Rad51 and DNA-PKcs are involved in the generation of specific telomere aberrations induced by the quadruplex ligand 360A that impair mitotic cell progression and lead to cell death

Functional telomeres are protected from non-homologous end-joining (NHEJ) and homologous recombination (HR) DNA repair pathways. Replication is a critical period for telomeres because of the requirement for reconstitution of functional protected telomere conformations, a process that involves DNA repair proteins. Using knockdown of DNA-PKcs and Rad51 expression in three different cell lines, we demonstrate the respective involvement of NHEJ and HR in the formation of telomere aberrations induced by the G-quadruplex ligand 360A during or after replication. HR contributed to specific chromatid-type aberrations (telomere losses and doublets) affecting the lagging strand telomeres, whereas DNA-PKcs-dependent NHEJ was responsible for sister telomere fusions as a direct consequence of G-quadruplex formation and/or stabilization induced by 360A on parental telomere G strands. NHEJ and HR activation at telomeres altered mitotic progression in treated cells. In particular, NHEJ-mediated sister telomere fusions were associated with altered metaphase-anaphase transition and anaphase bridges and resulted in cell death during mitosis or early G1. Collectively, these data elucidate specific molecular and cellular mechanisms triggered by telomere targeting by the G-quadruplex ligand 360A, leading to cancer cell death

Consequence of single break near telomere in human cells

Low dose radiation effects remain an open question, mainly due to the lack of models that permit to address it. Using human cells tagged with a plasmid integrated on end of a marker chromosome, we demonstrated that a single break near the telomere has dramatic consequences on the genome stability, inducing the panel of chromosomal instability detected in cancer cells including gene amplification and large chromosome imbalances (LOH up to 100 Mb), Sabatier et al, Mol Cancer Res 2005. One of the consequences of the chromosome instability induced by a single telomere loss is to let emerge cells with high proliferative advantages. An increase of the tumorigenicity of these cells is observed when transplanted on nude mice. Such cell models would be very informative to characterize the role of the modulation of DNA damage repair (NHEJ and HR) in the consequence of single chromosome break. First we tested the feasibility of using replicative small interfering vectors for efficient and long term silencing in human cells. We silenced NHEJ protein -DNAPK, LigIV, and XRCC4- and HR protein -Rad51/Rad52/Rad54- in Hela cells. Our data suggested that the major effect of NHEJ on telomere maintenance is indirect via misrepaired breaks in subtelomeric and telomeric whereas the major effect of HR on telomere maintenance is direct via telomere replication and telomere recombination. We are working on the invalidation of DNA repair genes in telomere tagged models. Having demonstrated that a single break near telomere could have such consequences we now address the question of radiation-induced chromosome breaks that will occur at low doses (25 mGy -> 1DSB per cell), stuying clonogenic survival and the emergence of resistant clones due to chromosome breakage