Temporal differences in DNA replication during the S phase using single fiber analysis of normal human fibroblasts and glioblastoma T98G cells.
Temporal differences in DNA replication during the S phase using single fiber analysis of normal human fibroblasts and glioblastoma T98G cells.
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DOI：
10.4161/cc.8.19.9682
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发表时间：
2009-10-01
期刊：
影响因子：
--
通讯作者：
Kaufman DG
中科院分区：
文献类型：
--
作者：
Frum RA;Khondker ZS;Kaufman DG
我们最近发现，复制叉在正常人成纤维细胞 (NHF1-hTERT) 的起点附近暂停，但在胶质母细胞瘤 T98G 细胞中则不然。这一观察结果使我们质疑这些细胞类型之间复制程序是否可能存在其他差异，这些差异可能与其遗传完整性有关。为了识别差异，我们在同步 S 期的每个小时开始时，通过免疫荧光检测了核苷酸类似物 IdU 和 CldU 顺序掺入复制 DNA 的情况。然后，我们对标记的复制 DNA 轨迹的模式进行了表征，并量化了每小时间隔发现的轨迹的百分比和长度。根据单个延伸复制 DNA 纤维中标记的方向性，轨迹被分类为单个双向起点、单向伸长、串联发射的起点簇或合并叉（终止）。我们的分析表明，NHF1-hTERT 细胞中 S 期的开始富含单一双向起点，随后在 S 期中期聚类增加，在 S 期后期合并叉增加。 T98G 细胞的早期 S 期也主要由单一双向起始起始组成；然而，簇的增加延迟到一个小时后，并且S期中/晚期的簇比NHF1-hTERT细胞中的簇更短。 T98G 细胞中的合并叉的峰值也直到一个小时后才出现。我们的观察结果提出了解释单个和集群起源的时间复制的模型，并表明正常细胞系和癌细胞系中复制程序的差异。
We have recently shown that replication forks pause near origins in normal human fibroblasts (NHF1-hTERT) but not glioblastoma T98G cells. This observation led us to question whether other differences in the replication program may exist between these cell types that may relate to their genetic integrity. To identify differences, we detected immunoflourescently the sequential incorporation of the nucleotide analogs IdU and CldU into replicating DNA at the start of every hour of a synchronized S phase. We then characterized the patterns of labeled replicating DNA tracks and quantified the percentages and lengths of the tracks found at these hourly intervals. From the directionality of labeling in single extended replicating DNA fibers, tracks were categorized as single bidirectional origins, unidirectional elongations, clusters of origins firing in tandem, or merging forks (terminations). Our analysis showed that the start of S phase is enriched in single bidirectional origins in NHF1-hTERT cells, followed by an increase in clustering during mid S phase and an increase in merging forks during late S phase. Early S phase in T98G cells also largely consisted of single bidirectional origin initiations; however, an increase in clustering was delayed until an hour later, and clusters were shorter in mid/late S phase than in NHF1-hTERT cells. The spike in merging forks also did not occur until an hour later in T98G cells. Our observations suggest models to explain the temporal replication of single and clustered origins, and suggest differences in the replication program in a normal and cancer cell line.
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影响因子：
4
作者：
Daboussi, Fayza;Courbet, Sylvain;Lopez, Bernard S.
通讯作者：
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DOI：
10.1073/pnas.94.9.4587
发表时间：
1997-04-29
影响因子：
11.1
作者：
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DOI：
10.1016/0006-291x(86)91125-3
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作者：
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作者：
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影响因子：
14.9
作者：
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通讯作者：
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