Regulation of 53BP1 Protein Stability by RNF8 and RNF168 Is Important for Efficient DNA Double-Strand Break Repair

Hu, Yiheng; Wang, Chao-Nan; Huang, Kun; Xia, Fang; Parvin, Jeffrey D.; Mondal, Neelima

doi:10.1371/journal.pone.0110522

Cited by 27 publications

(28 citation statements)

References 53 publications

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“…Following induction of DSBs and silencing of Wwox, NHEJ is measured by real time PCR using a probe that spans the junction of the break sites 26 . In 293/HW1 cells, silencing Wwox significantly decreased NHEJ by 50% ( P <0.05) (Figure 3c), indicating that, unlike HDR, Wwox expression enhanced NHEJ.…”

Section: Resultsmentioning

confidence: 99%

“…The NHEJ experiment was performed in Wwox-sufficient HEK293 cells with stably integrated NHEJ substrate, HW1. HDR, NHEJ, Alt-NHEJ and SSA assays were performed as previously described 22,26 . Wwox-deficient HeLa cells were transfected with 300 ng of myc-Wwox plasmid, empty myc vector, or myc-Wwox + siWwox where the myc-Wwox plasmid is resistant to silencing by the siRNAs.…”

Section: Methodsmentioning

confidence: 99%

“…For immunoblot analysis, cells were lysed with RIPA buffer (TFS, Waltham, MS) supplemented with Halt Protease Cocktail Inhibitors (TFS) or when detecting Brca1, lysed with low salt NP40 buffer as described 26 . When transfecting plasmids, ~75–85% confluent cells were overlayed with a mixture containing 2 μg of plasmid DNA, 12 μl Lipofectamine2000 (Invitrogen) in 200 μl Opti-Mem, and medium was changed 4–6 hrs later.…”

Section: Methodsmentioning

confidence: 99%

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Wwox–Brca1 interaction: role in DNA repair pathway choice

et al. 2016

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In this study, loss of expression of the fragile site encoded Wwox protein, was found to contribute to radiation and cisplatin resistance of cells, responses that could be associated with cancer recurrence and poor outcome. WWOX gene deletions occur in a variety of human cancer types and reduced Wwox protein expression can be detected early during cancer development. We find that Wwox loss is followed by mild chromosome instability in genomes of mouse embryo fibroblast cells from Wwox knockout mice. Human and mouse cells deficient for Wwox also exhibit significantly enhanced survival of ionizing radiation and bleomycin treatment, agents that induce double-strand breaks (DSBs). Cancer cells that survive radiation recur more rapidly in a xenograft model of irradiated breast cancer cells; Wwox-deficient cells exhibited significantly shorter tumor latencies vs Wwox-expressing cells. This Wwox effect has important consequences in human disease: in a cohort of cancer patients treated with radiation, Wwox-deficiency significantly correlated with shorter overall survival times. In examining mechanisms underlying Wwox-dependent survival differences, we found that Wwox-deficient cells exhibit enhanced homology directed repair (HDR), and decreased non-homologous end-joining repair, suggesting that Wwox contributes to DNA DSB repair pathway choice. Upon silencing of Rad51, a protein critical for HDR, Wwox-deficient cells were resensitized to radiation. We also demonstrated interaction of Wwox with Brca1, a driver of HDR, and show via immunofluorescent detection of repair proteins at ionizing radiation-induced DNA damage foci that Wwox expression suppresses DSB repair at the end-resection step of HDR. We propose a genome caretaker function for Wwox, in which Brca1-Wwox interaction supports non-homologous end-joining as the dominant DSB repair pathway in Wwox-sufficient cells. Together, the experimental results suggest that reduced Wwox expression, a common occurrence in cancers, dysregulates DSB repair, enhancing efficiency of likely mutagenic repair, and enabling radiation and cisplatin treatment resistance.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Wwox–Brca1 interaction: role in DNA repair pathway choice

et al. 2016

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“…However, the direct E3 ligase for 53BP1 remains elusive. Although RNF168 has been reported to regulate 53BP1 stability, it mediates K63‐linked, but not K48‐linked, ubiquitination of 53BP1 and is less likely to be the E3 ligase responsible for 53BP1 degradation . Besides, 53BP1 is sequentially phosphorylated by AuroraA and Plk1, and that the binding with Plk1 increases the stability of 53BP1 by accelerating its interaction with the deubiquitinase USP7 during mitosis .…”

