The Dispersal of Replication Proteins after Etoposide Treatment Requires the Cooperation of Nbs1 with the Ataxia Telangiectasia Rad3-Related/Chk1 Pathway

Abstract

In mammalian cells, DNA replication takes place in functional subnuclear compartments, called replication factories, where replicative factors accumulate. The distribution pattern of replication factories is diagnostic of the different moments (early, mid, and late) of the S phase. This dynamic organization is affected by different agents that induce cell cycle checkpoint activation via DNA damage or stalling of replication forks. Here, we explore the cell response to etoposide, an anticancer drug belonging to the topoisomerase II poisons. Etoposide does not induce an immediate block of DNA synthesis and progressively affects the distribution of replication proteins in S phase. First, it triggers the formation of large nuclear foci that contain the single-strand DNA binding protein replication protein A (RPA), suggesting that lesions produced by the drug are processed into extended singlestranded regions. These RPA foci colocalize with DNA replicated at the beginning of the treatment. Etoposide also triggers the dispersal of replicative proteins, proliferating cell nuclear antigen and DNA ligase I, from replication factories. This event requires the activity of the ataxia telangiectasia Rad3-related (ATR) checkpoint kinase. By comparing the effect of the drug in cell lines defective in different DNA repair and checkpoint pathways, we show that, along with the downstream kinase Chk1, the Nbs1 protein, mutated in the Nijmegen breakage syndrome, is also relevant for this response and for ATR-dependent phosphorylation. Finally, our analysis evidences a critical role of Nbs1 in the etoposideinduced inhibition of

Synergistic induction of apoptosis in human leukemia T cells by the Akt inhibitor perifosine and etoposide through activation of intrinsic and Fas-mediated extrinsic cell death pathways

“…Beside its well-known effects on G 2 -M phase of the cell cycle, etoposide has been shown to have an inhibitory mechanism also on S-phase progression, retaining cells in the latter half of the S phase (32,33). Accordingly, our data showed that etoposide (5 Amol/L) induced an accumulation of cells in both S phase (47%) and G 2 -M phase (35%).…”

DNA-PK promotes the survival of young neurons in the embryonic mouse retina

“…The topoisomerase II inhibitor etoposide provokes DSBs, as well as the rapid activation of ATM, ATR, DNA-PK and their corresponding substrates. [24][25][26] Exposing E14.5 retinas to etoposide in organotypic culture increased the accumulation of gH2AX and total p53 (Figure 3a), two common substrates of ATM, ATR and DNA-PK, 13,14,16 and indicative of the response of retinal cells to a genotoxic agent. Chk1, which is primarily activated by ATR, 16,27 also seemed to be phosphorylated in etoposide-treated retinas.…”

DNA ligase I and Nbs1 proteins associate in a complex and colocalize at replication factories

“…22 We have previously shown that bleomycin does not affect the association of replicative enzymes with replication factories. 23 Co-staining with α-LigI and α-Nbs1 antibodies demonstrate that after belomycin treatment Nbs1 and LigI colocalize at replication factories (Fig. 4D, Part a).…”