Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions

Kumar, Tadi Satish; Sebastian, Robin; Dahal, Sumedha; Babu, Ravi K; Choudhary, Bibha; Raghavan, Sathees C.

doi:10.1091/mbc.e15-05-0260

Cited by 108 publications

(143 citation statements)

References 84 publications

(110 reference statements)

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“…Previous studies have suggested that different cancer cells have varying levels of c‐NHEJ . Although it was correlated with differential expression of NHEJ proteins, levels of BCL2 expression were also responsible as it can interact with KU proteins . Observed robust expression of BCL2 in SUDHL8, further support our finding of weak c‐NHEJ in SUDHL8 cells (data not shown).…”

Section: Discussionsupporting

confidence: 88%

“…Importantly, background recombination due to bacterial proteins is subtracted from recombination catalyzed by DLBCL extracts in every experiment. Besides, previously such biochemical assay systems have been used extensively to assess HR mediated repair in human and other mammalian cells . Thus, it appears that HR mediated DSB repair is operational to maintain the integrity of the genome in cell cycle stages when the homologous chromosome partner is available.…”

Section: Discussionmentioning

confidence: 99%

“…There are evidences of low levels of MMEJ in normal cells, which is enhanced in the absence of c‐NHEJ. MMEJ is also shown to be the prominent repair pathway in mitochondria . Although MMEJ helps in the repair of DNA double‐strand breaks even in the absence of c‐NHEJ proteins, it can result in larger deletions, insertions, leading to chromosomal rearrangements.…”

Section: Discussionmentioning

confidence: 99%

“…Alternative NHEJ was studied using MMEJ assay as described earlier . A 13 nt microhomology substrate was incubated with cell‐free extracts prepared from SUDHL8, NALM6, and REH cells or shRNA treated DLBCL cells in MMEJ buffer (50 mM Tris‐HCl [pH 7.6], 1 mM DTT, 1 mM ATP, and 20 mM MgCl 2 ) for 1 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

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Characterization of DNA double‐strand break repair pathways in diffuse large B cell lymphoma

Gopalakrishnan

Dahal

Radha

et al. 2018

Molecular Carcinogenesis

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Efficient DNA repair is indispensable for maintaining genomic integrity in humans. Cancer associated deletions and mutations are mainly due to misrepaired DNA double‐strand breaks (DSBs). Classical nonhomologous end joining (c‐NHEJ) and homologous recombination (HR) are two major DSB repair pathways in humans. An error prone, alternative NHEJ pathway that utilizes microhomology was also reported in cancer cells and to a lesser extent in normal cells. In the present study, we evaluated the efficiency of various DSB repair pathways in the most common lymphoma, the diffuse large B cell lymphoma (DLBCL). Here we show that DNA repair through c‐NHEJ pathway is limited in SUDHL8, a cell line derived from a DLBCL patient. Unlike c‐NHEJ, microhomology mediated end joining (MMEJ) was predominant at physiological temperature. Consistent with the observation, expression level of repair proteins such as LIGASE I, LIGASE III, PARP1, CtIP, and MRE11 was higher in DLBCL cells when compared to c‐NHEJ proteins. Further, inhibition of LIGASE I or MRE11, led to reduction in the efficiency of MMEJ in DLBCL cells. Besides, HR‐mediated DSB repair occurring through gene conversion was observed. Thus, our results reveal the predominance of MMEJ over c‐NHEJ in repairing DSBs in DLBCL cells, while error‐free repair through HR was also evident.

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Section: Discussionsupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Characterization of DNA double‐strand break repair pathways in diffuse large B cell lymphoma

Gopalakrishnan

Dahal

Radha

et al. 2018

Molecular Carcinogenesis

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“…DNA Ligase IV/XRCC4 along with XLF and PAXX help in final ligation of DNA ends . In case of MMEJ, Ligase III and/or Ligase I along with other proteins help in repair of DSBs, whereas during HR, the Ligase involved is Ligase I [].…”

Section: Introductionmentioning

confidence: 99%

Autocyclized and oxidized forms ofSCR7 induce cancer cell death by inhibiting nonhomologousDNAend joining in a LigaseIVdependent manner

et al. 2018

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Nonhomologous DNA end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammals. Previously, we have described a small molecule inhibitor, SCR7, which can inhibit NHEJ in a Ligase IV-dependent manner. Administration of SCR7 within the cells resulted in the accumulation of DNA breaks, cell death, and inhibition of tumor growth in mice. In the present study, we report that parental SCR7, which is unstable, can be autocyclized into a stable form. Both parental SCR7 and cyclized SCR7 possess the same molecular weight (334.09) and molecular formula (C H N OS), whereas its oxidized form, SCR7-pyrazine, possesses a different molecular formula (C H N OS), molecular weight (332.07), and structure. While cyclized form of SCR7 showed robust inhibition of NHEJ in vitro, both forms exhibited efficient cytotoxicity. Cyclized and oxidized forms of SCR7 inhibited DNA end joining catalyzed by Ligase IV, whereas their impact was minimal on Ligase III, Ligase I, and T4 DNA Ligase-mediated joining. Importantly, both forms inhibited V(D)J recombination, although the effect was more pronounced for SCR7-cyclized. Both forms blocked NHEJ in a Ligase IV-dependent manner leading to the accumulation of DSBs within the cells. Although cytotoxicity due to SCR7-cyclized was Ligase IV specific, the pyrazine form exhibited nonspecific cytotoxicity at higher concentrations in Ligase IV-null cells. Finally, we demonstrate that both forms can potentiate the effect of radiation. Thus, we report that cyclized and oxidized forms of SCR7 can inhibit NHEJ in a Ligase IV-dependent manner, although SCR7-pyrazine is less specific to Ligase IV inside the cell.

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FEN1 inhibitor increases sensitivity of radiotherapy in cervical cancer cells

Wang

Chang

et al. 2019

Cancer Medicine

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BackgroundCervical cancer is one of the most common causes of cancer‐associated mortality among affected women in the world. At present, treatment with weekly cisplatin plus ionizing radiation (IR) therapy is the standard regimen for cervical cancer, especially for locally advanced cervical cancer. The purpose of this study is to determine whether FEN1 inhibitors could enhance the therapeutic effect of IR therapy.MethodsWestern blot was applied to determine the expression of FEN1‐ and apoptosis‐related proteins. Cell growth inhibition assay and colony formation assay were used to determine the effects of FEN1 inhibitor and IR exposure for Hela cells in vitro. CRISPR technology was used to knockdown FEN1 expression level of 293T cells, and tumor xenograft in nude mice was employed to determine the effects of FEN1 inhibitor and IR exposure on tumor growth in vivo.ResultsOur data revealed that FEN1 is overexpressed in HeLa cell and can be upregulated further by IR. We also demonstrated that FEN1 inhibitor enhances IR sensitivity of cervical cancer in vitro and in vivo.ConclusionFEN1 inhibitor SC13 could sensitize radiotherapy of cervical cancer cell.

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Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions

Cited by 108 publications

References 84 publications

Characterization of DNA double‐strand break repair pathways in diffuse large B cell lymphoma

Characterization of DNA double‐strand break repair pathways in diffuse large B cell lymphoma

Autocyclized and oxidized forms ofSCR7 induce cancer cell death by inhibiting nonhomologousDNAend joining in a LigaseIVdependent manner

FEN1 inhibitor increases sensitivity of radiotherapy in cervical cancer cells

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