Roles of Human AND-1 in Chromosome Transactions in S Phase

Coordinated execution of DNA replication, checkpoint activation, and postreplicative chromatid cohesion is intimately related to the replication fork machinery. Human AND-1/chromosome transmission fidelity 4 is localized adjacent to replication foci and is required for efficient DNA synthesis. In S phase, AND-1 is phosphorylated in response to replication arrest in a manner dependent on checkpoint kinase, ataxia telangiectasia-mutated, ataxia telangiectasia-mutated and Rad3-related protein, and Cdc7 kinase but not on Chk1. Depletion of AND-1 increases DNA damage, delays progression of S phase, leads to accumulation of late S and/or G₂ phase cells, and induces cell death in cancer cells. It also elevated UV-radioresistant DNA synthesis and caused premature recovery of replication after hydroxyurea arrest, indicating that lack of AND-1 compromises checkpoint activation. This may be partly due to the decreased levels of Chk1 protein in AND-1-depleted cells. Furthermore, AND-1 interacts with cohesin proteins Smc1, Smc3, and Rad21/Scc1, consistent with proposed roles of yeast counterparts of AND-1 in sister chromatid cohesion. Depletion of AND-1 leads to significant inhibition of homologous recombination repair of an I-SceI-driven double strand break. Based on these data, we propose that AND-1 coordinates multiple cellular events in S phase and G₂ phase, such as DNA replication, checkpoint activation, sister chromatid cohesion, and DNA damage repair, thus playing a pivotal role in maintenance of genome integrity.Replication fork is not only the site of DNA synthesis but also the center for coordinated execution of various chromosome transactions. The preparation for replication forks starts in the G₁ phase, when the prereplicative complex composed of origin recognition and minichromosome maintenance assembles on the chromosome. At the G₁-S boundary, Cdc45, GINS complex, and other factors join the prereplicative complex to generate a complex capable of initiating DNA replication. A series of phosphorylation events mediated by cyclin-dependent kinase and Cdc7 kinase play crucial roles in this process and facilitate the generation of active replication forks (1–6). Purification of the putative replisome complex in yeast indicated the presence of the checkpoint mediator Mrc1 and fork protection complex proteins Tof1 and Csm3 in the replication fork machinery (7), consistent with a previous report on the genome-wide analyses with chromatin immunoprecipitation analyses on chip (microarray) (8). Mcm10 is another factor present in the isolated complex, required for loading of replication protein A (RPA)² and primase-DNA polymerase α onto the replisome complex (7, 9, 10).Replication fork machinery can cope with various stresses, including shortage of the cellular nucleotide pool and replication fork blockages that interfere with its progression. Stalled replication forks activate checkpoint pathways, leading to cell cycle arrest, DNA repair, restart of DNA replication, or cell death in some cases (11–14). Single-stranded DNAs coated with RPA at the stalled replication forks are recognized by the ATR-ATR-interacting protein kinase complex and Rad17 for loading of the Rad9-Rad1-Hus1 checkpoint clamp (14–16). Factors present in the replisome complex are also known to be required for checkpoint activation. Claspin, Tim, and Tipin functionally and physically associate with sensor and effector kinases and serve as mediator/adaptors (17–23). Mcm7, a component of the replicative DNA helicase in eukaryotes, was reported to associate with the checkpoint clamp loader Rad17 (24) and to have a distinct function in checkpoint (24, 25). We recently reported that Cdc7 kinase, known to be required for DNA replication initiation, plays a role in activation of DNA replication checkpoint possibly through regulating Claspin phosphorylation (26). Thus, it appears that DNA replication and checkpoint activation functionally and physically interact with each other.Another crucial cellular event for maintenance of genome stability is sister chromatid cohesion. The cohesin complex, a conserved apparatus required