DNA Ligase IV Prevents Replication Fork Stalling and Promotes Cellular Proliferation in Triple Negative Breast Cancer.

IF 1.3 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Journal of Nucleic Acids Pub Date : 2019-01-31 eCollection Date: 2019-01-01 DOI:10.1155/2019/9170341
Rashmi R Joshi, Sk Imran Ali, Amanda K Ashley
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引用次数: 9

Abstract

DNA damage is a hallmark of cancer, and mutation and misregulation of proteins that maintain genomic fidelity are associated with the development of multiple cancers. DNA double strand breaks are arguably considered the most deleterious type of DNA damage. The nonhomologous end-joining (NHEJ) pathway is one mechanism to repair DNA double strand breaks, and proteins involved in NHEJ may also regulate DNA replication. We previously established that DNA-PKcs, a NHEJ protein, promotes genomic stability and cell viability following cellular exposure to replication stress; we wanted to discern whether another NHEJ protein, DNA ligase IV (Lig4), shares this phenotype. Our investigations focused on triple negative breast cancer cells, as, compared to nonbasal breast cancer, LIG4 is frequently amplified, and an increased gene dose is associated with higher Lig4 expression. We depleted Lig4 using siRNA and confirmed our knockdown by qPCR and western blotting. Cell survival diminished with Lig4 depletion alone, and this was associated with increased replication fork stalling. Checkpoint protein Chk1 activation and dephosphorylation were unchanged in Lig4-depleted cells. Lig4 depletion resulted in sustained DNA-PKcs phosphorylation following hydroxyurea exposure. Understanding the effect of Lig4 on genomic replication and the replication stress response will clarify the biological ramifications of inhibiting Lig4 activity. In addition, Lig4 is an attractive clinical target for directing CRISPR/Cas9-mediated repair towards homology-directed repair and away from NHEJ, thus understanding of how diminishing Lig4 impacts cell biology is critical.

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DNA连接酶IV阻止三阴性乳腺癌的复制叉停滞并促进细胞增殖。
DNA损伤是癌症的一个标志,维持基因组保真度的蛋白质的突变和错误调节与多种癌症的发展有关。DNA双链断裂被认为是最有害的DNA损伤类型。非同源末端连接(NHEJ)途径是修复DNA双链断裂的一种机制,参与NHEJ的蛋白也可能调控DNA复制。我们之前已经确定DNA-PKcs(一种NHEJ蛋白)在细胞暴露于复制应激后促进基因组稳定性和细胞活力;我们想要了解另一种NHEJ蛋白,DNA连接酶IV (Lig4)是否也具有这种表型。我们的研究重点是三阴性乳腺癌细胞,因为与非基底乳腺癌相比,LIG4经常被扩增,基因剂量的增加与LIG4的高表达相关。我们使用siRNA去除Lig4,并通过qPCR和western blotting证实了我们的敲除。仅Lig4耗竭就会降低细胞存活率,这与复制叉延迟增加有关。在lig4缺失的细胞中,检查点蛋白Chk1的激活和去磷酸化没有变化。Lig4缺失导致羟基脲暴露后DNA-PKcs持续磷酸化。了解Lig4对基因组复制和复制应激反应的影响将阐明抑制Lig4活性的生物学后果。此外,Lig4是一个有吸引力的临床靶点,可以将CRISPR/ cas9介导的修复从NHEJ引导到同源定向修复,因此了解减少Lig4如何影响细胞生物学是至关重要的。
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来源期刊
Journal of Nucleic Acids
Journal of Nucleic Acids BIOCHEMISTRY & MOLECULAR BIOLOGY-
CiteScore
3.10
自引率
21.70%
发文量
5
审稿时长
12 weeks
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