Dbf4 Is Direct Downstream Target of Ataxia Telangiectasia Mutated (ATM) and Ataxia Telangiectasia and Rad3-related (ATR) Protein to Regulate Intra-S-phase Checkpoint

Alan Yueh-Luen Lee,Takuya Chiba,Lan N Truong,An Ning Cheng,Johnny Do,Michael Jeffrey Cho,Longchuan Chen,Xiaohua Wu

doi:10.1074/jbc.m111.291104

Abstract

Dbf4/Cdc7 (Dbf4-dependent kinase (DDK)) is activated at the onset of S-phase, and its kinase activity is required for DNA replication initiation from each origin. We showed that DDK is an important target for the S-phase checkpoint in mammalian cells to suppress replication initiation and to protect replication forks. We demonstrated that ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) proteins directly phosphorylate Dbf4 in response to ionizing radiation and replication stress. We identified novel ATM/ATR phosphorylation sites on Dbf4 and showed that ATM/ATR-mediated phosphorylation of Dbf4 is critical for the intra-S-phase checkpoint to inhibit DNA replication. The kinase activity of DDK, which is not suppressed upon DNA damage, is required for fork protection under replication stress. We further demonstrated that ATM/ATR-mediated phosphorylation of Dbf4 is important for preventing DNA rereplication upon loss of replication licensing through the activation of the S-phase checkpoint. These studies indicate that DDK is a direct substrate of ATM and ATR to mediate the intra-S-phase checkpoint in mammalian cells.

Highlights

S-phase checkpoint is important for maintaining genome stability upon DNA damage in S-phase
Similar results were obtained following UV and aphidicolin treatment. These results suggest that ataxia telangiectasia mutated (ATM) and ATR play important roles in mediating Dbf4 hyperphosphorylation upon DNA damage
These results suggest that upon loss of the licensing control, S-phase checkpoint-mediated Dbf4 phosphorylation is an early response that is initially induced at the stage when DNA unwinding is uncoupled from DNA synthesis prior to double-stranded breaks (DSBs) formation

Summary

Background

S-phase checkpoint is important for maintaining genome stability upon DNA damage in S-phase. A Dbf4-related factor, Drf, was found to play a role more important than that of Dbf in mediating the S-phase checkpoint [32, 34] These studies raise a question as to whether DDK is a critical S-phase checkpoint target to inhibit replication initiation if its kinase activity is not altered after DNA damage in higher organisms. Dbf plays dual roles to mediate the S-phase checkpoint responses in mammalian cells It is a direct target of the S-phase checkpoint to inhibit DNA replication, DDK remains active to protect stalled replication forks. Consistent with its role in inhibiting replication initiation upon the activation of S-phase checkpoint, we demonstrated that ATM/ ATR-mediated phosphorylation of Dbf is important for the suppression of DNA rereplication when the licensing control is impaired

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION