome that have defects in ATR pathway signaling7,8, particularly with their disproportionately reduced head size. We therefore postulated that TRAIP could be a DDR protein acting in this pathway. Consistent with this, live-imaging of GFP-TRAIP protein demonstrated rapid re-localization from a pan-nuclear distribution to sites of DNA damage after UV laser microirradiation, irrespective of addition of BrdU as a DNA damage sensitizer (Fig. 3a). Colocalization was observed both with γH2AX and RFP-PCNA (Fig. 3b). Proximity ligation assays (PLA) detected GFP-TRAIP in close proximity (<40 nm) to PCNA in undamaged cells, suggesting it can be present at replication foci in undamaged cells. This association was substantially enriched after UV-C irradiation (Fig. 3c, d, p< 0.001), consistent with TRAIP localizing with PCNA at sites of DNA damage. Relocalisation of TRAIP to sites of UV-induced DNA damage was confirmed using localized UV-C irradiation through 3 μm isopore membrane filters, after which GFP-TRAIP was detected in irradiated regions, along with multiple DNA damage repair markers, including γH2AX and RPA2 (Fig. 3e). Notably, TRAIP was a highly significant hit in a recently reported high-throughput mass spectroscopy screen for proteins recruited to replication blocking DNA lesions27. As well as re-localizing to DNA damage sites, we also noted that TRAIP protein levels were regulated by DNA damage, with proteasome-mediated degradation observed at later time points after UV treatment (Supplementary Fig. 5), similar to certain DDR proteins involved in repair of UV lesions28.

Bottom Line: Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism.TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes.Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

View Article: PubMed Central - PubMed

Affiliation: MRC Human Genetics Unit, IGMM, University of Edinburgh, Edinburgh, UK.

ABSTRACT
DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

Show MeSH
Related in: MedlinePlus