for sister chromatid cohesion, contains Smc1, Smc3, and Rad21/Scc1/Mcd1 proteins. The assembled cohesin complexes are loaded onto chromatin prior to DNA replication in G₁ phase and link the sister chromosomes during S and G₂ phase until mitosis when they separate (27, 28). The mitotic cohesion defects are not rescued by supplementing cohesin in G₂ phase, and it has been suggested that establishment of sister chromatid cohesion is coupled with DNA replication (29, 30). Indeed, yeast mutants in some replisome components show defect in sister chromosome cohesion or undergo chromosome loss (31–33). Cdc7 kinase is also required for efficient mitotic chromosome cohesion (34, 35).Human AND-1 is the putative homolog of budding yeast CTF4/Pob1/CHL15 and fission yeast Mcl1/Slr3. The budding yeast counterpart was identified as a replisome component described above (7), which travels along with the replication fork (29). CTF4 is nonessential for viability, but its interactions with primase, Rad2 (FEN1 family of nuclease), and Dna2 have implicated CTF4 in lagging strand synthesis and/or Okazaki fragment processing (36–39). Yeast CTF4 and Mcl1 are involved in chromosome cohesion (33, 40, 41) and genetically interact with a cohesin, Mcd1/Rad21 (40, 42). Recently, it was reported that human AND-1 protein interacts with human primase-DNA polymerase α and Mcm10 and is required for DNA synthesis (43).Here we confirm that human AND-1 protein is required for DNA replication and efficient progression of S phase, and we further show that it facilitates replication checkpoint. Depletion of AND-1 causes accumulation of DNA damage and cell cycle arrest at late S to G₂ phase, ultimately leading to cell death. Furthermore, we also show that human AND-1 physically interacts with cohesin proteins Smc1, Smc3, Rad21/Scc1, suggesting a possibility that AND-1 may physically and functionally link replisome and cohesin complexes in vivo. Recent studies indicate that sister chromatid cohesion is required for recombinational DNA repair (44–47). Thus, we examined the requirement of AND-1 for repair of artificially induced double-stranded DNA breaks and showed that AND-1 depletion leads to significant reduction of the double strand break repair. Possible roles of AND-1 in coordination of various chromosome transactions at a replication fork and in maintenance of genome integrity during S phase will be discussed.     

In Vitro and In Vivo Biology of Recombinant Adenovirus Vectors with E1, E1/E2A,or E1/E4 Deleted

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.Replication-deficient human adenoviruses (Ad) have been widely investigated as ex vivo and in vivo gene delivery systems for human gene therapy. The ability of these vectors to mediate the efficient expression of candidate therapeutic or vaccine genes in a variety of cell types, including postmitotic cells, is considered an advantage over other gene transfer vectors (3, 28, 49). However, the successful application of currently available E1-defective Ad vectors in human gene therapy has been hampered by the fact that transgene expression is only transient in vivo (2, 15, 16, 33, 36, 46). This short-lived in vivo expression of the transgene has been explained, at least in part, by the induction in vivo of cytotoxic immune responses to cells infected with the Ad vector. Studies with rodent systems have suggested that cytotoxic T lymphocytes (CTLs) directed against virus antigens synthesized de novo in the transduced tissues play a major role in eliminating cells containing the E1-deleted viral genome (56–58, 61). Consistent with the concept of cellular antiviral immunity, expression of transgenes is significantly extended in experimental rodent systems that are deficient in various components of the cellular immune system or that have been rendered immunocompromised by administration of pharmacological agents (2, 33, 37, 48, 60, 64).Based on the assumption that further reduction of viral antigen expression may lower the immune response and thus extend persistence of transgene expression, previous studies have investigated the consequences of deleting both E1 and an additional viral regulatory region, such