Abstract
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.
Highlights
During normal cell proliferation DNA replication must be completed precisely once per cell cycle
We demonstrate that Cyclin A/CDK1 can hyperphosphorylate Cdt1 to inhibit maintenance complex (MCM) reloading in G2 phase
We found that the Cdt1 protein, one of the critical MCM loading factors, is inhibited
Summary
During normal cell proliferation DNA replication must be completed precisely once per cell cycle. The process of MCM loading is known as DNA replication origin licensing, and it is normally restricted to the G1 cell cycle phase [1,2,3]. Hundreds of thousands of replication origins are licensed in G1, a subset of these origins initiate replication in S phase. To achieve precise genome duplication, no origin should initiate more than once per cell cycle, and preventing re-initiation is achieved by preventing re-licensing [4,5,6,7]. Improper re-licensing in S, G2, or M phases leads to re-initiation and re-replication, a source of DNA damage and genome instability that can promote cell death or oncogenesis (reviewed in [7,8,9,10])
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