The dispersal of replication proteins after Etoposide treatment requires the cooperation of Nbs1 with the ataxia telangiectasia Rad3-related/Chk1 pathway.
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.
Rossi
Rossella
R
Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, via Abbiategrasso 207, I-27100 Pavia, Italy.
Lidonnici
Maria Rosa
MR
Soza
Samuela
S
Biamonti
Giuseppe
G
Montecucco
Alessandra
A
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects* / Q000502:physiology; D005047:Etoposide / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism; D016196:S Phase / Q000187:drug effects
D000972:Antineoplastic Agents, Phytogenic; D018797:Cell Cycle Proteins; D002843:Chromatin; C000607892:LIG1 protein, human; C112156:NBN protein, human; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A; D005047:Etoposide; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D011088:DNA Ligases; D000072481:DNA Ligase ATP