Abstract
Non-Homologous End Joining (NHEJ) is one of the two major pathways of DNA Double Strand Breaks (DSBs) repair. Mutations in human NHEJ genes can lead to immunodeficiency due to its role in V(D)J recombination in the immune system. In addition, most patients carrying mutations in NHEJ genes display developmental anomalies which are likely the result of a general defect in repair of endogenously induced DSBs such as those arising during normal DNA replication. Cernunnos/XLF is a recently identified NHEJ gene which is mutated in immunodeficiency with microcephaly patients. Here we aimed to investigate whether Cernunnos/XLF mutations disrupt the ability of patient cells to respond to replication stress conditions. Our results demonstrate that Cernunnos/XLF mutated cells and cells downregulated for Cernunnos/XLF have increased sensitivity to conditions which perturb DNA replication. In addition, under replication stress, these cells exhibit impaired DSB repair and increased accumulation of cells in G2/M. Moreover Cernunnos/XLF mutated and down regulated cells display greater chromosomal instability, particularly at fragile sites, under replication stress conditions. These results provide evidence for the role of Cernunnos/XLF in repair of DSBs and maintenance of genomic stability under replication stress conditions. This is the first study of a NHEJ syndrome showing association with impaired cellular response to replication stress conditions. These findings may be related to the clinical features in these patients which are not due to the V(D)J recombination defect. Additionally, in light of the emerging important role of replication stress in the early stages of cancer development, our findings may provide a mechanism for the role of NHEJ in preventing tumorigenesis.
Highlights
DNA Double Strand Breaks (DSBs) are one of the most hazardous forms of DNA damage to the integrity of the genome
We found that Cernunnos/XLF mutated cells are markedly more sensitive to aphidicolin than their complemented counterparts (Fig 1B)
Our results indicate that Cernunnos/XLF is required for cellular survival under conditions which perturb elongation of DNA replication and that Cernunnos/XLF mutations found in patients, disrutpt the cellular ability to cope with these conditions
Summary
DNA Double Strand Breaks (DSBs) are one of the most hazardous forms of DNA damage to the integrity of the genome. A failure to repair DSBs could lead to cell death or to chromosomal rearrangements [1]. There are two main DSB repair pathways, the Homologous Recombination (HR) and the Non-Homologous End Joining (NHEJ) which are conserved in eukaryotes from yeast to human [1,2]. The NHEJ repair process begins with binding of the DNA ends by the Ku70/Ku80 heterodimer and recruitment of DNA-PK catalytic subunit (DNA-PKcs). This is followed by processing of the DNA ends which involves Artemis and/or other DNA processing enzymes. The final step is the ligation of the two DNA ends by DNA Ligase IV, which acts in a complex together with XRCC4 and Cernunnos/XLF that stabilize it and stimulate its activity (for review see[3,4])
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