Cell cycle arrest at the initiation step of human chromosomal DNA replication causes DNA damage

Abstract

Cell cycle arrest in response to environmental effects can lead to DNA breaks. We investigated whether inhibition of DNA replication during the initiation step can lead to DNA damage and characterised a cell-cycle-arrest point at the replication initiation step before the establishment of active replication forks. This arrest can be elicited by the iron chelators mimosine, ciclopirox olamine or 2,2′-bipyridyl, and can be reversed by the removal of the drugs or the addition of excess iron. Iron depletion induces DNA double-strand breaks in treated cells, and activates a DNA damage response that results in focal phosphorylation of histone H2AX, focal accumulation of replication protein A (RPA) and ATR (ATM and Rad3-related kinase), and activation of CHK1 kinase. Abrogation of the checkpoint response does not abolish the cell cycle arrest before the establishment of active DNA replication forks. DNA breaks appear concomitantly with the arrival of cells at the arrest point and persist upon release from the cell cycle block. We conclude that DNA double-strand breaks are the consequence, and not the cause, of cell cycle arrest during the initiation step of DNA replication by iron chelation.