Characterisation of cell cycle checkpoint kinases in Toxoplasma gondii

Toxoplasma gondii is a protozoan parasite in the apicomplexan phylum. Apicomplexan parasites replicate using a variety of non-canonical cell division modes, distinct from binary fission, whose molecular regulation is incompletely understood. T. gondii replicates by endodyogeny in its intermediate hosts, and by endopolygeny in its definitive host. To improve our understanding of how these unusual, flexible cell division modes are regulated, we characterised the T. gondii homologues of the cell-cycle checkpoint kinases ATM and ATR. These phosphoinositol-3-kinase-like kinases are entirely absent in some related parasites including Plasmodium ; in T. gondii they are present but their putative checkpoint roles were previously uncharacterised. Both Tg ATM and Tg ATR were found to be dispersed throughout the parasite, rather than restricted to the nucleus, and they did not detectably relocate to the nucleus after DNA damage. Nevertheless, they were both required for checkpoint responses to DNA damage, including acute replication slowing and phosphorylation of the DNA damage marker histone H2AX. Unusually, the two kinases seemed to cooperate in the checkpoint response, with the loss of either one largely ablating the response, regardless of the type of DNA damage. Thus, T. gondii clearly retains a DNA-damage-responsive checkpoint, but some of its key features differ from the well-studied checkpoint in human cells.