RNF8-dependent and RNF8-independent Regulation of 53BP1 in Response to DNA Damage

Abstract

The DNA damage surveillance network orchestrates cellular responses to DNA damage through the recruitment of DNA damage-signaling molecules to DNA damage sites and the concomitant activation of protein phosphorylation cascades controlled by the ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) kinases. Activation of ATM/ATR triggers cell cycle checkpoint activation and adaptive responses to DNA damage. Recent studies suggest that protein ubiquitylation or degradation plays an important role in the DNA damage response. In this study, we examined the potential role of the proteasome in checkpoint activation and ATM/ATR signaling in response to UV light-induced DNA damage. HeLa cells treated with the proteasome inhibitor MG-132 showed delayed phosphorylation of ATM substrates in response to UV light. UV light-induced phosphorylation of 53BP1, as well as its recruitment to DNA damage foci, was strongly suppressed by proteasome inhibition, whereas the recruitment of upstream regulators of 53BP1, including MDC1 and H2AX, was unaffected. The ubiquitin-protein isopeptide ligase RNF8 was critical for 53BP1 focus targeting and phosphorylation in ionizing radiation-damaged cells, whereas UV light-induced 53BP1 phosphorylation and targeting exhibited partial dependence on RNF8 and the ubiquitin-conjugating enzyme UBC13. Suppression of RNF8 or UBC13 also led to subtle defects in UV light-induced G2/M checkpoint activation. These findings are consistent with in RNF8 ubiquitylation are for 53BP1 in response to ionizing whereas to 53BP1 targeting and phosphorylation in response to UV and of DNA The DNA damage surveillance network orchestrates cellular responses to DNA damage through the recruitment of DNA damage-signaling molecules to DNA damage sites and the concomitant activation of protein phosphorylation cascades controlled by the ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) kinases. Activation of ATM/ATR triggers cell cycle checkpoint activation and adaptive responses to DNA damage. Recent studies suggest that protein ubiquitylation or degradation plays an important role in the DNA damage response. In this study, we examined the potential role of the proteasome in checkpoint activation and ATM/ATR signaling in response to UV light-induced DNA damage. HeLa cells treated with the proteasome inhibitor MG-132 showed delayed phosphorylation of ATM substrates in response to UV light. UV light-induced phosphorylation of 53BP1, as well as its recruitment to DNA damage foci, was strongly suppressed by proteasome inhibition, whereas the recruitment of upstream regulators of 53BP1, including MDC1 and H2AX, was unaffected. The ubiquitin-protein isopeptide ligase RNF8 was critical for 53BP1 focus targeting and phosphorylation in ionizing radiation-damaged cells, whereas UV light-induced 53BP1 phosphorylation and targeting exhibited partial dependence on RNF8 and the ubiquitin-conjugating enzyme UBC13. Suppression of RNF8 or UBC13 also led to subtle defects in UV light-induced G2/M checkpoint activation. These findings are consistent with in RNF8 ubiquitylation are for 53BP1 in response to ionizing whereas to 53BP1 targeting and phosphorylation in response to UV and of DNA responses in cells that are for the of and cascades are to the DNA damage-signaling and of the important as DNA damage response of the the ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) signaling in response to of ATM by ionizing ionizing ubiquitin-protein isopeptide ionizing ubiquitin-protein isopeptide and that DNA whereas ATR to in DNA that DNA ATM and ATR and the and protein in cell cycle checkpoint through of ATM and ATR including and in DNA The of ATM and ATR to and studies suggest that the of ATM substrates of ATM and ATR are strongly by of to as checkpoint including MDC1 of DNA damage checkpoint and 53BP1 protein that are to DNA