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1.
In eukaryotic cells, replication of genomic DNA initiates from multiple replication origins distributed on multiple chromosomes. To ensure that each origin is activated precisely only once during each S phase, a system has evolved which features periodic assembly and disassembly of essential pre-replication complexes (pre-RCs) at replication origins. The pre-RC assembly reaction involves the loading of a presumptive replicative helicase, the MCM2-7 complexes, onto chromatin by the origin recognition complex (ORC) and two essential factors, CDC6 and Cdt1. The eukaryotic cell cycle is driven by the periodic activation and inactivation of cyclin-dependent kinases (Cdks) and assembly of pre-RCs can only occur during the low Cdk activity period from late mitosis through G1 phase, with inappropriate re-assembly suppressed during S, G2, and M phases. It was originally suggested that inhibition of Cdt1 function after S phase in vertebrate cells is due to geminin binding and that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance induces re-replication. However, recent progress has revealed that Cdt1 activity is more strictly regulated by two other mechanisms in addition to geminin: (1) functional and SCFSkp2-mediated proteolytic regulation through phosphorylation by Cdks; and (2) replication-coupled proteolysis mediated by the Cullin4-DDB1Cdt2 ubiquitin ligase and PCNA, an eukaryotic sliding clamp stimulating replicative DNA polymerases. The tight regulation implies that Cdt1 control is especially critical for the regulation of DNA replication in mammalian cells. Indeed, Cdt1 overexpression evokes chromosomal damage even without re-replication. Furthermore, deregulated Cdt1 induces chromosomal instability in normal human cells. Since Cdt1 is overexpressed in cancer cells, this could be a new molecular mechanism leading to carcinogenesis. In this review, recent insights into Cdt1 function and regulation in mammalian cells are discussed. 相似文献
2.
《Cell cycle (Georgetown, Tex.)》2013,12(21):4351-4363
The efficiency of metazoan origins of DNA replication is known to be enhanced by histone acetylation near origins. Although this correlates with increased MCM recruitment, the mechanism by which such acetylation regulates MCM loading is unknown. We show here that Cdt1 induces large scale chromatin decondensation that is required for MCM recruitment. This process occurs in G1, is suppressed by Geminin, and requires HBO1 HAT activity and histone H4 modifications. HDAC11, which binds Cdt1 and replication origins during S-phase, potently inhibits Cdt1-induced chromatin unfolding and re-replication, suppresses MCM loading, and binds Cdt1 more efficiently in the presence of Geminin. We also demonstrate that chromatin at endogenous origins is more accessible in G1 relative to S-phase. These results provide evidence that histone acetylation promotes MCM loading via enhanced chromatin accessibility. This process is regulated positively by Cdt1 and HBO1 in G1 and repressed by Geminin-HDAC11 association with Cdt1 in S-phase, and represents a novel form of replication licensing control. 相似文献
3.
Rakotomalala L Studach L Wang WH Gregori G Hullinger RL Andrisani O 《The Journal of biological chemistry》2008,283(42):28729-28740
Hepatitis B virus X protein (pX) is implicated in hepatocellular carcinoma pathogenesis by an unknown mechanism. Employing the tetracycline-regulated pX-expressing 4pX-1 cell line, derived from the murine AML12 hepatocyte cell line, we demonstrate that pX induces partial polyploidy (>4N DNA). Depletion of p53 in 4pX-1 cells increases by 5-fold the polyploid cells in response to pX expression, indicating that p53 antagonizes pX-induced polyploidy. Dual-parameter flow cytometric analyses show pX-dependent bromodeoxyuridine (BrdUrd) incorporation in 4pX-1 cells containing 4N and >4N DNA, suggesting pX induces DNA re-replication. Interestingly, pX increases expression of endogenous replication initiation factors Cdc6 and Cdtl while suppressing geminin expression, a negative regulator of rereplication. In comparison to a geminin knockdown 4pX-1 cell line used as DNA re-replication control, the Cdt1/geminin ratio is greater in 4pX-1 cells expressing pX, indicating that pX promotes DNA re-replication. In support of this conclusion, pX-expressing 4pX-1 cells, similar to the geminin knockdown 4pX-1 cells, continue to incorporate BrdUrd in the G2 phase and exhibit nuclear Cdc6 and MCM5 co-localization and the absence of geminin. In addition, pX expression activates the ATR kinase, the sensor of DNA re-replication, which in turn phosphorylates RAD17 and H2AX. Interestingly, phospho-H2AX-positive and BrdUrd -positive cells progress through mitosis, demonstrating a link between pX-induced DNA re-replication and polyploidy. Our studies high-light a novel function of pX that likely contributes to hepatocellular carcinoma pathogenesis. 相似文献
4.
