Root Heather, Larsen Andrew, Komosa Martin, Al-Azri Fakhriya, Li Ren, Bazett-Jones David P, Stephen Meyn M
Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto M5G 0A4, Canada.
School of Earth and Space Exploration, Arizona State University, Tempe 85281, USA.
Hum Mol Genet. 2016 Aug 1;25(15):3255-3268. doi: 10.1093/hmg/ddw175. Epub 2016 Jul 17.
Fanconi anemia and Bloom syndrome are genomic instability syndromes caused by mutations in proteins that participate in overlapping DNA repair and replication pathways. Here, we show that the monoubiquitinated form of the Fanconi Anemia protein FANCD2 acts in opposition to the BLM DNA helicase to restrain telomere replication and recombination in human cells that utilize the Alternative Lengthening of Telomeres (ALT) pathway. ALT relies on exchanges of telomeric DNA to maintain telomeres, a process that we show FANCD2 suppresses. Depletion of FANCD2 results in a hyper-ALT phenotype, including an increase in extrachromosomal telomeric repeat DNAs, putative recombinational byproducts that we show exist as intertwined complexes forming the nucleic acid component of ALT-associated PML bodies. Increases in telomeric DNA are suppressed by loss of BLM but not RAD51, occur without parallel upregulation of shelterin proteins TRF1 and TRF2, and are associated with increased frequencies of deprotected and fragile telomeres. Inactivation of the FA pathway does not trigger ALT, as FANCD2 depleted telomerase positive cells do not acquire ALT-like phenotypes. We observe frequent fragile telomeres in ALT cells, suggesting that telomere sequences are prone to replication problems. We propose that, in ALT cells, FANCD2 promotes intramolecular resolution of stalled replication forks in telomeric DNA while BLM facilitates their resection and subsequent involvement in the intermolecular exchanges that drive ALT.
范可尼贫血和布卢姆综合征是由参与重叠DNA修复和复制途径的蛋白质发生突变引起的基因组不稳定综合征。在这里,我们表明范可尼贫血蛋白FANCD2的单泛素化形式与BLM DNA解旋酶起相反作用,以抑制利用端粒替代延长(ALT)途径的人类细胞中的端粒复制和重组。ALT依赖于端粒DNA的交换来维持端粒,我们发现FANCD2抑制了这一过程。FANCD2的缺失导致高ALT表型,包括染色体外端粒重复DNA增加,我们发现这些假定的重组副产物以相互缠绕的复合物形式存在,构成了ALT相关PML小体的核酸成分。端粒DNA的增加被BLM的缺失所抑制,但不被RAD51抑制,在没有端粒保护蛋白TRF1和TRF2平行上调的情况下发生,并且与去保护和脆弱端粒的频率增加相关。FA途径的失活不会触发ALT,因为FANCD2缺失的端粒酶阳性细胞不会获得ALT样表型。我们在ALT细胞中观察到频繁的脆弱端粒,这表明端粒序列容易出现复制问题。我们提出,在ALT细胞中,FANCD2促进端粒DNA中停滞复制叉的分子内解析,而BLM促进其切除并随后参与驱动ALT的分子间交换。
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