Structure of the Cdt1 C‐terminal domain: Conservation of the winged helix fold in replication licensing factors

Khayrutdinov, Bulat I.; Bae, Won Jin; Yun, Young; Lee, Jie Hye; Tsuyama, Takashi; Kim, Jung-Joo; Hwang, Eunha; Ryu, Kyoung‐Seok; Cheong, Hae‐Kap; Cheong, Chaejoon; Ko, Jung-Soon; Enomoto, Takemi; Karplus, P. Andrew; Güntert, Peter; Tada, Shusuke; Jeon, Young Ho; Cho, Yunje

doi:10.1002/pro.236

Cited by 35 publications

(49 citation statements)

References 54 publications

(89 reference statements)

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“…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.…”

Section: Discussionmentioning

confidence: 61%

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

Chandrasekaran

Tan

Hall

et al. 2011

Molecular and Cellular Biology

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DNA replication is tightly coordinated both with cell cycle cues and with responses to extracellular signals to maintain genome stability. We discovered that human Cdt1, an essential origin licensing protein whose activity must be restricted to G 1 phase, is a substrate of the stress-activated mitogen-activated protein (MAP) kinases p38 and c-Jun N-terminal kinase (JNK). These MAP kinases phosphorylate Cdt1 both during unperturbed G 2 phase and during an acute stress response. Phosphorylation renders Cdt1 resistant to ubiquitinmediated degradation during S phase and after DNA damage by blocking Cdt1 binding to the Cul4 adaptor, Cdt2. Mutations that block normal cell cycle-regulated MAP kinase-mediated phosphorylation interfere with rapid Cdt1 reaccumulation at the end of S phase. Phosphomimetic mutations recapitulate the stabilizing effects of Cdt1 phosphorylation but also reduce the ability of Cdt1 to support origin licensing. Two other CRL4 Cdt2 targets, the cyclin-dependent kinase (CDK) inhibitor p21 and the methyltransferase PR-Set7/Set8, are similarly stabilized by MAP kinase activity. These findings support a model in which MAP kinase activity in G 2 promotes reaccumulation of a low-activity Cdt1 isoform after replication is complete.Precise and complete genome duplication presents a unique challenge during the cell division cycle. To permit efficient replication, DNA synthesis initiates at many chromosomal sites, known as origins of DNA replication. During G 1 phase, origins are loaded with an inactive form of the DNA helicase core, the minichromosome maintenance (MCM) complex. Origins with loaded MCM complexes are "licensed" because they are competent for replication initiation in the subsequent S phase. MCM loading is accomplished through recruitment of MCM complexes from the nucleoplasm by the Cdt1 protein to an origin-bound assembly of the origin recognition complex (ORC) and the Cdc6 protein. ORC and Cdc6 then load MCM onto DNA (48,57,58).Failure to properly control MCM loading can lead to replication errors and genome instability if insufficient origin licensing occurs in G 1 or if inappropriate origin relicensing occurs after the onset of S phase. For example, high levels of Cdt1 or Cdc6 activity in S or G 2 phase can promote origin relicensing, which leads to extensive rereplication and cell death; modest deregulation of either Cdc6 or Cdt1 promotes genome instability and tumorigenesis (5,28,44). Thus far, the best-understood mechanisms restricting origin licensing to G 1 phase are cell cycle-regulated accumulation and degradation of licensing proteins and inhibition of several licensing proteins after Sphase onset through phosphorylation by cyclin-dependent kinases (CDKs) (7,23,34).Given the critical need to maintain tight control and coordination of origin licensing, it is likely that additional important regulatory mechanisms have yet to be uncovered. Cell cycle progression is arrested in response to a variety of cellular stresses, including exposure to inflammatory cytokines, bacterial toxins,...

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Section: Discussionmentioning

confidence: 61%

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

Chandrasekaran

Tan

Hall

et al. 2011

Molecular and Cellular Biology

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“…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).…”

Section: Discussionmentioning

confidence: 99%

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

Shen

Chakraborty

Jain

et al. 2012

Molecular and Cellular Biology

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In eukaryotes, initiation of DNA replication requires the assembly of a multiprotein prereplicative complex (pre-RC) at the origins. We recently reported that a WD repeat-containing protein, origin recognition complex (ORC)-associated (ORCA/LRWD1), plays a crucial role in stabilizing ORC to chromatin. Here, we find that ORCA is required for the G 1 -to-S-phase transition in human cells. In addition to binding to ORC, ORCA associates with Cdt1 and its inhibitor, geminin. Single-molecule pulldown experiments demonstrate that each molecule of ORCA can bind to one molecule of ORC, one molecule of Cdt1, and two molecules of geminin. Further, ORCA directly interacts with the N terminus of Orc2, and the stability of ORCA is dependent on its association with Orc2. ORCA associates with Orc2 throughout the cell cycle, with Cdt1 during mitosis and G 1 , and with geminin in post-G 1 cells. Overexpression of geminin results in the loss of interaction between ORCA and Cdt1, suggesting that increased levels of geminin in post-G 1 cells titrate Cdt1 away from ORCA. We propose that the dynamic association of ORCA with pre-RC components modulates the assembly of its interacting partners on chromatin and facilitates DNA replication initiation.

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“…Addendum-While we were in preparation of the manuscript, a report describing the solution and crystal structures of C-terminal regions of mouse Cdt1 was published (55).…”

Section: Acknowledgments-we Thank Drs Toshihiko Eki For Constructingmentioning

confidence: 99%

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

Jee

Mizuno

Kamada

et al. 2010

Journal of Biological Chemistry

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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...

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Structure of the Cdt1 C‐terminal domain: Conservation of the winged helix fold in replication licensing factors

Cited by 35 publications

References 54 publications

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

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

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

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

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