Dataset Information

Cyclin F - EXO1 axis controls cell cycle dependent execution of double strand break repair

ABSTRACT: Ubiquitination is a crucial post-translational modification required for the proper repair of DNA double-strand breaks (DSBs) induced by ionising radiation (IR). DSBs are mainly repaired through homologous recombination (HR) when template DNA is present and non-homologous end joining (NHEJ) in its absence. Additionally, microhomology-mediated end joining (MMEJ) and single strand annealing (SSA) provide back-up DSBs repair pathways. However, the mechanisms controlling their use remain poorly understood. By employing a high-resolution CRISPR screen of the ubiquitin system after IR, we systematically uncover genes required for cell survival and elucidate a critical role of the E3 ubiquitin ligase SCFcyclin F in cell cycle dependent DSB repair. We show that SCFcyclin F -mediated EXO1 degradation prevents DNA end resection in mitosis, allowing MMEJ to take place. Moreover, we identify a conserved cyclin F recognition motif, distinct from the one used by other cyclins, with broad implications in cyclin specificity for cell cycle control.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Embryonic Stem Cell

SUBMITTER: iolanda Vendrell

LAB HEAD: Vincenzo D’Angiolella

PROVIDER: PXD051841 | Pride | 2024-10-17

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	FL1320_MSQ2101_20210316_PSmith_1_ccnf-nobiot_1.raw	Raw
	FL1320_MSQ2101_20210316_PSmith_2_ccnf-nobiot_2.raw	Raw
	FL1320_MSQ2101_20210316_PSmith_3_ccnf-nobiot_3.raw	Raw
	FL1320_MSQ2101_20210316_PSmith_4_ccnf_1.raw	Raw
	FL1320_MSQ2101_20210316_PSmith_5_ccnf_2.raw	Raw

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Publications

Cyclin F-EXO1 axis controls cell cycle-dependent execution of double-strand break repair.

Yang Hongbin H Fouad Shahd S Smith Paul P Bae Eun Young EY Ji Yu Y Lan Xinhui X Van Ess Ava A Buffa Francesca M FM Fischer Roman R Vendrell Iolanda I Kessler Benedikt M BM D'Angiolella Vincenzo V

Science advances 20240809 32

Ubiquitination is a crucial posttranslational modification required for the proper repair of DNA double-strand breaks (DSBs) induced by ionizing radiation (IR). DSBs are mainly repaired through homologous recombination (HR) when template DNA is present and nonhomologous end joining (NHEJ) in its absence. In addition, microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA) provide backup DSBs repair pathways. However, the mechanisms controlling their use remain poorly understo ...[more]

PMID: 39121215

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Project description:Classical non-homologous end-joining (C-NHEJ) is a major mammalian DNA double strand break (DSB) repair pathway. Core C-NHEJ factors, such as XRCC4, are required for joining DSB intermediates of the G1 phase-specific V(D)J recombination reaction in progenitor lymphocytes. Core factors also contribute to joining DSBs in cycling mature B-lineage cells, including DSBs generated during antibody class switch recombination (CSR) and DSBs generated by ionizing radiation (IR). The XLF C-NHEJ factor is dispensable for V(D)J recombination in normal cells, but, due to functional redundancy, is absolutely required for this process in cells deficient for the ATM DSB response factor. The recently identified PAralogue of XRCC4 and XLF (PAXX) factor has homology to these two proteins and variably contributes to IR-induced DSB repair in human and chicken cells. We now report that PAXX is dispensable for joining V(D)J recombination DSBs in G1-arrested mouse pro-B cell lines, dispensable for joining CSR-associated DSBs in a cycling mouse B cell line, and dispensable for normal IR-resistance in both G1-arrested and cycling pro-B lines. However, we find that combined deficiency for PAXX and XLF in G1-arrested pro-B lines abrogates DSB joining during V(D)J recombination and sensitizes the cells to IR exposure. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested Pro-B cell lines. Finally, we also find that PAXX-deficiency has no impact on V(D)J recombination DSB joining in ATM-deficient pro-B lines. We discuss implications of these findings with respect to potential PAXX and XLF functions in C-NHEJ. Examination of CSR Switch mu-to-alpha junctions from mu bait DSBs using LAM-HTGTS and Illumina Miseq. Two clones of PAXX-/- and XLF-/- and 1 clone of Ligase4-/- were derived from the parental CH12F3 line; the second Ligase4-/- clone was acquired from Keifei Yu. Two clones of XLF-/-PAXX-/-CH12 cells were derived from one of the PAXX-/- clones. Three biological replicates were analyzed for each clone.