Abstract

Satellite DNA is the main component of functional centromeres and forms the main structural constituent of heterochromatin. The presence of alkali-labile sites (ALSs) is a feature inherent to chromatin structure. Here we aimed to characterize ALSs in different satellite DNA loci in cervical epithelial cells using DNA breakage detection coupled with fluorescence in situ hybridization (DBD–FISH). Cervical epithelial cells embedded in an agarose matrix were deproteinized and exposed to alkaline denaturation, which generated single-stranded DNA (ssDNA) starting from the ends of spontaneous basal DNA breaks and ALSs. The amount of ssDNA produced within a specific sequence area could be detected by DBD–FISH using specific probes. The DBD–FISH signals, which were corrected for respective FISH signals during metaphase, were remarkably stronger in the 5 bp classical satellite DNA domains analyzed (D1Z1, D9Z3, and D16Z3) compared with alphoid satellite regions (D3Z1, D8Z2, and DXZ1). D1Z1 locus of chromome-1 being the most affected by alkali denaturation, and contrary, D3Z1 locus of chromosome–3 was the least sensitive to alkali treatment. These findings suggest a high density of constitutive ALSs—probably abasic sites—within the 5 bp satellite DNA sequences in cervical epithelial cells. The presence and relative abundance of ALSs might help explain the high frequency of spontaneous breakage and rearrangements in the pericentromeric heterochromatin of chromosomes 1, 9, and 16 when this chromatin region is undercondensed spontaneously or via induction, such as following viral infections. ALSs in these sequences could be useful tools to monitor DNA damage in cases of cervical carcinogenesis. ALSs on these sequences could be useful tools to monitor DNA damage in cases of cervical carcinogenesis.

中文翻译：

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通过DNA断裂与荧光原位杂交（DNA-FISH）结合检测卫星DNA上的碱不稳定位点，监测宫颈上皮细胞中的DNA损伤

摘要

卫星DNA是功能着丝粒的主要组成部分，是异染色质的主要结构组成部分。碱不稳定位点（ALS）的存在是染色质结构固有的特征。在这里，我们旨在利用DNA断裂检测与荧光原位杂交（DBD–FISH）来表征宫颈上皮细胞中不同卫星DNA基因座中的ALS。嵌入琼脂糖基质中的宫颈上皮细胞被脱蛋白并暴露于碱性变性下，这会从自然的基础DNA断裂和ALS的末端开始产生单链DNA（ssDNA）。DBD–FISH可使用特定探针检测特定序列区域内产生的ssDNA数量。DBD–FISH信号，已在中期对各个FISH信号进行了校正，与Alphoid卫星区域（D3Z1，D8Z2和DXZ1）相比，在分析的5 bp经典卫星DNA域（D1Z1，D9Z3和D16Z3）中，它们的强度显着增强。染色体1的D1Z1基因座受碱变性的影响最大，相反，染色体3的D3Z1基因座对碱处理的敏感性最低。这些发现表明，在宫颈上皮细胞的5 bp卫星DNA序列中存在高密度的组成性ALS（可能是无碱基位点）。ALS的存在和相对丰度可能有助于解释当染色体1、9和16的染色质区域自发或通过诱导（例如在病毒感染后）进行欠浓缩时，自发性破损和重排的频率很高。这些序列中的ALS可能是监测子宫颈癌发生时DNA损伤的有用工具。这些序列上的ALS可能是监测子宫颈癌发生情况中DNA损伤的有用工具。