Section: Discussionmentioning

confidence: 99%

Trip12 is an E3 ubiquitin ligase for USP7/HAUSP involved in the DNA damage response

Liu

Yang

et al. 2016

FEBS Letters

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The deubiquitinating enzyme, USP7/HAUSP (herpesvirus-associated ubiquitin-specific protease), is a key regulator of the tumor suppressor p53 and plays a major role in regulating genome stability. Here, we report that the protein stability of USP7 is regulated by the ubiquitin-proteasome pathway. We identified the thyroid hormone receptor interactor 12 (Trip12) as a ubiquitin E3 ligase for USP7. We also found that Trip12 affects USP7-mediated stabilization of p53 and the checkpoint proteins 53BP1 and Chk1. Knockdown of Trip12 leads to an increased cell population in G1 phase, mimicking USP7 overexpression. In contrast, Trip12 overexpression increased the number of cells in intra-S-phase, phenocopying the USP7 knockdown phenotype. Therefore, our data reveal an important modulatory role for Trip12 in the USP7-dependent DNA damage response.

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“…Digested DNA, 400 ng total DNA for input, and the C‐circle amplified products were dot‐blotted onto a nylon positively charged membrane (Amersham Hybond, GE Healthcare, Little Chalfont, United Kingdom) and crosslinked twice using a Strategene UV Stratalinker 2400. An end‐labeled P‐(CCCTAA) 3 probe was hybridized to membrane in Ultrahyb‐Oligo Hybridization Buffer (Ambion, Thermo Fisher Scientific) rotating at 42°C. Membranes were washed with 2× SSC, 0.5% SDS at 42°C and exposed to a phosphor screen (GE) overnight and imaged on a Typhoon FLA 7000 imager.…”

Section: Methodsmentioning

confidence: 99%

Differential requirements for DNA repair proteins in immortalized cell lines using alternative lengthening of telomere mechanisms

Martinez

Kaul

Parvin

et al. 2017

Genes Chromosomes & Cancer

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Cancer cells require telomere maintenance to enable uncontrolled growth. Most often telomerase is activated, although a subset of human cancers are telomerase-negative and depend on recombination-based mechanisms known as ALT (Alternative Lengthening of Telomeres). ALT depends on proteins that are essential for homologous recombination, including BLM and the MRN complex, to extend telomeres. This study surveyed the requirement for requisite homologous recombination proteins, yet to be studied in human ALT cell lines, by protein depletion using RNA interference. Effects on ALT were evaluated by measuring C-circle abundance, a marker of ALT. Surprisingly, several proteins essential for homologous recombination, BARD1, BRCA2, and WRN, were dispensable for C-circle production, while PALB2 had varying effects on C-circles among ALT cell lines. Depletion of homologous recombination proteins BRCA1 and BLM, which have been previously studied in ALT, decreased C-circles in all ALT cell lines. Depletion of the non-homologous end joining proteins 53BP1 and LIG4 had no effect on C-circles in any ALT cell line. Proteins such as chromatin modifiers that recruit double-strand break proteins, RNF8 and RNF168, and other proteins loosely grouped into excision DNA repair processes, XPA, MSH2, and MPG, reduced C-circles in some ALT cell lines. MSH2 depletion also reduced recombination at telomeres as measured by intertelomeric exchanges. Collectively, the requirement for DNA repair proteins varied between the ALT cell lines compared. In sum, our study suggests that ALT proceeds by multiple mechanisms that differ between cell lines and that some of these depend on DNA repair proteins not associated with homologous recombination pathways.

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Regulation of 53BP1 Protein Stability by RNF8 and RNF168 Is Important for Efficient DNA Double-Strand Break Repair

Cited by 27 publications

References 53 publications

Wwox–Brca1 interaction: role in DNA repair pathway choice

Wwox–Brca1 interaction: role in DNA repair pathway choice

Trip12 is an E3 ubiquitin ligase for USP7/HAUSP involved in the DNA damage response

Differential requirements for DNA repair proteins in immortalized cell lines using alternative lengthening of telomere mechanisms

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