damage sites and in DNA an H2AX, and MDC1 that ATM in response to damage In this ATM the of the H2AX, to the of MDC1 MDC1 ATM activation cycle in ATM the protein that to DNA damage sites The recruitment of 53BP1 that with and In 53BP1 with of DNA damage sites cells checkpoint defects and defects in through DNA whereas and defects in 53BP1 also by ATM and ATR DNA The that findings that 53BP1 in to DNA damage response regulators DNA damage this study, we to the that to the DNA damage with on the potential of the that proteasome important for phosphorylation and targeting of 53BP1 to damage sites in response to DNA and that of the ubiquitylation for the of proteasome on cellular and and cells in with with was for UV cells was in and of was in and of MG-132 and in to the of to DNA damage. for was in of and of was in and of UBC13 UBC13 was as RNF8 RNF8 was cells with and an was with the and and and and with and and by to and the was with in and with The was with ATM 53BP1 and UBC13 the with and by as on with or in for and with for The cells with in for 53BP1 and MDC1 by or cells treated with for and treated with for with and by to or was for as The cells with in and was to focus cells with as 53BP1 cells, and the was cells by the of 53BP1 cells by the of The in cells with or or UV The cells treated with for with and with cells of on DNA the of proteasome on the DNA damage we examined the of proteasome on of ATM and ATR HeLa cells with the proteasome inhibitor MG-132 for with and the phosphorylation of ATM/ATR was by MG-132 in the of ATM to UV and phosphorylation of or phosphorylation of the MG-132 UV light-induced signaling the UV phosphorylation and ATM by in UV cells to UV and to UV phosphorylation of 53BP1 suppressed by proteasome HeLa cells treated with UV or MG-132 or and cell with the HeLa cells with UV MG-132 or the and by with the HeLa cells with MG-132 or for or and for UV phosphorylation was by by DNA by In to the of MG-132 on ATM and on the UV and phosphorylation of 53BP1 was by MG-132 and 53BP1 phosphorylation by UV was on ATR UV light-induced phosphorylation was by that the of MG-132 on 53BP1 phosphorylation of or ATR activation. we also showed that proteasome including and UV light-induced 53BP1 phosphorylation in HeLa cells These suggest that proteasome the activation of in UV cells and that 53BP1 phosphorylation to proteasome to to DNA damage sites in cells the of MG-132 on 53BP1 focus HeLa cells with MG-132 or for to of or UV and with MG-132 UV light-induced 53BP1 focus and in the and of 53BP1 foci, the of cells was by and 53BP1 by also suppressed by MG-132 The ATM inhibitor on 53BP1 focus in response to 53BP1 phosphorylation and targeting to are suppressed by proteasome inhibition, in response to DNA UV light-induced checkpoint activation through ATR we to MG-132 of 53BP1 focus to HeLa cells with to UV and and with 53BP1 and of and cells for 53BP1 UV that DNA for 53BP1 focus targeting the MG-132 in 53BP1 focus in cells These suggest that the of MG-132 on 53BP1 focus in response to UV to 53BP1 of and of 53BP1 on and DNA damage MDC1 UV light-induced targeting of and MDC1 was in cells, that the in 53BP1 focus targeting was to defects in the of DNA damage and also examined the potential of 53BP1 phosphorylation and protein that in response to proteasome to HeLa cells, are for 53BP1 phosphorylation and focus targeting in cells in response to UV or also that phosphorylation of the of protein was in cells phosphorylation of in response to and and for checkpoint activation in cells of on the of 53BP1 phosphorylation by MG-132 of 53BP1 phosphorylation and of phosphorylation of proteasome in 53BP1 focus of and and MG-132 or MDC1 focus HeLa cells with UV with or MG-132 and for with or MG-132 53BP1 and phosphorylation in cells treated with UV or MG-132 or with the The the of MG-132 53BP1 phosphorylation in the of was or cells with UV MG-132 or and for 53BP1 phosphorylation and by potential of 53BP1 