Initiation of DNA synthesis involves the loading of the MCM2-7 helicase onto chromatin by Cdt1 (origin licensing). Geminin is thought to prevent relicensing by binding and inhibiting Cdt1. Here we show, using Xenopus egg extracts, that geminin binding to Cdt1 is not sufficient to block its activity and that a Cdt1-geminin complex licenses chromatin, but prevents rereplication, working as a molecular switch at replication origins. We demonstrate that geminin is recruited to chromatin already during licensing, while bulk geminin is recruited at the onset of S phase. A recombinant Cdt1-geminin complex binds chromatin, interacts with the MCM2-7 complex and licenses chromatin once per cell cycle. Accordingly, while recombinant Cdt1 induces rereplication in G1 or G2 and activates an ATM/ATR-dependent checkpoint, the Cdt1-geminin complex does not. We further demonstrate that the stoichiometry of the Cdt1-geminin complex regulates its activity. Our results suggest a model in which the MCM2-7 helicase is loaded onto chromatin by a Cdt1-geminin complex, which is inactivated upon origin firing by binding additional geminin. This origin inactivation reaction does not occur if only free Cdt1 is present on chromatin. 相似文献
5.
Cdt1 downregulation by proteolysis and geminin inhibition prevents DNA re-replication in Xenopus 总被引:4,自引:0,他引:4
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In late mitosis and G1, Mcm2-7 are assembled onto replication origins to 'license' them for initiation. At other cell cycle stages, licensing is inhibited, thus ensuring that origins fire only once per cell cycle. Three additional factors--the origin recognition complex, Cdc6 and Cdt1--are required for origin licensing. We examine here how licensing is regulated in Xenopus egg extracts. We show that Cdt1 is downregulated late in the cell cycle by two different mechanisms: proteolysis, which occurs in part due to the activity of the anaphase-promoting complex (APC/C), and inhibition by a protein called geminin. If both these regulatory mechanisms are abrogated, extracts undergo uncontrolled re-licensing and re-replication. The extent of re-replication is limited by checkpoint kinases that are activated as a consequence of re-replication itself. These results allow us to build a comprehensive model of how re-replication of DNA is prevented in Xenopus, with Cdt1 regulation being the key feature. The results also explain the original experiments that led to the proposal of a replication licensing factor. 相似文献
6.
Yoshiyuki Mizushina Toshifumi Takeuchi Yoichi Takakusagi Yuko Yonezawa Takeshi Mizuno Ken-ichiro Yanagi Naoko Imamoto Fumio Sugawara Kengo Sakaguchi Hiromi Yoshida Masatoshi Fujita 《Biochimica et Biophysica Acta (BBA)/General Subjects》2008
A human replication initiation protein Cdt1 is a very central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by directly binding to it. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1–geminin imbalance, for example by geminin silencing with siRNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. In the present study, we first established a high throughput screening system based on modified ELISA (enzyme linked immunosorbent assay) to identify compounds that interfere with human Cdt1–geminin binding. Using this system, we found that coenzyme Q10 (CoQ10) can inhibit Cdt1–geminin interaction in vitro. CoQ compound is an isoprenoid quinine that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. CoQ10, having a longer isoprenoid chain, was the strongest inhibitor of Cdt1–geminin binding in the tested CoQs, with 50% inhibition observed at concentrations of 16.2 μM. Surface plasmon resonance analysis demonstrated that CoQ10 bound selectively to Cdt1, but did not interact with geminin. Moreover, CoQ10 had no influence on the interaction between Cdt1 and mini-chromosome maintenance (MCM)4/6/7 complexes. These results suggested that CoQ10 inhibits Cdt1–geminin complex formation by binding to Cdt1 and thereby could liberate Cdt1 from inhibition by geminin. Using three-dimensional computer modeling analysis, CoQ10 was considered to interact with the geminin interaction interface on Cdt1, and was assumed to make hydrogen bonds with the residue of Arg243 of Cdt1. CoQ10 could prevent the growth of human cancer cells, although only at high concentrations, and it remains unclear whether such an inhibitory effect is associated with the interference with Cdt1–geminin binding. The application of inhibitors for the formation of Cdt1–geminin complex is discussed. 相似文献
7.