as E2A or E4. The E2A region encodes a DNA binding protein (DBP) with specific affinity for single-stranded Ad DNA. The DNA binding function is essential for the initiation and elongation of viral DNA synthesis during the early phase of Ad infection. During the late phase of infection, DBP plays a central role in the activation of the major late promoter (MLP) (for a recent review, see reference 44). The E4 region, located at the right end of the viral genome, encodes several regulatory proteins with pleiotropic functions which are involved in the accumulation, splicing, and transport of early and late viral mRNAs, in DNA replication, and in virus particle assembly (reviewed in reference 44). The simultaneous deletion of E1 and E2A or of E1 and E4 should therefore further reduce the replication of the virus genome and the expression of early and late viral genes. Such multidefective vectors have been generated and tested in vitro and in vivo (9, 12, 17, 19–21, 23, 24, 26, 34, 40, 52, 53, 59, 62, 63). Recombinant vectors with E1 deleted and carrying an E2A temperature-sensitive mutation (E2Ats) have been shown in vitro to express much smaller amounts of virus proteins, leading to extended transgene expression in cotton rats and mice (19, 20, 24, 59). To eliminate the risks of reversion of the E2Ats point mutation to a wild-type phenotype, improved vectors with both E1 and E2A deleted were subsequently generated in complementation cell lines coexpressing E1 and E2A genes (26, 40, 63). In vitro analysis of human cells infected by these viruses demonstrated that the double deletion completely abolished viral DNA replication and late protein synthesis (26). Similarly, E1/E4-deleted vectors have been generated in various in vitro complementation systems and tested in vitro and in vivo (9, 17, 23, 45, 52, 53, 62). These studies showed that deletion of both E1 and E4 did indeed reduce significantly the expression of early and late virus proteins (17, 23), leading to a decreased anti-Ad host immune response (23), reduced hepatotoxicity (17, 23, 52), and improved in vivo persistence of the transduced liver cells (17, 23, 52).Interpretation of these results is difficult, however, since all tested E1- and E1/E4-deleted vectors encoded the bacterial β-galactosidase (βgal) marker, whose strong immunogenicity is known to influence the in vivo persistence of Ad-transduced cells (32, 37). Moreover, the results described above are not consistent with the conclusions from other studies showing, in various immunocompetent mouse models, that cellular immunity to Ad antigens has no detectable impact on the persistence of the transduced cells (37, 40, 50, 51). Furthermore, in contrast to results of earlier studies (19, 20, 59), Fang et al. (21) demonstrated that injection of E1-deleted/E2Ats vectors into immunocompetent mice and hemophilia B dogs did not lead to an improvement of the persistence of transgene expression compared to that with isogenic E1-deleted vectors. Similarly, Morral et al. (40) did not observe any difference in persistence of transgene expression in mice injected with either vectors deleted in E1 only or vectors deleted in both E1 and E2A. Finally, the demonstration that some E4-encoded products can modulate transgene expression (1, 17, 36a) makes the evaluation of E1- and E1/E4-deleted vectors even more complex when persistence of transgene expression is used for direct comparison of the in vivo persistence of cells transduced by the two types of vectors.The precise influence of the host immune response to viral antigens on the in vivo persistence of the transduced cells, and hence the impact of further deletions in the virus genome, therefore still remains unclear. To investigate these questions, we generated a set of isogenic vectors with single deletions (AdE1°) and double deletions (AdE1°E2A° and AdE1°E4°) and their corresponding complementation cell lines and compared the biologies and immunogenicities of these vectors in vitro and in vivo. To eliminate any possible influence of transgene-encoded products on the interpretation of the in vivo results, we used E1-, E1/E2A-, and E1/E4-deleted vectors with no transgenes.     

