Structure and Mutagenesis Studies of the C-terminal Region of Licensing Factor Cdt1 Enable the Identification of Key Residues for Binding to Replicative Helicase Mcm Proteins

Abstract

In eukaryotes, DNA replication is fired once in a single cell cycle before cell division starts to maintain stability of the genome. This event is tightly controlled by a series of proteins. Cdt1 is one of the licensing factors and is involved in recruiting replicative DNA helicase Mcm2-7 proteins into the pre-replicative complex together with Cdc6. In Cdt1, the C-terminal region serves as a binding site for Mcm2-7 proteins, although the details of these interactions remain largely unknown. Here, we report the structure of the region and the key residues for binding to Mcm proteins. We determined the solution structure of the C-terminal fragment, residues 450 -557, of mouse Cdt1 by NMR. The structure consists of a winged-helix domain and shows unexpected similarity to those of the C-terminal domain of Cdc6 and the central fragment of Cdt1, thereby implying functionalandevolutionaryrelationships.Structure-basedmutagenesis and an in vitro binding assay enabled us to pinpoint the region that interacts with Mcm proteins. Moreover, by performing in vitro binding and budding yeast viability experiments, we showed that ϳ45 residues located in the N-terminal direction of the structural region are equally crucial for recognizing Mcm proteins. Our data suggest the possibility that winged-helix domain plays a role as a common module to interact with replicative helicase in the DNA replication-licensing process.In eukaryotes, DNA replication is highly coordinated to retain the integrity of the genome. Whereas DNA replication in prokaryotes begins at a single site and stops at the end of the genome, eukaryotic genomes consist of multiple replication origins where DNA replication starts. These origins are synchronized so that they are activated only once in a single division cycle. A series of proteins, the origin recognition complex (ORC), 6 cell division cycle 6 homolog (Cdc6), chromatin licensing and DNA replication factor 1 (Cdt1), and minichromosome maintenance 2-7 (Mcm2-7) are known to play correlated roles in licensing (1-5). Oncogenic proliferation often causes abnormal expression of the proteins involved in the DNA-licensing process, thus emphasizing the importance of harmonious adjustments between these proteins (6). A complicated interaction network between these proteins has been reported (7), although the details at residue and atom levels remain largely unknown.Formation of a pre-replication complex at each origin is the first event in the replication process. ORC proteins bind initially to each replication origin of DNA. The DNA sequences of the origins where ORC binds have not been identified, except for those in Saccharomyces cerevisiae (8), suggesting that there may be other factors involved in addition to the sequences (9). The DNA strand at the origin needs to be unpaired to begin replication; therefore, the existence of replicative helicase is essential. Mcm2-7 proteins are believed to function as the replicative helicase in eukaryotes. Each Mcm2-7 protein consists of a conserved AAAϩ ATPase type C-termin...

Cdt1p, through its interaction with Mcm6p, is required for the formation, nuclear accumulation and chromatin loading of the MCM complex

“…A recent study showed in budding yeast that Cdt1p C-terminal mutations, which include some of the mutation sites in Cdt1-5A cause a lethal phenotype (Jee et al, 2010), consistent with our results that the Mcm6-5A and Cdt1-5A mutations disrupt the Cdt1p-Mcm6p interaction. These results demonstrate that the interaction between Cdt1p and Mcm6p is crucial for the assembly and integrity of the Mcm2-7p complex in vivo.…”

Stress-Stimulated Mitogen-Activated Protein Kinases Control the Stability and Activity of the Cdt1 DNA Replication Licensing Factor

“…5C and data not shown). Four of the five phosphorylation sites (S391, T402, T406, and S411) lie in a flexible region of Cdt1 (36,39) that is sufficient for Mcm6 binding in vitro (70). Cdt1 is a highly dynamic protein both in vivo (72) and in a reconstituted MCM loading assay using purified Saccharomyces cerevisiae proteins (56) consistent with a model in which Cdt1 rapidly shuttles between the nucleoplasm and an origin-bound ORC-Cdc6 loading machine.…”

Dynamic Association of ORCA with Prereplicative Complex Components Regulates DNA Replication Initiation

“…Though the C terminus of Cdt1 binds to MCM2-7, the essential N terminus of Cdt1 has been shown to be critical for loading MCM2-7, along with Cdc45 and GINS, which stimulate helicase activity (51). The recent solution structure of the C-terminal domain of Cdt1 (aa 450 to 557 and aa 420 to 557 of mouse Cdt1) by X-ray crystallography and nuclear magnetic resonance (NMR) revealed the presence of a winged-helix domain that could possibly interact with MCM (23,24). Similarly, NMR studies have revealed the interaction between human Cdt1 (aa 410 to 440) and MCM6 (aa 708 to 821) (29).…”