Cdt1 is a conserved replication factor required in licensing the chromosome for a single round of DNA synthesis. The activity of Cdt1 is inhibited by geminin. The mechanism by which geminin interferes with Cdt1 activity is unknown. It is thought that geminin binds to and sequestrate Cdt1. We show that geminin does not interfere with the chromatin association of Cdt1 and that inhibition of DNA synthesis by geminin is observed following its accumulation on chromatin. The binding of geminin to chromatin has been investigated during S phase. We demonstrate that loading of geminin onto chromatin requires Cdt1, suggesting that geminin is targeted at replication origins. We also show that geminin binds chromatin at the transition from the pre-replication to pre-initiation complexes, which overlaps with the release of Cdt1. This regulation is strikingly different from that observed in somatic cells where the chromatin binding of these proteins is mutually exclusive. In contrast to somatic cells, we further show that geminin is stable during the early embryonic cell cycles. These results suggest a specific regulation of origin firing adapted to the rapid cell cycles of Xenopus and indicate that periodic degradation of geminin is not relevant to licensing during embryonic development. 相似文献
8.
Mizushina Y Takeuchi T Takakusagi Y Yonezawa Y Mizuno T Yanagi K Imamoto N Sugawara F Sakaguchi K Yoshida H Fujita M 《Biochimica et biophysica acta》2008,1780(2):203-213
A human replication initiation protein Cdt1 is a very central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by directly binding to it. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance, for example by geminin silencing with siRNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. In the present study, we first established a high throughput screening system based on modified ELISA (enzyme linked immunosorbent assay) to identify compounds that interfere with human Cdt1-geminin binding. Using this system, we found that coenzyme Q(10) (CoQ(10)) can inhibit Cdt1-geminin interaction in vitro. CoQ compound is an isoprenoid quinine that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. CoQ(10), having a longer isoprenoid chain, was the strongest inhibitor of Cdt1-geminin binding in the tested CoQs, with 50% inhibition observed at concentrations of 16.2 muM. Surface plasmon resonance analysis demonstrated that CoQ(10) bound selectively to Cdt1, but did not interact with geminin. Moreover, CoQ(10) had no influence on the interaction between Cdt1 and mini-chromosome maintenance (MCM)4/6/7 complexes. These results suggested that CoQ(10) inhibits Cdt1-geminin complex formation by binding to Cdt1 and thereby could liberate Cdt1 from inhibition by geminin. Using three-dimensional computer modeling analysis, CoQ(10) was considered to interact with the geminin interaction interface on Cdt1, and was assumed to make hydrogen bonds with the residue of Arg243 of Cdt1. CoQ(10) could prevent the growth of human cancer cells, although only at high concentrations, and it remains unclear whether such an inhibitory effect is associated with the interference with Cdt1-geminin binding. The application of inhibitors for the formation of Cdt1-geminin complex is discussed. 相似文献
9.
During late mitosis and early interphase, origins of replication become "licensed" for DNA replication by loading Mcm2-7 complexes. Mcm2-7 complexes are removed from origins as replication forks initiate replication, thus preventing rereplication of DNA in a single cell cycle. Premature origin licensing is prevented in metaphase by the action of geminin, which binds and inhibits Cdt1/RLF-B, a protein that is required for the loading of Mcm2-7. Recombinant geminin that is added to Xenopus egg extracts is efficiently degraded upon exit from metaphase. Here, we show that recombinant and endogenous forms of Xenopus geminin behave differently from one another, such that a significant proportion of endogenous geminin escapes proteolysis upon exit from metaphase. During late mitosis and early G1, the surviving population of endogenous geminin does not associate with Cdt1/RLF-B and does not inhibit licensing. Following nuclear assembly, geminin is imported into nuclei and becomes reactivated to bind Cdt1/RLF-B. This reactivated geminin provides the major nucleoplasmic inhibitor of origin relicensing during late interphase. Since the initiation of replication at licensed origins depends on nuclear assembly, our results suggest an elegant and novel mechanism for preventing rereplication of DNA in a single cell cycle. 相似文献
10.
