A CDK-catalysed regulatory phosphorylation for formation of the DNA replication complex Sld2–Dpb11

Abstract

Phosphorylation often regulates protein-protein interactions to control biological reactions. The Sld2 and Dpb11 proteins of budding yeast form a phosphorylation-dependent complex that is essential for chromosomal DNA replication. The Sld2 protein has a cluster of 11 cyclindependent kinase (CDK) phosphorylation motifs (Ser/ Thr-Pro), six of which match the canonical sequences Ser/Thr-Pro-X-Lys/Arg, Lys/Arg-Ser/Thr-Pro and Ser/ Thr-Pro-Lys/Arg. Simultaneous alanine substitution for serine or threonine in all the canonical CDK-phosphorylation motifs severely reduces complex formation between Sld2 and Dpb11, and inhibits DNA replication. Here we show that phosphorylation of these canonical motifs does not play a direct role in complex formation, but rather regulates phosphorylation of another residue, Thr84. This constitutes a non-canonical CDK-phosphorylation motif within a 28-amino-acid sequence that is responsible, after phosphorylation, for binding of Sld2-Dpb11. We further suggest that CDK-catalysed phosphorylation of sites other than Thr84 renders Thr84 accessible to CDK. Finally, we argue that this novel mechanism sets a threshold of CDK activity for formation of the essential Sld2 to Dpb11 complex and therefore prevents premature DNA replication.

Regulatory evolution in proteins by turnover and lineage-specific changes of cyclin-dependent kinase consensus sites

“…More recent analyses of phosphoregulation suggest alternative regulatory paradigms involving multiple phosphorylation sites that do not need to be conserved (24). Clusters of multiple phosphorylation sites can modulate interactions (25,(47)(48)(49) or provide specific dynamic properties (50)(51)(52) and these mechanisms may not depend on the specific locations or numbers of sites (24). Consistent with this model, we found statistical evidence for constraint at the level of the cluster of consensus sites in the linker region of ORC1, despite weak constraint at the amino acid level.…”

Mcm10 interacts with Rad4/Cut5TopBP1 and its association with origins of DNA replication is dependent on Rad4/Cut5TopBP1

“…These observations raise additional questions about the order of assembly of additional pre-initiation factors at origins of DNA replication. In budding yeast the Rad4 TopBP1 homologue Dpb11 has been shown to interact with both Sld2 and Sld3 and those events have been shown to be CDK-dependent and similar data have recently been reported in metazoans (6,7,8,10,11,12,49). Chromatin association of Cdc45 has been previously shown to be dependent upon Mcm10, Rad4 TopBP1 , Sld3 and the GINS complex (22,23,24,47).…”

“…In budding yeast Cdc45 and Sld3 associate with early firing origins in G1, prior to CDK and DDK activity, and with late firing origins in S phase (3,4). Evidence from budding yeast has demonstrated that Sld2 and Sld3 recruitment are pivotal events in replication initiation at origins and the two proteins are critical targets of the S phase CDK (5,6,7,8). Upon phosphorylation by CDK, Sld2 and Sld3 interact with distinct BRCT domains of a third protein, Dpb11, the Saccharomyces cerevisiae orthologue of human TopBP1, and that interaction enables recruitment to the pre-RC (6,7,8).…”

“…Evidence from budding yeast has demonstrated that Sld2 and Sld3 recruitment are pivotal events in replication initiation at origins and the two proteins are critical targets of the S phase CDK (5,6,7,8). Upon phosphorylation by CDK, Sld2 and Sld3 interact with distinct BRCT domains of a third protein, Dpb11, the Saccharomyces cerevisiae orthologue of human TopBP1, and that interaction enables recruitment to the pre-RC (6,7,8). Phosphorylation of Sld2 promotes formation of a pre-loading complex containing Dpb11, the heteromeric GINS complex, DNA Pol and Sld2 itself (9).…”

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

“…The dpb11-1 mutant is temperature sensitive for growth, has slow progression of DNA replication, and shows defects in both the S phase and G 2 /M checkpoint. The fourth BRCT repeat is involved in binding Sld2, and a two-hybrid analysis showed a strong reduction in the interaction signal between Sld2 and mutant Dpb11-1 compared with the wild type (29). The minimal fourth BRCT domain ends at Leu-566, however, extensive sequence conservation continues past amino acid 583, the point of truncation in dpb11-1, until position 586 (Fig.…”