focus we examined the of MG-132 on the of of 53BP1 of 53BP1 the 53BP1 to through with The focus targeting of in UV and cells was by MG-132 that MG-132 53BP1 focus targeting by an upstream for of 53BP1 to DNA damage through the was by MG-132 that proteasome this in the 53BP1 recruitment focus targeting of the 53BP1 of the of 53BP1, that to DNA damage HeLa cells with the and in the or of MG-132 as in the to and the cells with and RNF8 for 53BP1 The MG-132 of 53BP1 phosphorylation and targeting to that protein degradation by the in the 53BP1 activation The ubiquitin-conjugating enzyme UBC13 in DNA of DNA damage in with and UBC13 also for of in cells that UBC13 and the ligase RNF8 are for 53BP1 focus targeting in response to we UBC13 RNF8 was for 53BP1 focus targeting in response to UV light. that in to UV light-induced 53BP1 foci, of RNF8 UV light-induced 53BP1 targeting by and was to RNF8 the cell exhibited in 53BP1 focus targeting the of RNF8 with the MG-132 of 53BP1 focus in and UV UBC13 also led to partial in UV light-induced 53BP1 focus and and RNF8 are for 53BP1 in response to HeLa cells with or with to UBC13 or RNF8 was and of UBC13 and RNF8 by and cells to UV or 53BP1 are in 53BP1 The are in or UV light-induced 53BP1 and phosphorylation was by of cells with or RNF8 we examined the of RNF8 and UBC13 for DNA 53BP1 phosphorylation in HeLa 53BP1 phosphorylation was strongly suppressed by UBC13 or RNF8 whereas UV light-induced 53BP1 phosphorylation was by in UBC13 RNF8 53BP1 phosphorylation in UBC13 of 53BP1 phosphorylation in response to and UV whereas RNF8 53BP1 that MG-132 UV light-induced 53BP1 phosphorylation and UV light-induced 53BP1 focus findings suggest the that an ligase RNF8 53BP1 phosphorylation and targeting in response to UV for RNF8 and UBC13 for G2/M also the of UBC13 and RNF8 to G2/M checkpoint activation of HeLa cells with RNF8 led to of G2/M checkpoint of RNF8 also in the UV light-induced G2/M and UBC13 cells the of UBC13 G2/M checkpoint activation in of the suggest that UBC13 and RNF8 role in UV light-induced G2/M checkpoint activation and that for the G2/M Suppression of G2/M checkpoint activation by RNF8 consistent with the of cells, G2/M checkpoint activation in response to of and RNF8 the G2/M HeLa cells with the and for with or UV and or or UV and and with The of cells was The of are that of 53BP1 by DNA phosphorylation and focus are suppressed by of the proteasome in The of MG-132 on 53BP1 phosphorylation and targeting was in cells to the DNA UV and In the phosphorylation of suppressed by proteasome inhibition, whereas the phosphorylation of and substrates and The phosphorylation of in response to suppressed in cells that the phosphorylation in cells of 53BP1 The phosphorylation of sites by ATR in the of DNA UV that the in phosphorylation in cells to checkpoint findings are with by and that proteasome and focus of and 53BP1 In MG-132 53BP1 in response to whereas was in cells to the ATR UV and and and targeting of and MDC1 in response to UV was by MG-132 in the ubiquitin-conjugating enzyme UBC13 and ligase RNF8 as of the 53BP1 activation in cells UBC13 in and cells are also in of In and studies that UBC13 and RNF8 are regulators of 53BP1 focus the of RNF8 for 53BP1 focus targeting and showed that 53BP1 phosphorylation on 53BP1 phosphorylation and targeting in cells with G2/M checkpoint activation. The dependence of 53BP1 phosphorylation and targeting on RNF8 the that the are to RNF8 with MDC1 DNA damage through the RNF8 and the ubiquitylation of and the recruitment of we that of RNF8 ubiquitylation for 53BP1 UV light-induced phosphorylation and targeting of 53BP1 by that of In with RNF8 53BP1 in the of HeLa cells in response to UV light. the MG-132 UV light-induced 53BP1 foci, 53BP1 foci, and the of UV light-induced by MG-132 of or the cells in the of MG-132 and RNF8 was UV light-induced 53BP1 are of potential for the findings are consistent with in protein as to protein