Nishitani H Sugimoto N Roukos V Nakanishi Y Saijo M Obuse C Tsurimoto T Nakayama KI Nakayama K Fujita M Lygerou Z Nishimoto T 《The EMBO journal》2006,25(5):1126-1136
Replication licensing is carefully regulated to restrict replication to once in a cell cycle. In higher eukaryotes, regulation of the licensing factor Cdt1 by proteolysis and Geminin is essential to prevent re-replication. We show here that the N-terminal 100 amino acids of human Cdt1 are recognized for proteolysis by two distinct E3 ubiquitin ligases during S-G2 phases. Six highly conserved amino acids within the 10 first amino acids of Cdt1 are essential for DDB1-Cul4-mediated proteolysis. This region is also involved in proteolysis following DNA damage. The second E3 is SCF-Skp2, which recognizes the Cy-motif-mediated Cyclin E/A-cyclin-dependent kinase-phosphorylated region. Consistently, in HeLa cells cosilenced of Skp2 and Cul4, Cdt1 remained stable in S-G2 phases. The Cul4-containing E3 is active during ongoing replication, while SCF-Skp2 operates both in S and G2 phases. PCNA binds to Cdt1 through the six conserved N-terminal amino acids. PCNA is essential for Cul4- but not Skp2-directed degradation during DNA replication and following ultraviolet-irradiation. Our data unravel multiple distinct pathways regulating Cdt1 to block re-replication. 相似文献
11.
Hall JR Lee HO Bunker BD Dorn ES Rogers GC Duronio RJ Cook JG 《The Journal of biological chemistry》2008,283(37):25356-25363
The replication factors Cdt1 and Cdc6 are essential for origin licensing, a prerequisite for DNA replication initiation. Mechanisms to ensure that metazoan origins initiate once per cell cycle include degradation of Cdt1 during S phase and inhibition of Cdt1 by the geminin protein. Geminin depletion or overexpression of Cdt1 or Cdc6 in human cells causes rereplication, a form of endogenous DNA damage. Rereplication induced by these manipulations is however uneven and incomplete, suggesting that one or more mechanisms restrain rereplication once it begins. We find that both Cdt1 and Cdc6 are degraded in geminin-depleted cells. We further show that Cdt1 degradation in cells that have rereplicated requires the PCNA binding site of Cdt1 and the Cul4(DDB1) ubiquitin ligase, and Cdt1 can induce its own degradation when overproduced. Cdc6 degradation in geminin-depleted cells requires Huwe1, the ubiquitin ligase that regulates Cdc6 after DNA damage. Moreover, perturbations that specifically disrupt Cdt1 and Cdc6 degradation in response to DNA damage exacerbate rereplication when combined with geminin depletion, and this enhanced rereplication occurs in both human cells and in Drosophila melanogaster cells. We conclude that rereplication-associated DNA damage triggers Cdt1 and Cdc6 ubiquitination and destruction, and propose that this pathway represents an evolutionarily conserved mechanism that minimizes the extent of rereplication. 相似文献
12.
During late mitosis and early G1, replication origins are licensed for subsequent replication by loading heterohexamers of the mini-chromosome maintenance proteins (Mcm2-7). To prevent re-replication of DNA, the licensing system is down-regulated at other cell cycle stages. A small protein called geminin plays an important role in this down-regulation by binding and inhibiting the Cdt1 component of the licensing system. We examine here the organization of Xenopus Cdt1, delimiting regions of Cdt1 required for licensing and regions required for geminin interaction. The C-terminal 377 residues of Cdt1 are required for licensing and the extreme C-terminus contains a domain that interacts with an Mcm(2,4,6,7) complex. Two regions of Cdt1 interact with geminin: one at the N-terminus, and one in the centre of the protein. Only the central region binds geminin tightly enough to successfully compete with full-length Cdt1 for geminin binding. This interaction requires a predicted coiled-coil domain that is conserved amongst metazoan Cdt1 homologues. Geminin forms a homodimer, with each dimer binding one molecule of Cdt1. Separation of the domains necessary for licensing activity from domains required for a strong interaction with geminin generated a construct, whose licensing activity was partially insensitive to geminin inhibition. 相似文献
13.
