Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing.

All organisms need to ensure that no DNA segments are rereplicated in a single cell cycle. Eukaryotes achieve this through a process called origin licensing, which involves tight spatiotemporal control of the assembly of prereplicative complexes (pre-RCs) onto chromatin. Cdt1 is a key component and crucial regulator of pre-RC assembly. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent DNA rereplication. Here, we address the mechanism of DNA licensing inhibition by Geminin, by combining X-ray crystallography, small-angle X-ray scattering, and functional studies in Xenopus and mammalian cells. Our findings show that the Cdt1:Geminin complex can exist in two distinct forms, a "permissive" heterotrimer and an "inhibitory" heterohexamer. Specific Cdt1 residues, buried in the heterohexamer, are important for licensing. We postulate that the transition between the heterotrimer and the heterohexamer represents a molecular switch between licensing-competent and licensing-defective states.

Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins.

BACKGROUND: In order to ensure precise chromosome duplication, eukaryotes "license" their replication origins during late mitosis and early G1 by assembling complexes of Mcm2-7 onto them. Mcm2-7 are essential for DNA replication, but are displaced from origins as they initiate, thus ensuring that no origin fires more than once in a single cell cycle. RESULTS: Here we show that a combination of purified nucleoplasmin, the origin recognition complex (ORC), Cdc6, RLF-B/Cdt1 and Mcm2-7 can promote functional origin licensing and the assembly of Mcm2-7 onto Xenopus sperm nuclei. The reconstituted reaction is inhibited by geminin, a specific RLF-B/Cdt1 inhibitor. Interestingly, the purified ORC used in the reconstitution had apparently lost the Orc6 subunit, suggesting that Orc6 is not essential for replication licensing. We use the reconstituted system to make a preliminary analysis of the different events occurring during origin assembly, and examine their nucleotide requirements. We show that the loading of Xenopus ORC onto chromatin is strongly stimulated by both ADP, ATP and ATP-gamma-S whilst the loading of Cdc6 and Cdt1 is stimulated only by ATP or ATP-gamma-S. CONCLUSIONS: Nucleoplasmin, ORC, Cdc6, RLF-B/Cdt1 and Mcm2-7 are the only proteins required for functional licensing and the loading of Mcm2-7 onto chromatin. The requirement for nucleoplasmin probably only reflects a requirement to decondense sperm chromatin before ORC can bind to it. Use of this reconstituted system should allow a full biochemical analysis of origin licensing and Mcm2-7 loading.

DNA replication: stable driving prevents fatal smashes.

Cells respond to DNA damage during S phase by slowing chromosome replication. Recent results have shed light on the mechanism by which this 'intra-S phase' checkpoint is implemented.

Use of peptides from p21 (Waf1/Cip1) to investigate PCNA function in Xenopus egg extracts.

Cell-free systems derived from unfertilized Xenopus eggs have been particularly informative in the study of the regulation and biochemistry of DNA replication. We have developed a Xenopus-based system to analyze proliferating cell nuclear antigen (PCNA)-specific effects on the functional properties of egg extracts. To do this, we have coupled peptides derived from p21 (Waf1/Cip1) to beads and used these to deplete PCNA from Xenopus egg extracts. The effect on various aspects of DNA replication can be analyzed after the readdition of PCNA and other purified proteins. Using this system, we have shown that replication of single-stranded M13 DNA is entirely dependent upon PCNA. By adding exogenous T7 DNA polymerase to PCNA-depleted extracts, we have uncoupled processive DNA replication from PCNA activity and so created an experimental system to analyze the dependence of postreplicative processes on PCNA function. We have shown that successful chromatin assembly is specifically dependent on PCNA. However, systems for analyzing the far more complex mechanisms required for the replication of nuclear double-stranded DNA have proved so far to be refractory to specific PCNA depletion.

Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin.

Eukaryotic replication origins are 'licensed' for replication early in the cell cycle by loading Mcm(2-7) proteins. As chromatin replicates, Mcm(2-7) are removed, thus preventing the origin from firing again. Here we report the purification of the RLF-B component of the licensing system and show that it corresponds to Cdt1. RLF-B/Cdt1 was inhibited by geminin, a protein that is degraded during late mitosis. Immunodepletion of geminin from metaphase extracts allowed them to assemble licensed replication origins. Inhibition of CDKs in metaphase stimulated origin assembly only after the depletion of geminin. These experiments suggest that geminin-mediated inhibition of RLF-B/Cdt1 is essential for repressing origin assembly late in the cell cycle of higher eukaryotes.

