Arnoult Nausica, Saintome Carole, Ourliac-Garnier Isabelle, Riou Jean-François, Londoño-Vallejo Arturo
Telomeres and Cancer Laboratory, Institut Curie, Paris 75248, France.
Genes Dev. 2009 Dec 15;23(24):2915-24. doi: 10.1101/gad.544009.
Mechanisms of telomere replication remain poorly defined. It has been suggested that G-rich telomeric strand replication by lagging mechanisms requires, in a stochastic way, the WRN protein. Here we show that this requirement is more systematic than previously thought. Our data are compatible with a situation in which, in the absence of WRN, DNA synthesis at replication forks is uncoupled, thus allowing replication to continue on the C strand, while single G strands accumulate. We also show that in cells in which both WRN and POT1 are limiting, both G- and C-rich telomeric strands shorten, suggesting a complete replication block. Under this particular condition, expression of a fragment spanning the two POT1-OB (oligonucleotide-binding) fold domains is able to restore C (but not G) strand replication, suggesting that binding of POT1 to the lagging strand allows DNA synthesis uncoupling in the absence of WRN. Furthermore, in vitro experiments indicate that purified POT1 has a higher affinity for the telomeric G-rich strand than purified RPA. We propose a model in which the relative enrichments of POT1 versus RPA on the telomeric lagging strand allows or does not allow uncoupling of DNA synthesis at the replication fork. Our study reveals an unanticipated role for hPOT1 during telomere replication.
端粒复制的机制仍不清楚。有人提出，通过滞后机制进行富含G的端粒链复制随机需要WRN蛋白。在此我们表明，这种需求比之前认为的更具系统性。我们的数据与以下情况相符：在没有WRN的情况下，复制叉处的DNA合成解偶联，从而使C链上的复制得以继续，而单链G链则积累。我们还表明，在WRN和POT1均有限的细胞中，富含G和C的端粒链都会缩短，这表明存在完全的复制阻滞。在这种特殊条件下，表达跨越两个POT1-OB（寡核苷酸结合）折叠结构域的片段能够恢复C链（而非G链）复制，这表明POT1与滞后链的结合能在没有WRN的情况下使DNA合成解偶联。此外，体外实验表明，纯化的POT1对富含G的端粒链的亲和力高于纯化的RPA。我们提出了一个模型，其中POT1与RPA在端粒滞后链上的相对富集情况决定了复制叉处的DNA合成是否解偶联。我们的研究揭示了hPOT1在端粒复制过程中一个意想不到的作用。
