The Cdk8/19-cyclin C transcription regulator functions in genome replication through metazoan Sld7

Abstract

Accurate genome duplication underlies genetic homeostasis. Metazoan Mdm2 binding protein (MTBP) forms a main regulatory platform for origin firing together with Treslin/TICRR and TopBP1 (Topoisomerase II binding protein 1 (TopBP1)–interacting replication stimulating protein/TopBP1-interacting checkpoint and replication regulator). We report the first comprehensive analysis of MTBP and reveal conserved and metazoa-specific MTBP functions in replication. This suggests that metazoa have evolved specific molecular mechanisms to adapt replication principles conserved with yeast to the specific requirements of the more complex metazoan cells. We uncover one such metazoa-specific process: a new replication factor, cyclin-dependent kinase 8/19–cyclinC (Cdk8/19-cyclin C), binds to a central domain of MTBP. This interaction is required for complete genome duplication in human cells. In the absence of MTBP binding to Cdk8/19-cyclin C, cells enter mitosis with incompletely duplicated chromosomes, and subsequent chromosome segregation occurs inaccurately. Using remote homology searches, we identified MTBP as the metazoan orthologue of yeast synthetic lethal with Dpb11 7 (Sld7). This homology finally demonstrates that the set of yeast core factors sufficient for replication initiation in vitro is conserved in metazoa. MTBP and Sld7 contain two homologous domains that are present in no other protein, one each in the N and C termini. In MTBP the conserved termini flank the metazoa-specific Cdk8/19-cyclin C binding region and are required for normal origin firing in human cells. The N termini of MTBP and Sld7 share an essential origin firing function, the interaction with Treslin/TICRR or its yeast orthologue Sld3, respectively. The C termini may function as homodimerisation domains. Our characterisation of broadly conserved and metazoa-specific initiation processes sets the basis for further mechanistic dissection of replication initiation in vertebrates. It is a first step in understanding the distinctions of origin firing in higher eukaryotes.

Binding of the Treslin-MTBP Complex to Specific Regions of the Human Genome Promotes the Initiation of DNA Replication

“…Auxin-treated cells with two alleles of MTBP-mAID did not incorporate EdU above background levels at t = 6 h in comparison with cells incubated without auxin ( Figures 1B and S1C ). This observation underscores the importance of MTBP for entry into S-phase ( Boos et al, 2013 ; Köhler et al, 2019 ; Kumagai and Dunphy, 2017 ). On the other hand, the MTBP-mAID/MTBP-WT-FLAG cells displayed similar incorporation of EdU at t = 6 h with and without auxin ( Figures 1B and S1C ).…”

Evolutionary repair: Changes in multiple functional modules allow meiotic cohesin to support mitosis

“…In budding yeast, genes of the Cdk8 complex have genetic interactions with genes that trigger replication initiation (DBF4, DPB11, SLD3, and CDC7) and core helicase components (SLD5) [73]. In fission yeast [74] and mammalian cells [75], mutations of the Cdk8 complex are reported to alter genome replication, suggesting our finding that the Cdk8 module is involved in genome replication is not species specific, although the detailed mechanism remains unknown.…”

Origin Firing Regulations to Control Genome Replication Timing

“…Instead, an essential origin firing factor, the MTBP protein, for which no orthologue in yeast could be found and that binds to Treslin/TICRR Sld3 was described [88]. Only later remote homology of MTBP with Sld7 in two protein regions could be detected [89]. MTBP Sld7 completed the list of core origin firing factors in higher eukaryotes that had been defined in yeast.…”

“…The metazoan orthologues of the SDS complex, TMT complex proteins Treslin/TICRR Sld3 , MTBP Sld7 and TopBP1 Dpb11 , have metazoa-specific protein domains in addition to the core domains conserved with yeast [88,89]. These metazoa-specific domains may well constitute regulatory units that mediate proper control of origin firing in the complex metazoan cells, in which appropriately controlled origin firing is probably particularly important.…”