Reconstitution of Rad53 Activation by Mec1 through Adaptor Protein Mrc1

Upon DNA replication stress, stalled DNA replication forks serve as a platform to recruit many signaling proteins, leading to the activation of the DNA replication checkpoint. Activation of Rad53, a key effector kinase in the budding yeast Saccharomyces cerevisiae, is essential for stabilizing DNA replication forks during replication stress. Using an activity-based assay for Rad53, we found that Mrc1, a replication fork-associated protein, cooperates with Mec1 to activate Rad53 directly. Reconstitution of Rad53 activation using purified Mec1 and Mrc1 showed that the addition of Mrc1 stimulated a more than 70-fold increase in the ability of Mec1 to activate Rad53. Instead of increasing the catalytic activity of Mec1, Mrc1 was found to facilitate the phosphorylation of Rad53 by Mec1 via promotion of a stronger enzyme-substrate interaction between them. Further, the conserved C-terminal domain of Mrc1 was found to be required for Rad53 activation. These results thus provide insights into the role of the adaptor protein Mrc1 in activating Rad53 in the DNA replication checkpoint.Faithful replication of the genome is important for the survival of all organisms. During DNA replication, replication stress can arise from a variety of situations, including intrinsic errors made by DNA polymerases, difficulties in replicating repeated DNA sequences, and failures to repair damaged DNA caused by either endogenous oxidative agents or exogenous mutagens such as UV light and DNA-damaging chemicals (1–3). In eukaryotes, there is an evolutionarily conserved DNA replication checkpoint that becomes activated in response to DNA replication stress. It helps to stabilize DNA replication forks, block late replication origin firing, and delay mitosis and ultimately helps recovery from stalled replication forks after DNA repair (4–7). Defects in the DNA replication checkpoint could result in elevated genomic instabilities, cancer development, or cell death (8, 9).Aside from replicating the genome, the DNA replication forks also provide a platform to assemble many signaling proteins that function in the DNA replication checkpoint. In the budding yeast Saccharomyces cerevisiae, Mec1, an ortholog of human ATR,² is a phosphoinositide 3-kinase-like kinase (PIKK) involved in sensing stalled DNA replication forks. Mec1 forms a protein complex with Ddc2 (ortholog of human ATRIP). The Mec1-Ddc2 complex is recruited to stalled replication forks through replication protein A (RPA)-coated single-stranded DNA (10, 11). The Mec3-Rad17-Ddc1 complex, a proliferating cell nuclear antigen (PCNA)-like checkpoint clamp and ortholog of the human 9-1-1 complex, was shown to be loaded onto the single- and double-stranded DNA junction of the stalled replication forks by the clamp loader Rad24-RFC complex (12). Once loaded, the Mec3-Rad17-Ddc1 complex stimulates Mec1 kinase activity (13). Dbp11 and its homolog TopBP1 in vertebrates are known components of the replication machinery (14). In addition to regulating the initiation of DNA replication, they were found to play a role in the DNA replication checkpoint (15–17). They interact with the 9-1-1 complex and directly stimulate Mec1/ATR activity in vitro (18–20). Thus, the assembly of multiple protein complexes at stalled DNA replication forks appears to facilitate activation of the DNA replication checkpoint (13, 18).Mrc1 (for mediator of replication checkpoint) was originally identified to be important for cells to respond to hydroxyurea in S. cerevisiae and Schizosaccharomyces pombe (21, 22). Mrc1 is a component of the DNA replisome and travels with the replication forks along chromosome during DNA synthesis (23–25). Deletion of MRC1 causes defects in DNA replication, indicating its role in the normal progression of DNA replication (23). Interestingly, when DNA replication is blocked by hydroxyurea, Mrc1 undergoes Mec1- and Rad3 (S. pombe ortholog of Mec1)-dependent phosphorylation (21, 22). In S. cerevisiae, mutations of Mrc1 at the (S/T)Q sites, which are consensus phosphorylation sites of the Mec1/ATR family kinases, abolishes hydroxyurea-induced Mrc1 phosphorylation in vivo, suggesting a direct phosphorylation of Mrc1 by Mec1 (21, 22).Rad53 and Cds1, homologs of human Chk2, are the major effector kinases in the DNA replication checkpoints in S. cerevisiae and S. pombe, respectively. Activation of Rad53 is a hallmark of DNA replication checkpoint activation and is