Gocha April Renee Sandy, Acharya Samir, Groden Joanna
Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, Ohio, United States of America.
PLoS One. 2014 Apr 7;9(4):e93991. doi: 10.1371/journal.pone.0093991. eCollection 2014.
Telomere maintenance can occur in the presence of telomerase or in its absence, termed alternative lengthening of telomeres (ALT). ALT adds telomere repeats using recombination-based processes and DNA repair proteins that function in homologous recombination. Our previous work reported that the RecQ-like BLM helicase is required for ALT and that it unwinds telomeric substrates in vitro. WRN is also a RecQ-like helicase that shares many biochemical functions with BLM. WRN interacts with BLM, unwinds telomeric substrates, and co-localizes to ALT-associated PML bodies (APBs), suggesting that it may also be required for ALT processes. Using long-term siRNA knockdown of WRN in three ALT cell lines, we show that some, but not all, cell lines require WRN for telomere maintenance. VA-13 cells require WRN to prevent telomere loss and for the formation of APBs; Saos-2 cells do not. A third ALT cell line, U-2 OS, requires WRN for APB formation, however WRN loss results in p53-mediated apoptosis. In the absence of WRN and p53, U-2 OS cells undergo telomere loss for an intermediate number of population doublings (50-70), at which point they maintain telomere length even with the continued loss of WRN. WRN and the tumor suppressor BRCA1 co-localize to APBs in VA-13 and U-2 OS, but not in Saos-2 cells. WRN loss in U-2 OS is associated with a loss of BRCA1 from APBs. While the loss of WRN significantly increases telomere sister chromatid exchanges (T-SCE) in these three ALT cell lines, loss of both BRCA1 and WRN does not significantly alter T-SCE. This work demonstrates that ALT cell lines use different telomerase-independent maintenance mechanisms that variably require the WRN helicase and that some cells can switch from one mechanism to another that permits telomere elongation in the absence of WRN. Our data suggest that BRCA1 localization may define these mechanisms.
端粒维持可在端粒酶存在或不存在的情况下发生,后者被称为端粒的替代延长(ALT)。ALT利用基于重组的过程和在同源重组中起作用的DNA修复蛋白来添加端粒重复序列。我们之前的工作报道,类RecQ的BLM解旋酶是ALT所必需的,并且它在体外能解开端粒底物。WRN也是一种类RecQ解旋酶,与BLM具有许多生化功能。WRN与BLM相互作用,解开端粒底物,并共定位于ALT相关的PML小体(APB),这表明它可能也是ALT过程所必需的。通过在三种ALT细胞系中使用长期siRNA敲低WRN,我们发现部分而非全部细胞系在端粒维持方面需要WRN。VA - 13细胞需要WRN来防止端粒丢失和APB的形成;Saos - 2细胞则不需要。第三种ALT细胞系U - 2 OS需要WRN来形成APB,然而WRN缺失会导致p53介导的细胞凋亡。在没有WRN和p53的情况下,U - 2 OS细胞在经过中间数量的群体倍增(50 - 70次)后会发生端粒丢失,此时即便WRN持续缺失,它们仍能维持端粒长度。WRN和肿瘤抑制因子BRCA1在VA - 13和U - 2 OS细胞中共定位于APB,但在Saos - 2细胞中并非如此。U - OS细胞中WRN的缺失与APB中BRCA1的丢失有关。虽然WRN的缺失在这三种ALT细胞系中显著增加了端粒姐妹染色单体交换(T - SCE),但BRCA 和WRN两者的缺失并未显著改变T - SCE。这项工作表明,ALT细胞系使用不同的不依赖端粒酶的维持机制,这些机制对WRN解旋酶的需求各不相同,并且一些细胞可以从一种机制切换到另一种机制,从而在没有WRN的情况下实现端粒延长。我们的数据表明,BRCA1的定位可能决定了这些机制。
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