ATR kinase inhibition induces unscheduled origin firing through a Cdc7-dependent association between GINS and And-1

Tatiana Moiseeva,Christopher J Bakkenist,Mark J O’Connor,Brian Hood,Thomas P Conrads,Sandy Schamus

doi:10.1038/s41467-017-01401-x

Tatiana Moiseeva, Christopher J Bakkenist + Show 4 more

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https://doi.org/10.1038/s41467-017-01401-x

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ATR kinase activity slows replication forks and prevents origin firing in damaged cells. Here we describe proteomic analyses that identified mechanisms through which ATR kinase inhibitors induce unscheduled origin firing in undamaged cells. ATR-Chk1 inhibitor-induced origin firing is mediated by Cdc7 kinase through previously undescribed phosphorylations on GINS that induce an association between GINS and And-1. ATR-Chk1 inhibitor-induced origin firing is blocked by prior exposure to DNA damaging agents showing that the prevention of origin firing does not require ongoing ATR activity. In contrast, ATR-Chk1 inhibitor-induced origins generate additional replication forks that are targeted by subsequent exposure to DNA damaging agents. Thus, the sequence of administration of an ATR kinase inhibitor and a DNA damaging agent impacts the DNA damage induced by the combination. Our experiments identify competing ATR and Cdc7 kinase-dependent mechanisms at replication origins in human cells.

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ATR kinase activity slows replication forks and prevents origin firing in damaged cells
While ATR and RPA34 accumulate in the chromatin after treatments with DNA damaging agents, neither of these proteins were recruited to the chromatin in cells treated with ATRi alone (Fig. 1e)
Studying the effect of ATR inhibition allowed us to detect a Cdc7dependent interaction between And-1. The interaction of Ctf4 (And-1) and GINS complex in human cells and other markers of origin firing such as MCM4 hyper-phosphorylation and the accumulation of replication fork component in the chromatin

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ATR kinase activity slows replication forks and prevents origin firing in damaged cells. The complexity of identified DNA damage signaling indicates that >10% of the proteome may be modified by ATR kinase-dependent signaling after exposure to clinically relevant doses of DNA damaging chemotherapy, ionizing radiation (IR), or ultraviolet radiation (UV) Modifications embedded within this complex system induce the DNA replication checkpoint that protects stalled and collapsed replication forks and inhibits DNA replication origin firing. ATR kinase activity is implicated in the regulation of unperturbed DNA replication by recent observations that ATR kinase inhibition induces unscheduled origin firing and reduces replication fork velocity by an unknown mechanism(s)[10, 11]. To our knowledge, these observations have not been pursued further.

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