important for the maintenance of DNA replication forks in response to DNA replication stress (5, 6). Thus, it is important to understand how Rad53 activity is controlled. Interestingly, mutation of all the (S/T)Q sites of Mrc1 not only abolishes the phosphorylation of Mrc1 by Mec1 but also compromises hydroxyurea-induced Rad53 activation in S. cerevisiae (21). Similarly, mutation of the TQ sites of Mrc1 in S. pombe was shown to abolish the binding between Cds1 and Mrc1 as well as Cds1 activation (22). Further, mutation of specific TQ sites of Mrc1 in S. pombe abolishes its binding to Cds1 in vitro and the activation of Cds1 in vivo (26). Thus, Mec1/Rad3-dependent phosphorylation of Mrc1 is responsible for Mrc1 binding to Rad53/Cds1, which is essential for Rad53/Cds1 activation.An intriguing property of the Chk2 family kinases is their ability to undergo autophosphorylation and activation in the absence of other proteins in vitro (27, 28). First, autophosphorylation of a conserved threonine residue in the activation loop of Chk2 family kinase was found to be an essential part of their activation processes (26, 29–31). Second, a direct and trans-phosphorylation of the N-terminal TQ sites of the Chk2 family kinases by the Mec1/ATR family kinases is also important for their activation in vivo. Analogous to the requirement of N-terminal TQ site phosphorylation of Chk2 by ATR in human (32), the activation of Rad53/Cds1 in vivo requires phosphorylation of TQ sites in their N termini by Mec1/Rad3 (33, 34).Considering that Mec1, Mrc1, and many other proteins are recruited at stalled DNA replication forks and have been shown to be involved in DNA replication checkpoint activation, a key question remains unresolved: what is the minimal system that is capable of activating Rad53 directly? Given the direct physical interaction between Mrc1 and Rad53 and the requirement of Mrc1 and Mec1 in vivo, it is likely that they both play a role in Rad53 activation. Furthermore, what is the molecular mechanism of Rad53 activation by its upstream activators? To address these questions, a faithful reconstitution of the activation of Rad53 using purified proteins is necessary. In this study, we developed an activity-based assay consisting of the Dun1 kinase, a downstream substrate of Rad53, and Sml1, as a substrate of Dun1, to quantitatively measure the activity of Rad53. Using this coupled kinase assay from Rad53 to Dun1 and then to Sml1, we screened for Mrc1 and its associated factors to see whether they could directly activate Rad53 in vitro. Our results showed that Mec1 and Mrc1 collaborate to constitute a minimal system in direct activation of Rad53.     

Two Regions of Simian Virus 40 T Antigen Determine Cooperativity of Double-Hexamer Assembly on the Viral Origin of DNA Replication and Promote Hexamer Interactions during Bidirectional Origin DNA Unwinding

Phosphorylation of simian virus 40 large tumor (T) antigen on threonine 124 is essential for viral DNA replication. A mutant T antigen (T124A), in which this threonine was replaced by alanine, has helicase activity, assembles double hexamers on viral-origin DNA, and locally distorts the origin DNA structure, but it cannot catalyze origin DNA unwinding. A class of T-antigen mutants with single-amino-acid substitutions in the DNA binding domain (class 4) has remarkably similar properties, although these proteins are phosphorylated on threonine 124, as we show here. By comparing the DNA binding properties of the T124A and class 4 mutant proteins with those of the wild type, we demonstrate that mutant double hexamers bind to viral origin DNA with reduced cooperativity. We report that T124A T-antigen subunits impair the ability of double hexamers containing the wild-type protein to unwind viral origin DNA, suggesting that interactions between hexamers are also required for unwinding. Moreover, the T124A and class 4 mutant T antigens display dominant-negative inhibition of the viral DNA replication activity of the wild-type protein. We propose that interactions between hexamers, mediated through the DNA binding domain and the N-terminal phosphorylated region of T antigen, play a role in double-hexamer assembly and origin DNA unwinding. We speculate that one surface of the DNA binding domain in each subunit of one hexamer may form a docking site that can interact with each subunit in the other hexamer, either directly with the N-terminal phosphorylated region or with another region that is regulated by phosphorylation.