important for the UV light-induced 53BP1 response. In this RNF8 and to UV light-induced 53BP1 activation through degradation of showed that UBC13 also to UV light-induced 53BP1 phosphorylation and targeting that UBC13 and RNF8 to UV light-induced 53BP1 phosphorylation and to as for and are for of cell and The that to 53BP1 and phosphorylation are by proteasome for the of cell by and proteasome of responses in cells that are for the of and cascades are to the DNA damage-signaling and of the important as DNA damage response of the the ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) signaling in response to of ATM by ionizing ionizing ubiquitin-protein isopeptide ionizing ubiquitin-protein isopeptide and that DNA whereas ATR to in DNA that DNA ATM and ATR and the and protein in cell cycle checkpoint through of ATM and ATR including and in DNA The of ATM and ATR to and studies suggest that the of ATM substrates The of ATM and ATR are strongly by of to as checkpoint including MDC1 of DNA damage checkpoint and 53BP1 protein that are to DNA damage sites and in DNA an H2AX, and MDC1 that ATM in response to damage In this ATM the of the H2AX, to the of MDC1 MDC1 ATM activation cycle in ATM the 53BP1 protein that to DNA damage sites The recruitment of 53BP1 that with and In 53BP1 with of DNA damage sites cells checkpoint defects and defects in through DNA whereas and defects in 53BP1 also by ATM and ATR DNA The that findings that 53BP1 in to DNA damage response regulators DNA damage In this study, we to the that to the DNA damage with on the potential of the that proteasome important for phosphorylation and targeting of 53BP1 to damage sites in response to DNA and that of the ubiquitylation for the of proteasome on cellular and and cells in with with was for UV cells was in and of was in and of MG-132 and in to the of to DNA damage. for was in of and of was in and of UBC13 UBC13 was as RNF8 RNF8 was cells with and an was with the and and and and with and and by to and the was with in and with The was with ATM 53BP1 and UBC13 the with and by as on with or in for and with for The cells with in for 53BP1 and MDC1 by or cells treated with for and treated with for with and by to or was for as The cells with in and was to focus cells with as 53BP1 cells, and the was cells by the of 53BP1 cells by the of The in cells with or or UV The cells treated with for with and with cells and and cells in with with was for UV cells was in and of was in and of MG-132 and in to the of to DNA damage. for was in of and of was in and of UBC13 UBC13 was as RNF8 RNF8 was cells with and an was with the and and and and with and and by to and the was with in and with The was with ATM 53BP1 and UBC13 the with and by as on with or in for and with for The cells with in for 53BP1 and MDC1 by or cells treated with for and treated with for with and by to or was for as The cells with in and was to focus cells with as 53BP1 cells, and the was cells by the of 53BP1 cells by the of The in cells with or or UV The cells treated with for with and with cells of on DNA the of proteasome on the DNA damage we examined the of proteasome on of ATM and ATR HeLa cells with the proteasome inhibitor MG-132 for with and the phosphorylation of ATM/ATR was by MG-132 in the of ATM to UV and phosphorylation of or phosphorylation of the MG-132 UV light-induced signaling the UV phosphorylation and ATM by in UV cells to UV and to UV by DNA by In to the of MG-132 on ATM and on the UV and phosphorylation of 53BP1 was by MG-132 and 53BP1 phosphorylation by UV was on ATR UV light-induced phosphorylation was by that the of MG-132 on 53BP1 phosphorylation of or ATR activation. we also showed that proteasome including and UV light-induced 53BP1 phosphorylation in HeLa cells These suggest that proteasome the activation of in UV cells and that 53BP1 phosphorylation to proteasome to to DNA damage sites in cells the of MG-132 on 53BP1 focus HeLa cells with MG-132 or for to