Biology subjects

Boos, D.

Publications and source records attributed to Boos, D..

5 recordsLinked to original sources

bioRxiv2025

CDC7 and CDK8 kinases cooperate to support DNA replication origin firing in human cells

The coordinated activation of DNA replication origins is important for efficient DNA synthesis and genome stability. S-phase cyclin dependent kinases (CDKs) together with CDC7 kinase, are essential to origin activation by converting the pre-replicative complex into a fully active helicase. To identify genes that tune DNA replication, we have performed a chemo-genetic genome-wide CRISPR-KO screen with cells challenged with the CDC7 inhibitor XL413. By developing a methodology based on genetic coessentiality to functionally cluster the hits, we uncover the transcriptional CDK8/CCNC kinase in a cluster with replication initiation factors. We find that CDK8 depletion further reduces the rate of DNA synthesis imposed by CDC7 inhibitors. DNA fibre experiments provide compelling evidence that CDK8 and CDC7 cooperate in origin activation. Suppression of DNA synthesis by CDK8 inhibition requires the binding of CDK8/CCNC to the MDM2 Binding Protein (MTBP) and we show that CDC7 and CDK8 individually contribute to the phosphorylation of MCM4 subunit of the replicative helicase. Thus, this work identifies CDK8 as the third protein kinase directly involved in origin activation in human cells.

biochemistry↗

bioRxiv2025

MTBP allosterically activates Cdk8-CycC kinase activity

How the approximately 300 human protein kinases identify their dedicated substrates despite the significant overlap of their phosphorylation consensus sequences is relevant for nearly all cellular processes. We show here that the Cdk8/19-CycC kinase uses mutually exclusive targeting and activation factors to facilitate distinct cellular roles. The Med12 protein is known to target Cdk8/19-CycC to the mediator of transcription complex to control the transcription of specific gene sets upon respective stimuli. We describe that a second Cdk8/19-CycC targeting factor exists, the replication origin firing regulator MTBP that targets the kinase to Med12-independent cellular roles. Both Med12 and MTBP constitute allosteric activators of the enzymatic Cdk8/19-CycC kinase activity in vitro. We describe the structural basis of this activation that involves distinct mechanisms how Med12 and MTBP reposition the T-loop of the kinase independently of T-loop phosphorylation - the canonical mechanism of CDK kinase activation. Our results support the following model: the Cdk8/19-CycC dimer alone has low enzymatic activity, which may help avoid off-target phosphorylation. Med12, MTBP and potentially other as yet unidentified accessory factors, target the kinase to distinct molecular environments, at the same time activating kinase activity for efficient substrate phosphorylation. Our work establishes an unusual mechanism of CDK kinase control and change the current paradigm how Cdk8/19-CycC selects its substrates. Substrate selection of the kinase may be relevant for cancer biology and therapy because Cdk8 is a well-established colorectal cancer promoting factor.

biochemistry↗

bioRxiv2023

TopBP1 utilises a bipartite GINS binding mode to activate the replicative helicase

Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies demonstrate that the helicase activator GINS interacts with TopBP1 through two separate binding surfaces, the first involving a stretch of highly conserved amino acids in the TopBP1-GINI region, the second a surface on TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS subunit Psf1. Mutation analysis reveals that either surface is individually able to support TopBP1-GINS interaction, albeit with reduced affinity. Consistently, either surface is sufficient for replication origin firing in Xenopus egg extracts and becomes essential in the absence of the other. The TopBP1-GINS interaction appears sterically incompatible with simultaneous binding of DNA polymerase epsilon (Pol{varepsilon}) to GINS when bound to Mcm2-7-Cdc45, although TopBP1-BRCT4 and the Pol{varepsilon} subunit PolE2 show only partial competitivity in binding to Psf1. Our TopBP1-GINS model improves the understanding of the recently characterised metazoan pre-loading complex. It further predicts the coordination of three molecular origin firing processes, DNA polymerase epsilon arrival, TopBP1 ejection and GINS integration into Mcm2-7-Cdc45.

molecular biology↗

bioRxiv2021

Refining the domain architecture model of the replication origin firing factor Treslin/TICRR

Faithful genome duplication requires appropriately controlled replication origin firing. The metazoan Treslin/TICRR origin firing factor and its yeast orthologue Sld3 are regulation hubs of origin firing. They share the Sld3-Treslin domain (STD) and the adjacent TopBP1/Dpb11 interaction domain (TDIN). We report a revised domain architecture model of Treslin/TICRR. Complementary protein sequence analyses uncovered Ku70-homologous {beta}-barrel folds in the Treslin/TICRR middle domain (M domain) and in Sld3. Thus, the Sld3-homologous Treslin/TICRR core comprises its three central domains, M domain, STD and TDIN. This Sld3-core is flanked by non-conserved terminal domains, the CIT (conserved in Treslins) and the C-terminus. We also identified Ku70-like {beta}-barrels in MTBP and Sld7. Our binding experiments showed that the Treslin {beta}-barrel mediates interaction with the MTBP {beta}-barrel, reminiscent of the homotypic Ku70-Ku80 dimerization. This binding mode is conserved in the Sld3-Sld7 dimer. We used Treslin/TICRR domain mutants to show that all Sld3-core domains and the non-conserved terminal domains fulfil important functions during origin firing in human cells. Thus, metazoa-specific and widely conserved molecular processes cooperate during origin firing in metazoa.

molecular biology↗

bioRxiv2018

N-terminal BRCT domains of the DNA damage checkpoint proteins TOPBP1/Rad4 display distinct specificities for phosphopeptide ligands

TOPBP1 and its fission yeast homologue Rad4, are critical players in a range of DNA replication, repair and damage signalling processes. They are composed of multiple BRCT domains, some of which have the capacity to bind phosphorylated motifs in other proteins. They thus act as multi-point adaptors bringing proteins together into functional combinations, dependent on post-translational modifications downstream of cell cycle and DNA damage signals. We have now structurally and/or biochemically characterised a sufficient number of high-affinity complexes for the conserved N-terminal region of TOPBP1 and Rad4 in complex with diverse phospho-ligands - which include human RAD9 and Treslin, as well as S.pombe Crb2 and Sld3 - to define the key determinants of BRCT domain specificity. We use this information to identify and characterise previously unknown phosphorylation-dependent TOPBP1/Rad4-binding motifs in human RHNO1 and the fission yeast homologue of MDC1, Mdb1. These results provide important insights into how multiple BRCT domains within TOPBP1/Rad4 achieve selective and combinatorial binding of their multiple partner proteins.

biochemistry↗