Replication origin firing regulation to ensure genome stability in metazoan cells
Final Report Abstract
Accurate and complete genome duplication is essential for faithful genetic and epigenetic inheritance, with replication of origin firing being a critical regulatory step. Our research has contributed to defining the MTBP-Treslin-TopBP1 complex as a central regulatory hub of origin firing. We previously identified MTBP as an essential replication factor targeted by cell cycle and DNA damage kinases and recently discovered its interaction with the CDK8-cyclin C kinase (CDK8) via a metazoan-specific MTBP domain. While this interaction is implicated in preventing replication stress, its precise cellular function and regulatory mechanisms remain unclear. First, we confirmed that the MTBP-CDK8 interaction is critical for DNA replication. Unexpectedly, we found that MTBP mutants lacking canonical CDK consensus phosphorylation sites still incorporated radioactive phosphate in a CDK8-dependent manner, suggesting that CDK8 docking enables phosphorylation at non-consensus sites. This led us to analysing the MTBP-CDK8 structure using Alphafold2 prediction. The resulting high-confidence prediction showed that MTBP interacts primarily with cyclin C with additional contacts with Cdk8’s active site. These interaction patterns resembled those of Med12, a known CDK8 activator, raising the hypothesis that MTBP functions as an alternative activator of CDK8. To test this, we biochemically reconstituted CDK8-cyclin C complexes with MTBP or Med12 and established kinase assays. Our results demonstrated that both MTBP and Med12 enhance CDK8-dependent phosphorylation of RNA polymerase II (Pol II-CTD), with Med12 being the more potent activator. Moreover, Med12-bound CDK8 phosphorylated additional Pol II-CTD residues, suggesting distinct substrate specificities compared to MTBP-CDK8. Molecular dynamics simulations (in collaboration) supported that MTBP and Med12 activate CDK8 through distinct mechanisms, likely by differentially stabilizing the kinase T-loop. Given Med12’s role in cancer-associated CDK8 activation, our findings highlight the need to consider pathway-specific activation in therapeutic targeting of CDK8. Our current efforts include: • Detailed biochemical and structural characterization of the MTBP-CDK8 complex. • Investigation of the cellular CDK8 function through pathway-specific activation by MTBP and Med12. • Identification of MTBP-dependent CDK8 substrates relevant for DNA replication initiation. Through these investigations, we aim to define CDK8’s regulatory role in genome replication that has implications for cancer biology and targeted therapies.