of or UV and with MG-132 UV light-induced 53BP1 focus and in the and of 53BP1 foci, the of cells was by and 53BP1 by also suppressed by MG-132 The ATM inhibitor on 53BP1 focus in response to 53BP1 phosphorylation and targeting to are suppressed by proteasome inhibition, in response to DNA UV light-induced checkpoint activation through ATR we to MG-132 of 53BP1 focus to HeLa cells with to UV and and with 53BP1 and of and cells for 53BP1 UV that DNA for 53BP1 focus targeting the MG-132 in 53BP1 focus in cells These suggest that the of MG-132 on 53BP1 focus in response to UV to 53BP1 of and of 53BP1 on and DNA damage MDC1 UV light-induced targeting of and MDC1 was in cells, that the in 53BP1 focus targeting was to defects in the of DNA damage and also examined the potential of 53BP1 phosphorylation and protein that in response to proteasome to HeLa cells, are for 53BP1 phosphorylation and focus targeting in cells in response to UV or also that phosphorylation of the of protein was in cells phosphorylation of in response to and and for checkpoint activation in cells of on the of 53BP1 phosphorylation by MG-132 of 53BP1 phosphorylation and of phosphorylation of proteasome in 53BP1 focus of and and MG-132 or MDC1 focus HeLa cells with UV with or MG-132 and for with or MG-132 53BP1 and phosphorylation in cells treated with UV or MG-132 or with the The the of MG-132 53BP1 phosphorylation in the of was or cells with UV MG-132 or and for 53BP1 phosphorylation and by potential of 53BP1 focus we examined the of MG-132 on the of of 53BP1 of 53BP1 the 53BP1 to through with The focus targeting of in UV and cells was by MG-132 that MG-132 53BP1 focus targeting by an upstream for of 53BP1 to DNA damage through the was by MG-132 that proteasome this in the 53BP1 recruitment focus targeting of the 53BP1 of the of 53BP1, that to DNA damage HeLa cells with the and in the or of MG-132 as in the to and the cells with and RNF8 for 53BP1 The MG-132 of 53BP1 phosphorylation and targeting to that protein degradation by the in the 53BP1 activation The ubiquitin-conjugating enzyme UBC13 in DNA of DNA damage in with and UBC13 also for of in cells that UBC13 and the ligase RNF8 are for 53BP1 focus targeting in response to we UBC13 RNF8 was for 53BP1 focus targeting in response to UV light. that in to UV light-induced 53BP1 foci, of RNF8 UV light-induced 53BP1 targeting by and was to RNF8 the cell exhibited in 53BP1 focus targeting the of RNF8 with the MG-132 of 53BP1 focus in and UV UBC13 also led to partial in UV light-induced 53BP1 focus and and RNF8 are for 53BP1 in response to HeLa cells with or with to UBC13 or RNF8 was and of UBC13 and RNF8 by and cells to UV or 53BP1 are in 53BP1 The are in or UV light-induced 53BP1 and phosphorylation was by of cells with or RNF8 we examined the of RNF8 and UBC13 for DNA 53BP1 phosphorylation in HeLa 53BP1 phosphorylation was strongly suppressed by UBC13 or RNF8 whereas UV light-induced 53BP1 phosphorylation was by in UBC13 RNF8 53BP1 phosphorylation in UBC13 of 53BP1 phosphorylation in response to and UV whereas RNF8 53BP1 that MG-132 UV light-induced 53BP1 phosphorylation and UV light-induced 53BP1 focus findings suggest the that an ligase RNF8 53BP1 phosphorylation and targeting in response to UV for RNF8 and UBC13 for G2/M also the of UBC13 and RNF8 to G2/M checkpoint activation of HeLa cells with RNF8 led to of G2/M checkpoint of RNF8 also in the UV light-induced G2/M and UBC13 cells the of UBC13 G2/M checkpoint activation in of the suggest that UBC13 and RNF8 role in UV light-induced G2/M checkpoint activation and that for the G2/M Suppression of G2/M checkpoint activation by RNF8 consistent with the of cells, G2/M checkpoint activation in response to of and RNF8 the G2/M HeLa cells with the and for with or UV and or or UV and and with The of cells was The of are that of 53BP1 by DNA phosphorylation and focus are suppressed by of the proteasome in The of MG-132 on 53BP1 phosphorylation and targeting was in cells to the DNA UV and In the phosphorylation of suppressed by proteasome inhibition, whereas the phosphorylation of and substrates and The phosphorylation of in response to suppressed in cells that the phosphorylation in cells of 53BP1 The phosphorylation of sites by ATR in the of DNA UV that the in phosphorylation in cells to checkpoint findings are with by and that proteasome and focus of and 53BP1 In MG-132 53BP1 in response to whereas was in cells to the ATR UV and and and targeting of and MDC1 in response to UV was by MG-132 in the ubiquitin-conjugating enzyme UBC13 and ligase RNF8 as of the 53BP1 activation in cells UBC13 in and cells are also in of In and studies that UBC13 and RNF8 are regulators of 53BP1 focus the of RNF8 for 53BP1 focus targeting and showed that 53BP1 phosphorylation on 53BP1 phosphorylation and targeting in cells with G2/M checkpoint activation. The dependence of 53BP1 phosphorylation and targeting on RNF8 the that the are to RNF8 with MDC1 DNA damage through the RNF8 and the ubiquitylation of and the recruitment of we that of RNF8 ubiquitylation for 53BP1 UV light-induced phosphorylation and targeting of 53BP1 by that of In with RNF8 53BP1 in the of HeLa cells in response to UV light. the MG-132 UV light-induced 53BP1 foci, 53BP1 foci, and the of UV light-induced by MG-132 of or the cells in the of MG-132 and RNF8 was UV light-induced 53BP1 are of potential for the findings are consistent with in protein as to protein important for the UV light-induced 53BP1 response. In this RNF8 and to UV light-induced 53BP1 activation through degradation of showed that UBC13 also to UV light-induced 53BP1 phosphorylation and targeting that UBC13 and RNF8 to UV light-induced 53BP1 phosphorylation and to as for and are for of cell and The that to 53BP1 and phosphorylation are by proteasome for the of cell by and proteasome of of on DNA the of proteasome on the DNA damage we examined the of proteasome on of ATM and ATR HeLa cells with the proteasome inhibitor MG-132 for with and the phosphorylation of ATM/ATR was by MG-132 in the of ATM to UV and phosphorylation of or phosphorylation of the MG-132 UV light-induced signaling the UV phosphorylation and ATM by in UV cells to UV and to UV 53BP1 by DNA by In to the of MG-132 on ATM and on the UV and phosphorylation of 53BP1 was by MG-132 and 53BP1 phosphorylation by UV was on ATR UV light-induced phosphorylation was by that the of MG-132 on 53BP1 phosphorylation of or ATR activation. we also showed that proteasome including and UV light-induced 53BP1 phosphorylation in HeLa cells These suggest that proteasome the activation of in UV cells and that 53BP1 phosphorylation to proteasome 53BP1 to to DNA damage sites in cells the of MG-132 on 53BP1 focus HeLa cells with MG-132 or for to of or UV and with MG-132 UV light-induced 53BP1 focus and in the and of 53BP1 foci, the of cells was by and 53BP1 by also suppressed by MG-132 The ATM inhibitor on 53BP1 focus in response to 53BP1 phosphorylation and targeting to are suppressed by proteasome inhibition, in response to DNA UV light-induced checkpoint activation through ATR we to MG-132 of 53BP1 focus to HeLa cells with to UV and and with 53BP1 and of and cells for 53BP1 UV that DNA for 53BP1 focus targeting the MG-132 in 53BP1 focus in cells These suggest that the of MG-132 on 53BP1 focus in response to UV to MG-132 53BP1 of and of 53BP1 on and DNA damage MDC1 UV light-induced targeting of and MDC1 was in cells, that the in 53BP1 focus targeting was to defects in the of DNA damage and also examined the potential of 53BP1 phosphorylation and protein that in response to proteasome to HeLa cells, are for 53BP1 phosphorylation and focus targeting in cells in response to UV or also that phosphorylation of the of protein was in cells phosphorylation of in response to and and for checkpoint activation in cells of on the of 53BP1 phosphorylation by MG-132 of 53BP1 phosphorylation