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.

Author summary

Efficient and well-regulated DNA replication origin firing is central to ensure complete and accurate genome duplication before cell division. We here use bioinformatics and cultured human cells to understand the role of the essential origin firing factor Mdm2 binding protein (MTBP). We prove that MTBP is orthologous to yeast Sld7. Their homologous N-terminal domains bind the orthologous firing factors Treslin/TICRR and Sld3, respectively. The homologous C termini may constitute MTBP/Sld7 homodimerisation domains. Because Sld7 was the only yeast core origin firing factor for which no metazoan orthologue had been found, our work proves that the complement of core firing factors is conserved from yeast to man. We also find that MTBP contains a central region that is not found in yeast, and that is important for replication in human cells, showing that—despite fundamental conservation—higher eukaryotes have also evolved specific origin firing processes. The Cdk8/19-cyclin C transcription kinase that has not previously been implicated in DNA replication binds the central MTBP region, and this is required for complete DNA replication and proper chromosome segregation in human cells. Our work suggests that MTBP helps integrate universal conserved eukaryotic origin firing processes into the complex metazoan cells.