TY - JOUR
T1 - Ectopic RNF168 expression promotes break-induced replication-like DNA synthesis at stalled replication forks
AU - Krais, John J.
AU - Johnson, Neil
N1 - Funding Information:
US National Institutes of Health (NIH) Grants [P50 CA083638, 5P30 CA006927, R01CA214799]; Susan Komen [CCRCR17499048]; Department of Defense [OC130212 to N.J.]; American Cancer Society––Tri State CEOs Against Cancer Postdoctoral Fellowship [PF-19-097-01-DMC to J.J.K.]; Ovarian Cancer Research Alliance and Phil and Judy Messing [597484], NIH [T32 CA009035]. Funding for open access charge: National Cancer Institute [R01CA214799]. Conflict of interest statement. None declared.
Funding Information:
US National Institutes of Health (NIH) Grants [P50 CA083638, 5P30 CA006927, R01CA214799]; Susan Komen [CCRCR17499048]; Department of Defense [OC130212 to N.J.]; American Cancer Society??Tri State CEOs Against Cancer Postdoctoral Fellowship [PF-19-097-01-DMC to J.J.K.]; Ovarian Cancer Research Alliance and Phil and Judy Messing [597484], NIH [T32 CA009035]. Funding for open access charge: National Cancer Institute [R01CA214799].
Publisher Copyright:
© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.
PY - 2021
Y1 - 2021
N2 - The RNF168 E3 ubiquitin ligase is activated in response to double stranded DNA breaks (DSBs) where it mono-ubiquitinates γH2AX (ub-H2AX). RNF168 protein expression and ubiquitin signaling are finely regulated during the sensing, repair and resolution of DNA damage in order to avoid excessive spreading of ubiquitinated chromatin. Supra-physiological RNF168 protein expression levels have been shown to block DNA end resection at DSBs and increase PARP inhibitor (PARPi) sensitivity. In this study, we examined the impact of ectopic RNF168 overexpression on hydroxyurea (HU)-induced stalled replication forks in the setting of BRCA1 deficiency. Surprisingly, RNF168 overexpression resulted in the extension of DNA fibers, despite the presence of HU, in BRCA1 deficient cells. Mechanistically, RNF168 overexpression recruited RAD18 to ub-H2AX at HU-induced DNA breaks. Subsequently, a RAD18-SLF1 axis was responsible for initiating DNA synthesis in a manner that also required the break-induced replication (BIR) factors RAD52 and POLD3. Strikingly, the presence of wild-type BRCA1 blocked RNF168-induced DNA synthesis. Notably, BIR-like repair has previously been linked with tandem duplication events found in BRCA1-mutated genomes. Thus, in the absence of BRCA1, excessive RNF168 expression may drive BIR, and contribute to the mutational signatures observed in BRCA1-mutated cancers.
AB - The RNF168 E3 ubiquitin ligase is activated in response to double stranded DNA breaks (DSBs) where it mono-ubiquitinates γH2AX (ub-H2AX). RNF168 protein expression and ubiquitin signaling are finely regulated during the sensing, repair and resolution of DNA damage in order to avoid excessive spreading of ubiquitinated chromatin. Supra-physiological RNF168 protein expression levels have been shown to block DNA end resection at DSBs and increase PARP inhibitor (PARPi) sensitivity. In this study, we examined the impact of ectopic RNF168 overexpression on hydroxyurea (HU)-induced stalled replication forks in the setting of BRCA1 deficiency. Surprisingly, RNF168 overexpression resulted in the extension of DNA fibers, despite the presence of HU, in BRCA1 deficient cells. Mechanistically, RNF168 overexpression recruited RAD18 to ub-H2AX at HU-induced DNA breaks. Subsequently, a RAD18-SLF1 axis was responsible for initiating DNA synthesis in a manner that also required the break-induced replication (BIR) factors RAD52 and POLD3. Strikingly, the presence of wild-type BRCA1 blocked RNF168-induced DNA synthesis. Notably, BIR-like repair has previously been linked with tandem duplication events found in BRCA1-mutated genomes. Thus, in the absence of BRCA1, excessive RNF168 expression may drive BIR, and contribute to the mutational signatures observed in BRCA1-mutated cancers.
UR - http://www.scopus.com/inward/record.url?scp=85084188912&partnerID=8YFLogxK
U2 - 10.1093/NAR/GKAA154
DO - 10.1093/NAR/GKAA154
M3 - Article
C2 - 32182354
AN - SCOPUS:85084188912
SN - 0305-1048
VL - 48
SP - 4298
EP - 4308
JO - Nucleic acids research
JF - Nucleic acids research
IS - 8
ER -