and of phosphorylation of proteasome in potential of 53BP1 focus we examined the of MG-132 on the of of 53BP1 of 53BP1 the 53BP1 to through with The focus targeting of in UV and cells was by MG-132 that MG-132 53BP1 focus targeting by an upstream for of 53BP1 to DNA damage through the was by MG-132 that proteasome this in the 53BP1 recruitment UBC13 and RNF8 for 53BP1 The MG-132 of 53BP1 phosphorylation and targeting to that protein degradation by the in the 53BP1 activation The ubiquitin-conjugating enzyme UBC13 in DNA of DNA damage in with and UBC13 also for of in cells that UBC13 and the ligase RNF8 are for 53BP1 focus targeting in response to we UBC13 RNF8 was for 53BP1 focus targeting in response to UV light. that in to UV light-induced 53BP1 foci, of RNF8 UV light-induced 53BP1 targeting by and was to RNF8 the cell exhibited in 53BP1 focus targeting the of RNF8 with the MG-132 of 53BP1 focus in and UV UBC13 also led to partial in UV light-induced 53BP1 focus and we examined the of RNF8 and UBC13 for DNA 53BP1 phosphorylation in HeLa 53BP1 phosphorylation was strongly suppressed by UBC13 or RNF8 whereas UV light-induced 53BP1 phosphorylation was by in UBC13 RNF8 53BP1 phosphorylation in UBC13 of 53BP1 phosphorylation in response to and UV whereas RNF8 53BP1 that MG-132 UV light-induced 53BP1 phosphorylation and UV light-induced 53BP1 focus findings suggest the that an ligase RNF8 53BP1 phosphorylation and targeting in response to UV damage. for RNF8 and UBC13 for G2/M also the of UBC13 and RNF8 to G2/M checkpoint activation of HeLa cells with RNF8 led to of G2/M checkpoint of RNF8 also in the UV light-induced G2/M and UBC13 cells the of UBC13 G2/M checkpoint activation in of the suggest that UBC13 and RNF8 role in UV light-induced G2/M checkpoint activation and that for the G2/M Suppression of G2/M checkpoint activation by RNF8 consistent with the of cells, G2/M checkpoint activation in response to of that of 53BP1 by DNA phosphorylation and focus are suppressed by of the proteasome in The of MG-132 on 53BP1 phosphorylation and targeting was in cells to the DNA UV and In the phosphorylation of suppressed by proteasome inhibition, whereas the phosphorylation of and substrates and The phosphorylation of in response to suppressed in cells that the phosphorylation in cells of 53BP1 The phosphorylation of sites by ATR in the of DNA UV that the in phosphorylation in cells to checkpoint findings are with by and that proteasome and focus of and 53BP1 In MG-132 53BP1 in response to whereas was in cells to the ATR UV and and and targeting of and MDC1 in response to UV was by MG-132 in the ubiquitin-conjugating enzyme UBC13 and ligase RNF8 as of the 53BP1 activation in cells UBC13 in and cells are also in of In and studies that UBC13 and RNF8 are regulators of 53BP1 focus the of RNF8 for 53BP1 focus targeting and showed that 53BP1 phosphorylation on 53BP1 phosphorylation and targeting in cells with G2/M checkpoint activation. The dependence of 53BP1 phosphorylation and targeting on RNF8 the that the are to RNF8 with MDC1 DNA damage through the RNF8 and the ubiquitylation of and the recruitment of we that of RNF8 ubiquitylation for 53BP1 The UV light-induced phosphorylation and targeting of 53BP1 by that of In with RNF8 53BP1 in the of HeLa cells in response to UV light. the MG-132 UV light-induced 53BP1 foci, 53BP1 foci, and the of UV light-induced by MG-132 of or the cells in the of MG-132 and RNF8 was UV light-induced 53BP1 are of potential for the findings are consistent with in protein as to protein important for the UV light-induced 53BP1 response. In this RNF8 and to UV light-induced 53BP1 activation through degradation of showed that UBC13 also to UV light-induced 53BP1 phosphorylation and targeting that UBC13 and RNF8 to UV light-induced 53BP1 phosphorylation and to as for and are for of cell and The that to 53BP1 and phosphorylation are by proteasome for the of cell by and proteasome of with with