The Unstructured C-terminal Tail of Yeast Dpb11 (Human TopBP1) Protein Is Dispensable for DNA Replication and the S Phase Checkpoint but Required for the G2/M Checkpoint

Abstract

Background: Specific activators turn on Mec1/ATR kinase during DNA damage or replication stress, mediating cell cycle arrest. Results: We have separated the replication function of multifunctional Dpb11 from its checkpoint activation function. Conclusion: Dpb11 functions primarily during the G 2 /M phase. Significance: Our results suggest that each phase of the cell cycle may use specific Mec1 activators.

Lagging strand maturation factor Dna2 is a component of the replication checkpoint initiation machinery

“…1C, lane 6). As previously observed for Dpb11-mediated activation of Mec1 (Mordes et al 2008;Navadgi-Patil et al 2011), DNA is not required for Dna2 to stimulate Mec1 kinase (Fig. 1C, cf.…”

“…Furthermore, mutants lacking the Mec1 stimulatory activity of DDC1 and DPB11 that completely abrogated the G1 and G2/M DNA damage checkpoints still showed the presence of a robust replication and S-phase DNA damage checkpoint (Navadgi-Patil et al 2011). Even after potential contributions by Tel1 were eliminated (this study), a replication checkpoint remained, suggesting the presence of another Mec1 stimulatory factor that is specific for the S phase of the cell cycle.…”

“…Specific sensors transduce the DNA damage response signal to Mec1 and stimulate its kinase activity. Two sensor proteins identified to date are the PCNA-like heterotrimeric checkpoint clamp 9-1-1 (Saccharomyces cerevisiae Ddc1-Mec3-Rad17 and human Rad9-Rad1-Hus1) (Majka et al 2006a) and the replication initiation factor Dpb11 (human TopBP1) (Kumagai et al 2006;Choi et al 2007;Mordes et al 2008;Navadgi-Patil et al 2011). These factors stimulate Mec1/ATR to phosphorylate many downstream proteins, including the principal downstream effector kinase Rad53 (the functional homolog of human Chk1), which propagates the checkpoint pathway (Sanchez et al 1996).…”

Direct Role for the Replication Protein Treslin (Ticrr) in the ATR Kinase-mediated Checkpoint Response

“…In addition to its checkpoint function, TopBP1 is also a replication protein (12). Studies with conditional and separation-of-function mutants show that the checkpoint function of TopBP1 can be separated from its replication function (13,14). The N-terminal part of TopBP1 was shown to be both necessary and sufficient for DNA replication, whereas domains in the C-terminal half of TopBP1 are involved in the DNA damage and replication stress response (13)(14)(15).…”

“…Studies with conditional and separation-of-function mutants show that the checkpoint function of TopBP1 can be separated from its replication function (13,14). The N-terminal part of TopBP1 was shown to be both necessary and sufficient for DNA replication, whereas domains in the C-terminal half of TopBP1 are involved in the DNA damage and replication stress response (13)(14)(15). Specifically, a region between BRCT domains 6 and 7 of human TopBP1, which is conserved in Xenopus and yeast, has been shown to be important for ATR-dependent phosphorylation of Chk1 and is thus called the ATR activation domain (AAD) 2 (9, 16 -21).…”