Abstract

Rad26 is the homolog of human ATRIP and budding yeast Ddc2 in Schizosaccharomyces pombe . Like ATRIP and Ddc2, Rad26 works with Rad3 ATR/Mec1 to initiate checkpoint signalling in response to perturbed DNA replication and various types of DNA damage. To better understand the checkpoint initiation mechanism in fission yeast, we carried out genetic and biochemical analyses on the N-terminus of Rad26. Although Rad26 homologs do not share much sequence similarity, we demonstrate that, like ATRIP and Ddc2, Rad26 possesses a replication protein A (RPA) binding domain (RBD) in its N-terminus, suggesting a highly conserved mechanism. Elimination of the RBD in Rad26, however, only moderately affects the checkpoint signalling and cellular resistance to genotoxins. Rad26 has a short KKRK sequence in the N-terminal region, a motif conserved in Ddc2 that binds DNA and is crucial for the checkpoint function in budding yeast. Mutations of this motif in Rad26 cause only a minor defect in the checkpoint. However, simultaneous mutations of the RBD and the KKRK motif nearly eliminate the Rad3 ATR kinase signalling at the perturbed replication fork. This suggests that the two functional units of Rad26 cooperate to initiate the DNA replication checkpoint. On the contrary, the simultaneous mutations of Rad26 only moderately or minimally sensitize the cell to different types of DNA damage. We hypothesize that the checkpoint initiation at the DNA damage site in fission yeast may follow a different mechanism that depends less on the two functional units of Rad26. Rad26 ATRIP is a checkpoint sensor protein in the fission yeast S. pombe . Like its homologous proteins in other model organisms, it collaborates with the checkpoint sensor kinase Rad3 ATR to trigger Rad3 ATR -mediated checkpoint signaling at perturbed DNA replication forks, activating the DNA replication checkpoint, or at the sites of DNA damage, initiating the DNA damage checkpoint. The current checkpoint model suggests t