RESUMEN
Achieving complete and precise genome duplication requires that each genomic segment be replicated only once per cell division cycle. Protecting large eukaryotic genomes from re-replication requires an overlapping set of molecular mechanisms that prevent the first DNA replication step, the DNA loading of MCM helicase complexes to license replication origins, after S phase begins. Previous reports have defined many such origin licensing inhibition mechanisms, but the temporal relationships among them are not clear, particularly with respect to preventing re-replication in G2 and M phases. Using a combination of mutagenesis, biochemistry, and single cell analyses in human cells, we define a new mechanism that prevents re-replication through hyperphosphorylation of the essential MCM loading protein, Cdt1. We demonstrate that Cyclin A/CDK1 can hyperphosphorylate Cdt1 to inhibit MCM re-loading in G2 phase. The mechanism of inhibition is to block Cdt1 binding to MCM independently of other known Cdt1 inactivation mechanisms such as Cdt1 degradation during S phase or Geminin binding. Moreover, our findings suggest that Cdt1 dephosphorylation at the mitosis-to-G1 phase transition re-activates Cdt1. We propose that multiple distinct, non-redundant licensing inhibition mechanisms act in a series of sequential relays through each cell cycle phase to ensure precise genome duplication.
Asunto(s)
Replicación del ADN/genética , Genoma Humano/genética , Origen de Réplica/genética , Duplicaciones Segmentarias en el Genoma/genética , Proteína Quinasa CDC2/genética , Proteínas de Ciclo Celular/genética , Ciclina A/genética , Fase G2/genética , Geminina/genética , Genes Duplicados/genética , Células HEK293 , Humanos , Proteínas de Mantenimiento de Minicromosoma/genética , Fosforilación/genética , Fase S/genéticaRESUMEN
The earliest step in DNA replication is origin licensing, which is the DNA loading of minichromosome maintenance (MCM) helicase complexes. The Cdc10-dependent transcript 1 (Cdt1) protein is essential for MCM loading during the G1 phase of the cell cycle, but the mechanism of Cdt1 function is still incompletely understood. We examined a collection of rare Cdt1 variants that cause a form of primordial dwarfism (the Meier-Gorlin syndrome) plus one hypomorphic Drosophila allele to shed light on Cdt1 function. Three hypomorphic variants load MCM less efficiently than wild-type (WT) Cdt1, and their lower activity correlates with impaired MCM binding. A structural homology model of the human Cdt1-MCM complex positions the altered Cdt1 residues at two distinct interfaces rather than the previously described single MCM interaction domain. Surprisingly, one dwarfism allele (Cdt1-A66T) is more active than WT Cdt1. This hypermorphic variant binds both cyclin A and SCFSkp2 poorly relative to WT Cdt1. Detailed quantitative live-cell imaging analysis demonstrated no change in the stability of this variant, however. Instead, we propose that cyclin A/CDK inhibits the Cdt1 licensing function independent of the creation of the SCFSkp2 phosphodegron. Together, these findings identify key Cdt1 interactions required for both efficient origin licensing and tight Cdt1 regulation to ensure normal cell proliferation and genome stability.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Ciclina A/metabolismo , Replicación del ADN/fisiología , Genoma Humano , Proteínas de Mantenimiento de Minicromosoma/fisiología , Alelos , Sitios de Unión , Proteínas de Ciclo Celular/genética , Línea Celular , Microtia Congénita/genética , Microtia Congénita/metabolismo , Variación Genética , Trastornos del Crecimiento/genética , Trastornos del Crecimiento/metabolismo , Células HEK293 , Humanos , Micrognatismo/genética , Micrognatismo/metabolismo , Mutación Missense , Rótula/anomalías , Rótula/metabolismo , Unión Proteica , Fase S , Proteínas Quinasas Asociadas a Fase-S/metabolismoRESUMEN
Successful cell proliferation requires efficient and precise genome duplication followed by accurate chromosome segregation. The Cdc10-dependent transcript 1 protein (Cdt1) is required for the first step in DNA replication, and in human cells Cdt1 is also required during mitosis. Tight cell cycle controls over Cdt1 abundance and activity are critical to normal development and genome stability. We review here recent advances in elucidating Cdt1 molecular functions in both origin licensing and kinetochore-microtubule attachment, and we describe the current understanding of human Cdt1 regulation.