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 single-stranded 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
etoposide-induced inhibition of DNA replication in early S phase.