The initiation of simian virus 40 (SV40) DNA replication by the viral T antigen is a complex series of events that begins when T antigen binds specifically to a palindromic arrangement of four GAGGC pentanucleotide sequences in the minimal origin of viral DNA replication (recently reviewed in references 1, 2, 3, 22, and 48). In the presence of Mg-ATP, T antigen assembles cooperatively on the two halves of the palindrome as a double hexamer (10, 11, 13, 24, 30, 38, 51, 53). The DNA conformation flanking the T-antigen binding sites is locally distorted upon hexamer assembly (reference 7 and references therein). One pair of pentanucleotides is sufficient to direct double-hexamer assembly and local distortion of the origin DNA but not to initiate DNA replication (25). ATP hydrolysis by T-antigen hexamers then catalyzes bidirectional unwinding of the parental DNA (reference 53 and references therein). A mutant origin with a single nucleotide insertion in the center of the palindromic T-antigen binding site prevents cooperative interactions between hexamers and cannot support bidirectional origin unwinding (8, 51), suggesting that both processes require interactions between T-antigen hexamers. After assembly of the two replication forks, bidirectional replication is carried out by 10 cellular proteins and T antigen, which remains at the forks as the only essential helicase (reviewed in references 3, 22, and 48).The phosphorylation state of SV40 T antigen governs its ability to initiate viral DNA replication (reviewed in references 15 to 17 and 39). T antigen contains two clusters of phosphorylation sites located at the N and C termini (40, 41). Phosphorylation of T antigen on threonine 124 in the N-terminal cluster was shown to be essential for viral DNA replication in monkey cells and in vitro (5, 14, 32–36, 44). Efforts to define what step in viral DNA replication requires modification of threonine 124 revealed that Mg-ATP-induced hexamer formation of T antigen in solution and DNA helicase activity of T antigen did not require phosphorylation at this site (33, 36). Origin DNA binding of T antigen lacking the modification at residue 124 was weaker than that of the modified T antigen (33, 34, 36, 44), but the reduction in binding was modest under the conditions used for SV40 DNA replication in vitro (36). Moreover, a mutant T antigen containing alanine in place of the phosphorylated threonine (T124A) assembled as a double hexamer on the viral origin and altered the conformation of the early palindrome and AT-rich sequences flanking the T-antigen binding sites in the viral origin in the same manner as the wild-type protein, except that higher concentrations were required (36). However, even at an elevated concentration, these mutant double hexamers were unable to unwind closed circular duplex DNA containing the viral origin (33, 36), suggesting that the defect in unwinding was responsible for the inability of T124A T antigen to replicate SV40 DNA. One possible explanation for the unwinding defect of the mutant T antigen, despite its helicase activity, was that some essential interaction between the two hexamers during bidirectional unwinding depended upon phosphorylation of threonine 124. Electron micrographs of SV40 DNA unwinding intermediates, which showed two single-stranded DNA loops protruding between two hexamers of T antigen, provided support for this explanation, implying that a double hexamer pulled the parental duplex DNA into the protein complex and spooled the single-stranded DNA out (53). Furthermore, double-hexamer formation significantly enhanced the helicase activity of T antigen (47, 47a).Most of the T antigen isolated from mammalian cells is in a hyperphosphorylated form, containing multiple phosphoserines, as well as two phosphothreonines, and supports SV40 DNA replication in vitro poorly but can be stimulated by treatment with alkaline phosphatase or protein phosphatase 2A (19, 28, 37, 42, 49, 50). Hyperphosphorylated T antigen is unable to unwind duplex closed circular duplex DNA harboring the viral origin (4, 6, 51). Dephosphorylation of serines 120 and 123 restores its ability to unwind origin DNA (14, 43, 51). Studies of double-hexamer assembly on the origin indicate that phosphorylation of T antigen on serines 120 and 123 also impairs the cooperativity of double-hexamer assembly (14, 51). These results demonstrate that hyperphosphorylation of T