SETDB1, HP1 and SUV39 promote repositioning of 53BP1 to extend resection during homologous recombination in G2 cells

Recent studies have shown that homologous recombination (HR) requires chromatin repression as well as relaxation at DNA double strand breaks (DSBs). HP1 and SUV39H1/2 are repressive factors essential for HR. Here, we identify SETDB1 as an additional compacting factor promoting HR. Depletion of HP1, SUV39, SETDB1 or BRCA1 confer identical phenotypes. The repressive factors, like BRCA1, are dispensable for the initiation of resection but promote the extension step causing diminished RPA or RAD51 foci and HR in irradiated G2 cells. Depletion of the compacting factors does not inhibit BRCA1 recruitment but at 8 h post IR, BRCA1 foci are smaller and aberrantly positioned compared to control cells. BRCA1 promotes 53BP1 repositioning to the periphery of enlarged foci and formation of a devoid core with BRCA1 becoming enlarged and localised internally to 53BP1. Depletion of the compacting factors precludes these changes at irradiation-induced foci. Thus, the repressive factors are required for BRCA1 function in promoting the repositioning of 53BP1 during HR. Additionally, depletion of these repressive factors in undamaged cells causes diminished sister chromatid association at centromeric sequences. We propose a model for how these findings may be functionally linked

Mouse Slfn8 and Slfn9 genes complement human cells lacking SLFN11 during the replication stress response

The Schlafen (SLFN)11 gene has been implicated in various biological processes such as suppression of HIV replication, replication stress response, and sensitization of cancer cells to chemotherapy. Due to the rapid diversification of the SLFN family members, it remains uncertain whether a direct ortholog of human SLFN11 exists in mice. Here we show that mSLFN8/9 and hSLFN11 were rapidly recruited to microlaser-irradiated DNA damage tracks. Furthermore, Slfn8/9 expression could complement SLFN11 loss in human SLFN11⁻⁄⁻ cells, and as a result, reduced the growth rate to wild-type levels and partially restored sensitivity to DNA-damaging agents. In addition, both Slfn8/9 and SLFN11 expression accelerated stalled fork degradation and decreased RPA and RAD51 foci numbers after DNA damage. Based on these results, we propose that mouse Slfn8 and Slfn9 genes may share an orthologous function with human SLFN11. This notion may facilitate understanding of SLFN11’s biological role through in vivo studies via mouse modeling

Co-operation of BRCA1 and POH1 relieves the barriers posed by 53BP1 and RAP80 to resection

In G2 phase cells, DNA double-strand break repair switches from DNA non-homologous end-joining to homologous recombination. This switch demands the promotion of resection. We examine the changes in 53BP1 and RAP80 ionizing radiation induced foci (IRIF) in G2 phase, as these are factors that restrict resection. We observed a 2-fold increase in the volume of 53BP1 foci by 8 h, which is not seen in G1 cells. Additionally, an IRIF core devoid of 53BP1 arises where RPA foci form, with BRCA1 IRIF forming between 53BP1 and replication protein A (RPA). Ubiquitin chains assessed using a-FK2 antibodies are similarly repositioned. Repositioning of all these components requires BRCA1’s BRCT but not the ring finger domain. 53BP1, RAP80 and ubiquitin chains are enlarged following POH1 depletion by small interfering RNA, but a devoid core does not form and RPA foci formation is impaired. Co-depletion of POH1 and RAP80, BRCC36 or ABRAXAS allows establishment of the 53BP1 and ubiquitin chain-devoid core. Thus, the barriers posed by 53BP1 and RAP80 are relieved by BRCA1 and POH1, respectively. Analysis of combined depletions shows that these represent distinct but interfacing barriers to promote loss of ubiquitin chains in the IRIF core, which is required for subsequent resection. We propose a model whereby BRCA1 impacts on 53BP1 to allow access of POH1 to RAP80. POH1-dependent removal of RAP80 within the IRIF core enables degradation of ubiquitin chains, which promotes loss of 53BP1. Thus, POH1 represents a novel component regulating the switch from nonhomologous end-joining to homologous recombination

RNF168 E3 ligase participates in ubiquitin signaling and recruitment of SLX4 during DNA crosslink repair

遺伝性血液疾患の原因タンパク質を制御する新規のユビキチン経路を解明 --ファンコニ貧血にかかわる新たな関連因子群の同定--. 京都大学プレスリリース. 2021-10-28.SLX4/FANCP is a key Fanconi anemia (FA) protein and a DNA repair scaffold for incision around a DNA interstrand crosslink (ICL) by its partner XPF nuclease. The tandem UBZ4 ubiquitin-binding domains of SLX4 are critical for the recruitment of SLX4 to damage sites, likely by binding to K63-linked polyubiquitin chains. However, the identity of the ubiquitin E3 ligase that mediates SLX4 recruitment remains unknown. Using small interfering RNA (siRNA) screening with a GFP-tagged N-terminal half of SLX4 (termed SLX4-N), we identify the RNF168 E3 ligase as a critical factor for mitomycin C (MMC)-induced SLX4 foci formation. RNF168 and GFP-SLX4-N colocalize in MMC-induced ubiquitin foci. Accumulation of SLX4-N at psoralen-laser ICL tracks or of endogenous SLX4 at Digoxigenin-psoralen/UVA ICL is dependent on RNF168. Finally, we find that RNF168 is epistatic with SLX4 in promoting MMC tolerance. We conclude that RNF168 is a critical component of the signal transduction that recruits SLX4 to ICL damage

Early G2/M checkpoint failure as a molecular mechanism underlying etoposide-induced chromosomal aberrations

Topoisomerase II (Topo II) inhibitors are cell cycle–specific DNA-damaging agents and often correlate with secondary leukemia with chromosomal translocations involving the mixed-lineage leukemia/myeloid lymphoid leukemia (MLL) gene on chromosome 11 band q23 (11q23). In spite of the clinical importance, the molecular mechanism for this chromosomal translocation has yet to be elucidated. In this study, we employed 2-color FISH and detected intracellular chromosomal translocations induced by etoposide treatment. Cells such as ataxia-telangiectasia mutated–deficient fibroblasts and U2OS cells, in which the early G2/M checkpoint after treatment with low concentrations of etoposide has been lost, executed mitosis with etoposide-induced DNA double-strand breaks, and 2-color FISH signals located on either side of the MLL gene were segregated in the postmitotic G1 phase. Long-term culture of cells that had executed mitosis under etoposide treatment showed frequent structural abnormalities of chromosome 11. These findings provide convincing evidence for Topo II inhibitor–induced 11q23 translocation. Our study also suggests an important role of the early G2/M checkpoint in preventing fixation of chromosomal abnormalities and reveals environmental and genetic risk factors for the development of chromosome 11 translocations, namely, low concentrations of Topo II inhibitors and dysfunctional early G2/M checkpoint control