Philip G Wong Michele A Glozak Thinh V Cao Cyrus Vaziri Edward Seto Mark G Alexandrow 《Cell cycle (Georgetown, Tex.)》2010,9(21):4351-4363
The efficiency of metazoan origins of DNA replication is known to be enhanced by histone acetylation near origins. Although this correlates with increased MCM recruitment, the mechanism by which such acetylation regulates MCM loading is unknown. We show here that Cdt1 induces large-scale chromatin decondensation that is required for MCM recruitment. This process occurs in G1, is suppressed by Geminin and requires HBO1 HAT activity and histone H4 modifications. HDAC11, which binds Cdt1 and replication origins during S phase, potently inhibits Cdt1-induced chromatin unfolding and re-replication, suppresses MCM loading and binds Cdt1 more efficiently in the presence of Geminin. We also demonstrate that chromatin at endogenous origins is more accessible in G1 relative to S phase. These results provide evidence that histone acetylation promotes MCM loading via enhanced chromatin accessibility. This process is regulated positively by Cdt1 and HBO1 in G1 and repressed by Geminin-HDAC11 association with Cdt1 in S phase and represents a novel form of replication licensing control.Key words: Cdt1, HBO1, HDAC11, chromatin, DNA replication 相似文献
14.
Mouse geminin inhibits not only Cdt1-MCM6 interactions but also a novel intrinsic Cdt1 DNA binding activity 总被引:7,自引:0,他引:7
DNA replication is controlled by the stepwise assembly of a pre-replicative complex and the replication apparatus. Cdt1 is a novel component of the pre-replicative complex and plays a role in loading the minichromosome maintenance (MCM) 2-7 complex onto chromatin. Cdt1 activity is inhibited by geminin, which is essential for the G(2)/M transition in metazoan cells. To understand the molecular basis of the Cdt1-geminin regulatory mechanism in mammalian cells, we cloned and expressed the mouse Cdt1 homologue cDNA in bacterial cells and purified mouse Cdt1 to near homogeneity. We found by yeast two-hybrid analysis that mouse Cdt1 associates with geminin, MCM6, and origin recognition complex 2. MCM6 interacts with the Cdt1 carboxyl-terminal region (amino acids 407-477), which is conserved among eukaryotes, whereas geminin associates with the Cdt1 central region (amino acids 177-380), which is conserved only in metazoans. In addition, we found that Cdt1 can bind DNA in a sequence-, strand-, and conformation-independent manner. The Cdt1 DNA binding domain overlaps with the geminin binding domain, and the binding of Cdt1 to DNA is inhibited by geminin. Taken together, we have defined structural domains and novel biochemical properties for mouse Cdt1 that suggest that Cdt1 behaves as an intrinsic DNA binding factor in the pre-replicative complex. 相似文献
15.
Initiation of DNA replication in eukaryotes is exquisitely regulated to ensure that DNA replication occurs exactly once in each cell division. A conserved and essential step for the initiation of eukaryotic DNA replication is the loading of the mini-chromosome maintenance 2-7 (MCM2-7) helicase onto chromatin at replication origins by Cdt1. To elucidate the molecular mechanism of this event, we determined the structure of the human Cdt1-Mcm6 binding domains, the Cdt1(410-440)/MCM6(708-821) complex by NMR. Our structural and site-directed mutagenesis studies showed that charge complementarity is a key determinant for the specific interaction between Cdt1 and Mcm2-7. When this interaction was interrupted by alanine substitutions of the conserved interacting residues, the corresponding yeast Cdt1 and Mcm6 mutants were defective in DNA replication and the chromatin loading of Mcm2, resulting in cell death. Having shown that Cdt1 and Mcm6 interact through their C-termini, and knowing that Cdt1 is tethered to Orc6 during the loading of MCM2-7, our results suggest that the MCM2-7 hexamer is loaded with its C terminal end facing the ORC complex. These results provide a structural basis for the Cdt1-mediated MCM2-7 chromatin loading. 相似文献
16.
17.
