Opresko Patricia L, Sowd Gregory, Wang Hong
Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania, United States of America.
PLoS One. 2009;4(3):e4825. doi: 10.1371/journal.pone.0004825. Epub 2009 Mar 13.
RecQ DNA helicases are critical for preserving genome integrity. Of the five RecQ family members identified in humans, only the Werner syndrome protein (WRN) possesses exonuclease activity. Loss of WRN causes the progeroid disorder Werner syndrome which is marked by cancer predisposition. Cellular evidence indicates that WRN disrupts potentially deleterious intermediates in homologous recombination (HR) that arise in genomic and telomeric regions during DNA replication and repair. Precisely how the WRN biochemical activities process these structures is unknown, especially since the DNA unwinding activity is poorly processive. We generated biologically relevant mobile D-loops which mimic the initial DNA strand invasion step in HR to investigate whether WRN biochemical activities can disrupt this joint molecule. We show that WRN helicase alone can promote branch migration through an 84 base pair duplex region to completely displace the invading strand from the D-loop. However, substrate processing is altered in the presence of the WRN exonuclease activity which degrades the invading strand both prior to and after release from the D-loop. Furthermore, telomeric D-loops are more refractory to disruption by WRN, which has implications for tighter regulation of D-loop processing at telomeres. Finally, we show that WRN can recognize and initiate branch migration from both the 5' and 3' ends of the invading strand in the D-loops. These findings led us to propose a novel model for WRN D-loop disruption. Our biochemical results offer an explanation for the cellular studies that indicate both WRN activities function in processing HR intermediates.
RecQ DNA解旋酶对于维持基因组完整性至关重要。在人类中鉴定出的五个RecQ家族成员中，只有沃纳综合征蛋白（WRN）具有核酸外切酶活性。WRN的缺失会导致早衰性疾病沃纳综合征，其特征是易患癌症。细胞证据表明，WRN会破坏DNA复制和修复过程中在基因组和端粒区域出现的同源重组（HR）中潜在有害的中间体。目前尚不清楚WRN的生化活性究竟如何处理这些结构，特别是因为DNA解旋活性的持续性很差。我们生成了生物学上相关的可移动D环，模拟HR中的初始DNA链侵入步骤，以研究WRN的生化活性是否能破坏这种连接分子。我们发现，单独的WRN解旋酶可以促进通过一个84碱基对的双链区域进行分支迁移，从而将侵入链从D环中完全置换出来。然而，在存在WRN核酸外切酶活性的情况下，底物处理会发生改变，该活性会在从D环释放之前和之后降解侵入链。此外，端粒D环比WRN破坏更具抗性，这对端粒处D环加工的更严格调控具有重要意义。最后，我们表明WRN可以识别并从D环中侵入链的5'和3'末端启动分支迁移。这些发现使我们提出了一个关于WRN破坏D环的新模型。我们的生化结果为细胞研究提供了解释，表明WRN的两种活性在处理HR中间体中都起作用。