antigen interferes with interactions between hexamers that are required for origin unwinding and raise the question of whether the phosphorylation state of threonine 124 might also affect the cooperativity of double-hexamer assembly on the viral origin.One class of T antigen mutants with single-amino-acid substitutions in the DNA binding domain (class 4) has been reported to display properties similar to those of the T124A mutant and the hyperphosphorylated form of T antigen (54). Class 4 mutant proteins are defective in viral DNA replication in vivo and in vitro, bind to the viral origin as double hexamers and alter the local DNA conformation, and have helicase activity but do not unwind closed circular duplex viral DNA. The replication and unwinding defects could be due to faulty phosphorylation patterns or to other malfunctions not dependent on phosphorylation status.The work presented here was undertaken to reevaluate the assembly of wild-type and T124A T antigen on SV40 origin DNA by using more-sensitive quantitative assays and to compare them with the class 4 mutants. We report that cooperativity of T124A T antigen in double-hexamer assembly on the viral origin is impaired. The class 4 mutant T antigens were also found to have defects in cooperativity of double-hexamer assembly. T124A T antigen inhibited the ability of the wild-type protein to unwind closed circular duplex origin DNA. Both T124A and the class 4 mutants displayed dominant-negative phenotypes in viral DNA replication in vitro. Based on these observations, we propose that the N-terminal cluster of phosphorylation sites and the DNA binding domain mediate cooperative hexamer-hexamer interactions during assembly on the viral origin and speculate that these regions of T antigen may interact during origin DNA unwinding.     

Geminin Stabilizes Cdt1 during Meiosis in Xenopus Oocytes

During the mitotic cell cycle, Geminin can act both as a promoter and inhibitor of initiation of DNA replication. As a promoter, Geminin stabilizes Cdt1 and facilitates its accumulation leading to the assembly of the pre-replication complex on DNA. As an inhibitor, Geminin prevents Cdt1 from loading the mini-chromosome maintenance complex onto pre-replication complexes in late S, G₂, and M phases. Here we show that during meiosis Geminin functions as a stabilizer of Cdt1 promoting its accumulation for the early division cycles of the embryo. Depletion of Geminin in Xenopus immature oocytes leads to a decrease of Cdt1 protein levels during maturation and after activation of these oocytes. Injection of exogenous recombinant Geminin into the depleted oocytes rescues Cdt1 levels demonstrating that Geminin stabilizes Cdt1 during meiosis and after fertilization. Furthermore, Geminin-depleted oocytes did not replicate their DNA after meiosis I indicating that Geminin does not act as an inhibitor of initiation of DNA replication between meiosis I and meiosis II.In eukaryotes, initiation of DNA replication involves the formation and activation of the pre-replication complex (pre-RC)³ at the origins of replication. Pre-RCs are formed by the sequential binding of the origin recognition complex components, Cdc6, Cdt1, and mini-chromosome maintenance complex (MCM 2–7) proteins, to DNA. After loading the MCM complex, the pre-RCs are activated by S phase kinases (Dbf4-dependent kinase and Cdks) to initiate DNA replication (1). Replication of DNA, limited to only once per cell cycle, is critical to maintain genomic stability. Redundant mechanisms exist to ensure that DNA replication is tightly regulated during the cell cycle (1, 2). A small protein named Geminin has been shown to play a significant role in such regulatory mechanisms during mitosis (2–6). Geminin, a multifunctional 25-kDa protein, was first identified in a screen for proteins degraded during mitosis in Xenopus laevis egg extracts (7). Geminin is present in higher eukaryotes, but its presence in yeast has not yet been reported (7–10). Geminin plays a major role in regulating the function of Cdt1, one of the pre-RC components (8, 11–13). Numerous studies suggest that in higher eukaryotes the interaction between Geminin and Cdt1 is pivotal to restrict DNA replication to only once per cell cycle (6, 14–22). Furthermore, in Xenopus egg extracts, the Geminin/Cdt1 ratio seems to control the assembly of pre-RCs at replication origins and to determine whether the origins are licensed