Senga T Sivaprasad U Zhu W Park JH Arias EE Walter JC Dutta A 《The Journal of biological chemistry》2006,281(10):6246-6252
Cdt1, a protein essential in G1 for licensing of origins for DNA replication, is inhibited in S-phase, both by binding to geminin and degradation by proteasomes. Cdt1 is also degraded after DNA damage to stop licensing of new origins until after DNA repair. Phosphorylation of Cdt1 by cyclin-dependent kinases promotes its binding to SCF-Skp2 E3 ubiquitin ligase, but the Cdk2/Skp2-mediated pathway is not essential for the degradation of Cdt1. Here we show that the N terminus of Cdt1 contains a second degradation signal that is active after DNA damage and in S-phase and is dependent on the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) through a PCNA binding motif. The degradation involves N-terminal ubiquitination and requires Cul4 and Ddb1 proteins, components of an E3 ubiquitin ligase implicated in protein degradation after DNA damage. Therefore PCNA, the matchmaker for many proteins involved in DNA and chromatin metabolism, also serves to promote the targeted degradation of associated proteins in S-phase or after DNA damage. 相似文献
18.
Elizabeth S. Dorn Paul D. Chastain II Jonathan R. Hall Jeanette Gowen Cook 《Nucleic acids research》2009,37(1):60-69
A major challenge each human cell-division cycle is to ensure that DNA replication origins do not initiate more than once, a phenomenon known as re-replication. Acute deregulation of replication control ultimately causes extensive DNA damage, cell-cycle checkpoint activation and cell death whereas moderate deregulation promotes genome instability and tumorigenesis. In the absence of detectable increases in cellular DNA content however, it has been difficult to directly demonstrate re-replication or to determine if the ability to re-replicate is restricted to a particular cell-cycle phase. Using an adaptation of DNA fiber spreading we report the direct detection of re-replication on single DNA molecules from human chromosomes. Using this method we demonstrate substantial re-replication within 1 h of S phase entry in cells overproducing the replication factor, Cdt1. Moreover, a comparison of the HeLa cancer cell line to untransformed fibroblasts suggests that HeLa cells produce replication signals consistent with low-level re-replication in otherwise unperturbed cell cycles. Re-replication after depletion of the Cdt1 inhibitor, geminin, in an untransformed fibroblast cell line is undetectable by standard assays but readily quantifiable by DNA fiber spreading analysis. Direct evaluation of re-replicated DNA molecules will promote increased understanding of events that promote or perturb genome stability. 相似文献
19.
A human replication initiation protein, Cdt1, is a central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by direct binding. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance, for example, by geminin silencing with small interfering RNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. We established a high throughput screening system based on a modified enzyme linked immunosorbent assay to identify compounds that interfere with human Cdt1-geminin binding. Using this system, we screened inhibitors from natural materials containing food components, and found that a glycolipid, sulfoquinovosyl diacylglycerol (SQDG), from spinach can inhibit Cdt1-geminin interaction in vitro, with 50% inhibition observed at concentrations of 1.79mug/ml. Other major glycolipids, such as monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) from spinach, had no influence. Surface plasmon resonance analysis demonstrated that SQDG bound selectively to Cdt1, but did not interact with geminin. Using three-dimensional computer modeling analysis, SQDG was considered to interact with the geminin interaction interface on Cdt1, and the sulfate group of SQDG was assumed to make hydrogen bonds with the residue of Arg346 of Cdt1. These data could help to further understanding of the structure and function of Cdt1. In addition, SQDG could be a clue to developing more appropriate inhibitors of Cdt1-geminin interactions. 相似文献
20.
Prereplication complexes (pre-RCs) define potential origins of DNA replication and allow the recruitment of the replicative DNA helicase MCM2-7. Here, we characterize MCM9, a member of the MCM2-8 family. We demonstrate that MCM9 binds to chromatin in an ORC-dependent manner and is required for the recruitment of the MCM2-7 helicase onto chromatin. Its depletion leads to a block in pre-RC assembly, as well as DNA replication inhibition. We show that MCM9 forms a stable complex with the licensing factor Cdt1, preventing an excess of geminin on chromatin during the licensing reaction. Our data suggest that MCM9 is an essential activating linker between Cdt1 and the MCM2-7 complex, required for loading the MCM2-7 helicase onto DNA replication origins. Thus, Cdt1, with its two opposing regulatory binding factors MCM9 and geminin, appears to be a major platform on the pre-RC to integrate cell-cycle signals. 相似文献