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Author:
Date:
2020-08-05
[Abstract] DNA double strand breaks (DSBs) are among the most toxic lesions affecting genome integrity. DSBs are mainly repaired through non-homologous end joining (NHEJ) and homologous recombination (HR). A crucial step of the HR process is the generation, through DNA end-resection, of a long 3′ single-strand DNA stretch, necessary to prime DNA synthesis using a homologous region as a template, following DNA strand invasion. DNA end resection inhibits NHEJ and triggers homology-directed DSB repair, ultimately guaranteeing a faithful DNA repair. Established methods to evaluate the DNA end-resection process are the immunofluorescence analysis of the phospho-S4/8 RPA32 protein foci, a marker of DNA end-resection, or of the phospho-S4/8 RPA32 protein levels by Western blot. Recently, the Single ...
[摘要] [摘要] DNA双链断裂（dsb）是影响基因组完整性的最具毒性的损伤之一。dsb主要通过非同源末端连接（NHEJ）和同源重组（HR）进行修复。HR过程的一个关键步骤是通过DNA末端切除，产生一个长的3′单链DNA链，这是在DNA链入侵后，以同源区域为模板进行DNA合成所必需的。DNA末端切除抑制NHEJ并触发同源定向的DSB修复，最终保证DNA的可靠修复。已建立的评价DNA末端切除过程的方法是免疫荧光法分析磷酸化S4/8rpa32蛋白病灶（DNA末端切除的标志物）或磷酸化S4/8rpa32蛋白水平。近年来，切除轨迹单分子分析（SMART）被认为是一种可靠的方法，可以通过免疫荧光法观察S期特异性DNA损伤剂（如喜树碱）处理细胞后产生的长3′单链DNA尾。然后，通过图像分析软件（如Photoshop）测量DNA束长度，评价DNA末端切除机的处理能力。DNA纤维的制备是在非变性条件下进行的，因此免疫荧光只检测DSB处理产生的特定的3′单链DNA尾。
[背景] ...
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Author:
Date:
2017-06-05
[Abstract] Genome stability is continuously challenged by a wide range of DNA damaging factors. To promote a correct DNA repair and cell survival, cells orchestrate a coordinated and finely tuned cascade of events collectively known as the DNA Damage Response (DDR). Ultra Violet (UV) rays are among the main environmental sources of DNA damage and a well recognized cancer risk factor. UV rays induce the formation of toxic cyclobutane-type pyrimidine dimers (CPD) and [6-4]pyrimidine-pyrimidone (6-4PP) photoproducts which trigger the activation of the intra-S phase cell cycle checkpoint (Kaufmann, 2010) aimed at preventing replication fork collapse, late origin firing, and stabilizing fragile sites (Branzei and Foiani, 2009). To monitor the activation of the intra-S phase checkpoint in response to UV ...
[摘要] 基因组稳定性不断受到DNA损伤因素的广泛影响。为了促进正确的DNA修复和细胞存活，细胞协调统一称为DNA损伤反应（DDR）的协调和精细调整的事件级联。超紫外线（UV）是DNA损伤的主要环境来源之一，也是公认的癌症危险因素。紫外线诱导形成毒性环丁烷型嘧啶二聚体（CPD）和[6-4]嘧啶嘧啶酮（6-4PP）光产物，其触发S期细胞周期检查点的活化（Kaufmann，2010），目的在于防止复制叉崩溃，晚期起火和稳定脆弱场所（Branzei和Foiani，2009）。为了监测响应于UV型C（UVC）暴露的S-S相检查点的激活，DNA纤维测定可用于分析新的起始点和DNA合成速率（Jackson等， ，1998; Merrick等人，2004; Alfano等人，2016）。 DNA纤维测定技术在90年代被设想，然后通过使用并入新生DNA链中的胸苷类似物（如CldU和IdU）进一步开发。通过用这些类似物以连续模式处理细胞，可以通过携带不同荧光团的特异性抗体来观察细胞，可以监测复制叉活性并评估其如何受到紫外辐射或其他试剂的影响。
背景 ...
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