or not (23). The positive and negative roles of Geminin in origin licensing and DNA replication are made possible by their temporal separation during the cell cycle. Pre-RC formation occurs during late M and early G₁ phase, whereas pre-RC inhibition occurs from late S to mid M phase.As a positive regulator of DNA replication, Geminin has been shown to stabilize Cdt1. In human osteosarcoma cells, silencing of GEMININ expression limits CDT1 accumulation during mitosis and therefore the formation of pre-RCs in the subsequent cell cycle. This stabilizing effect is the result of a direct interaction between CDT1 and GEMININ preventing CDT1 ubiquitination and degradation (13). Similar findings were also recently observed in normal human cells and various cancer cells (24). However, in both human normal and tumor cells, the low level of CDT1, generated by the absence of GEMININ, did not always prevent cellular proliferation or re-replication of the genome (5, 24, 25). Therefore, one might question the importance of the role of GEMININ in stabilizing CDT1 in human cells. Beyond its role as a stabilizer of Cdt1 levels, Geminin has also been shown to participate directly in the formation of pre-RCs in Xenopus egg extracts. A complex between Cdt1 and Geminin binds to chromatin and supports pre-RC assembly. However, the recruitment of additional Geminin molecules to this complex on the chromatin blocks further pre-RC formation. These results indicate that the stoichiometry of Cdt1 and Geminin in this complex regulates its activity as a promoter or inhibitor of pre-RC assembly and DNA replication (23, 26). Several mechanisms have been shown to modulate the Geminin/Cdt1 balance on the chromatin. In Xenopus the binding of Cdt1 to the MCM9 protein seems to block the recruitment of an excess of Geminin to the chromatin and therefore favors pre-RC assembly (27). Similarly, the inactivation of Geminin by either ubiquitination or degradation also has a positive effect on pre-RC assembly (8, 11, 28–30). On the other hand, the replication-dependent degradation of Cdt1 has the opposite effect and prevents refiring of replication origins during S and G₂ phases of the mitotic cell cycle (18, 20, 31).Although the role of Geminin during mitosis has been extensively studied, not much is known about its function during meiosis. The expression pattern of Geminin during oocyte maturation is unclear. The presence of Geminin in immature stage VI Xenopus oocytes is controversial, but the protein is fully expressed in mature oocytes arrested in metaphase of meiosis II (7, 32). To form haploid gametes, DNA replication has to be inhibited between meiosis I (MI) and meiosis II (MII). In Xenopus oocytes, cyclin B-dependent kinase 1 (Cdk1) also known as maturation-promoting factor (MPF) plays a role in preventing DNA replication between the two meiotic divisions (33–36). Inhibition of Cdk1 activity between MI and MII leads to the formation of interphase nucleus and DNA replication. However, the role of Geminin in preventing DNA replication between meiotic divisions has not been tested so far. Finally, the possibility that Geminin stabilizes Cdt1 during meiosis and ensures its accumulation for the early embryonic divisions has not been formally examined.Here we show that the levels of Geminin and Cdt1 proteins increase significantly during meiosis in Xenopus oocytes and that the primary role of geminin is to promote the accumulation of Cdt1 and not to repress DNA replication between meiosis I and meiosis II. Depletion of Geminin in Xenopus immature oocytes does not lead to DNA replication after the first meiotic division but to a decrease in Cdt1 stability during the maturation and activation of these oocytes. Rescue of Cdt1 levels in these Geminin-depleted oocytes is achieved by injection of exogenous recombinant Geminin protein confirming the role of Geminin as a stabilizer of Cdt1 during meiosis and the early embryonic division cycles. These results provide further support for the idea that Geminin functions universally in stabilizing Cdt1. Although the stabilizing role of Geminin might not be its most important function in somatic cells, we show here that stabilizing Cdt1 is a dominant function for Geminin in Xenopus oocytes undergoing meiosis. This stabilizing role of Geminin is essential for the stockpiling of Cdt1 before fertilization that is required to sustain the rapid divisions of the early embryo.