Alterations of DNA and chromatin structures at telomeres and genetic instability in mouse cells defective in DNA polymerase α
Alterations of DNA and chromatin structures at telomeres and genetic instability in mouse cells defective in DNA polymerase α
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DOI：
10.1128/mcb.25.24.11073-11088.2005
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发表时间：
2005-12-01
影响因子：
5.3
通讯作者：
Ishikawa, F
中科院分区：
文献类型：
--
作者：
Nakamura, M;Nabetani, A;Ishikawa, F
端粒长度由涉及端粒酶、端粒相关蛋白和传统复制机制的稳态机制控制。具体来说，滞后链合成和端粒酶的协调作用一直存在争议。尽管 DNA 聚合酶 α（一种对滞后链合成很重要的酶）已被证明在酵母和纤毛虫的端粒代谢中发挥作用，但尚未在高等真核生物中对其进行表征。在这里，我们使用含有温度敏感聚合酶 α 突变等位基因的 tsFT20 小鼠突变细胞研究了聚合酶 α 活性受损对端粒的影响。当聚合酶α被温度诱导失活时，我们观察到一系列连续事件，包括G尾的初始延伸，随后分别以不依赖端粒酶和依赖端粒酶的方式发生整体端粒长度的显着增加。 TRF1 和 POT1 的定量染色质免疫沉淀和免疫荧光分析表明，端粒 DNA 的这些改变伴随着端粒染色质结构的改变。出乎意料的是，聚合酶和抑制导致罗伯逊染色体融合的发生率显着升高，而其他类型的染色体畸变却没有明显增加。这些结果表明，尽管 DNA 聚合酶 α 对于全基因组 DNA 复制至关重要，但低等位性活性会导致一系列相当特殊的染色体异常。
Telomere length is controlled by a homeostatic mechanism that involves telomerase, telomere-associated proteins, and conventional replication machinery. Specifically, the coordinated actions of the lagging strand synthesis and telomerase have been argued. Although DNA polymerase alpha, an enzyme important for the lagging strand synthesis, has been indicated to function in telomere metabolism in yeasts and ciliates, it has not been characterized in higher eukaryotes. Here, we investigated the impact of compromised polymerase alpha activity on telomeres, using tsFT20 mouse mutant cells harboring a temperature-sensitive polymerase alpha mutant allele. When polymerase alpha was temperature-inducibly inactivated, we observed sequential events that included an initial extension of the G-tail followed by a marked increase in the overall telomere length occurring in telomerase-independent and -dependent manners, respectively. These alterations of telomeric DNA were accompanied by alterations of telomeric chromatin structures as revealed by quantitative chromatin immunoprecipitation and immunofluorescence analyses of TRF1 and POT1. Unexpectedly, polymerase et inhibition resulted in a significantly high incidence of Robertsonian chromosome fusions without noticeable increases in other types of chromosomal aberrations. These results indicate that although DNA polymerase alpha is essential for genome-wide DNA replication, hypomorphic activity leads to a rather specific spectrum of chromosomal abnormality.