PMC:4829102 / 5045-7881
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4829102","sourcedb":"PMC","sourceid":"4829102","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4829102","text":"EXD2 promotes DNA end resection and the generation of ssDNA\nCtIP is essential for efficient DNA end processing during DSB repair, with cells depleted for this factor showing a defect in the generation of single stranded DNA (ssDNA) and the subsequent formation of RPA foci 16, 25, 26. Thus we hypothesized that EXD2 may promote DNA end resection. To test this, we analysed RPA focus formation in response to both CPT and IR in WT and EXD2-depleted cells. Strikingly, cells depleted for EXD2 showed severely impaired kinetics of RPA focus formation in response to both treatments (Fig. 2a and b and Supplementary Fig. 2c and d). RPA2 phosphorylation at S4 and S8 has been widely used as a marker for the generation of single-stranded DNA by DNA-end resection 27. Consistent with the data above, EXD2 depleted cells showed impaired RPA S4/S8 phosphorylation in response to DNA damage (Fig. 2c and Supplementary Fig. 2e). Treatment with both agents resulted in a robust phosphorylation of histone H2AX and CHK2 (Fig. 2c and Supplementary Fig. 2e), confirming induction of DNA damage in cells. Moreover, since these responses were intact in EXD2-depleted cells, EXD2 is most likely not required for initial sensing of the DNA damage. Failure to generate RPA foci could be associated with either a defect in exonucleolytic processing of the DSB into ssDNA, or with impaired recruitment of RPA itself to ssDNA. To distinguish between these two possibilities we tested the efficiency of ssDNA generation in EXD2-depleted cells exposed to DNA damage. To this end, we labelled cells with BrdU and then employed immunofluorescence microscopy using an anti-BrdU antibody under non-denaturing conditions to detect stretches of ssDNA. Depletion of EXD2 significantly reduced formation of ssDNA foci (Fig. 2d and e) suggesting impaired resection. This is reminiscent of the phenotype observed in cells depleted for essential components of DNA end resection machinery, such as MRE11 or CtIP 6, 16. Importantly, these phenotypes were not due to changes in the cell cycle, as EXD2-depletion had little effect on the overall cell-cycle distribution profile (Supplementary Fig. 2f). Despite multiple attempts, we were unable to visualise EXD2 recruitment to DNA damage foci. In this regard, we note that certain other proteins involved in DNA repair do not readily form cytologically discernible foci in mammalian cells i.e. Ku70, Ku80 or DNA2. However, EXD2 was recruited to chromatin in HeLa cells upon DNA damage as assayed by subcellular fractionation (Fig. 2f), supporting its putative role in the processing of DSBs. A similar recruitment was also observed for the key resection factor MRE11 (Fig. 2f). Taken together, these results indicate that EXD2 is a putative component of the resection machinery required for the efficient processing of DSBs.","divisions":[{"label":"Title","span":{"begin":0,"end":59}}],"tracks":[{"project":"2_test","denotations":[{"id":"26807646-17965729-73828166","span":{"begin":273,"end":275},"obj":"17965729"},{"id":"26807646-17965729-73828166","span":{"begin":273,"end":275},"obj":"17965729"},{"id":"26807646-17936710-73828167","span":{"begin":277,"end":279},"obj":"17936710"},{"id":"26807646-17936710-73828167","span":{"begin":277,"end":279},"obj":"17936710"},{"id":"26807646-19357644-73828168","span":{"begin":281,"end":283},"obj":"19357644"},{"id":"26807646-19357644-73828168","span":{"begin":281,"end":283},"obj":"19357644"},{"id":"26807646-22733999-73828169","span":{"begin":758,"end":760},"obj":"22733999"},{"id":"26807646-22733999-73828169","span":{"begin":758,"end":760},"obj":"22733999"},{"id":"26807646-21227759-73828170","span":{"begin":1976,"end":1977},"obj":"21227759"},{"id":"26807646-21227759-73828170","span":{"begin":1976,"end":1977},"obj":"21227759"},{"id":"26807646-17965729-73828171","span":{"begin":1979,"end":1981},"obj":"17965729"},{"id":"26807646-17965729-73828171","span":{"begin":1979,"end":1981},"obj":"17965729"}],"attributes":[{"subj":"26807646-17965729-73828166","pred":"source","obj":"2_test"},{"subj":"26807646-17965729-73828166","pred":"source","obj":"2_test"},{"subj":"26807646-17936710-73828167","pred":"source","obj":"2_test"},{"subj":"26807646-17936710-73828167","pred":"source","obj":"2_test"},{"subj":"26807646-19357644-73828168","pred":"source","obj":"2_test"},{"subj":"26807646-19357644-73828168","pred":"source","obj":"2_test"},{"subj":"26807646-22733999-73828169","pred":"source","obj":"2_test"},{"subj":"26807646-22733999-73828169","pred":"source","obj":"2_test"},{"subj":"26807646-21227759-73828170","pred":"source","obj":"2_test"},{"subj":"26807646-21227759-73828170","pred":"source","obj":"2_test"},{"subj":"26807646-17965729-73828171","pred":"source","obj":"2_test"},{"subj":"26807646-17965729-73828171","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ecc993","default":true}]}]}}