Replication origins in Xenopus egg extract Are 5-15 kilobases apart and are activated in clusters that fire at different times.

When Xenopus eggs and egg extracts replicate DNA, replication origins are positioned randomly with respect to DNA sequence. However, a completely random distribution of origins would generate some unacceptably large interorigin distances. We have investigated the distribution of replication origins in Xenopus sperm nuclei replicating in Xenopus egg extract. Replicating DNA was labeled with [(3)H]thymidine or bromodeoxyuridine and the geometry of labeled sites on spread DNA was examined. Most origins were spaced 5-15 kb apart. This regular distribution provides an explanation for how complete chromosome replication can be ensured although origins are positioned randomly with respect to DNA sequence. Origins were grouped into small clusters (typically containing 5-10 replicons) that fired at approximately the same time, with different clusters being activated at different times in S phase. This suggests that a temporal program of origin firing similar to that seen in somatic cells also exists in the Xenopus embryo. When the quantity of origin recognition complexes (ORCs) on the chromatin was restricted, the average interorigin distance increased, and the number of origins in each cluster decreased. This suggests that the binding of ORCs to chromatin determines the regular spacing of origins in this system.

Xenopus cdc7 function is dependent on licensing but not on XORC, XCdc6, or CDK activity and is required for XCdc45 loading.

The assembly and disassembly of protein complexes at replication origins play a crucial role in the regulation of chromosomal DNA replication. The sequential binding of the origin recognition complex (ORC), Cdc6, and the minichromosome maintenance (MCM/P1) proteins produces a licensed replication origin. Before the initiation of replication can occur, each licensed origin must be acted upon by S phase-inducing CDKs and the Cdc7 protein kinase. In the present report we describe the role of Xenopus Cdc7 (XCdc7) in DNA replication using cell-free extracts of Xenopus eggs. We show that XCdc7 binds to chromatin during G(1) and S phase. XCdc7 associates with chromatin only once origins have been licensed, but this association does not require the continued presence of XORC or XCdc6 once they have fulfilled their essential role in licensing. Moreover, XCdc7 is required for the subsequent CDK-dependent loading of XCdc45 but is not required for the destabilization of origins that occurs once licensing is complete. Finally, we show that CDK activity is not necessary for XCdc7 to associate with chromatin, induce MCM/P1 phosphorylation, or perform its essential replicative function. From these results we suggest a simple model for the assembly of functional initiation complexes in the Xenopus system.

Interaction of Xenopus Cdc2 x cyclin A1 with the origin recognition complex.

The initiation of DNA replication in eukaryotes is regulated in a minimum of at least two ways. First, several proteins, including origin recognition complex (ORC), Cdc6 protein, and the minichromosome maintenance (MCM) protein complex, need to be assembled on chromatin before initiation. Second, cyclin-dependent kinases regulate DNA replication in both a positive and a negative way by inducing the initiation of DNA replication at G(1)/S transition and preventing further rounds of origin firing within the same cell cycle. Here we characterize a link between the two levels. Immunoprecipitation of Xenopus origin recognition complex with anti-XOrc1 or anti-XOrc2 antibodies specifically co-immunoprecipitates a histone H1 kinase activity. The kinase activity is sensitive to several inhibitors of cyclin-dependent kinases including 6-dimethylaminopurine (6-DMAP), olomoucine, and p21(Cip1). This kinase activity also copurifies with ORC over several fractionation steps and was identified as a complex of the Cdc2 catalytic subunit and cyclin A1. Neither Cdk2 nor cyclin E could be detected in ORC immunoprecipitations. Reciprocal immunoprecipitations with anti-Xenopus Cdc2 or anti-Xenopus cyclin A1 antibodies specifically co-precipitate XOrc1 and XOrc2. Our results indicate that Xenopus ORC and Cdc2 x cyclin A1 physically interact and demonstrate a physical link between an active cyclin-dependent kinase and proteins involved in the initiation of DNA replication.

The replication capacity of intact mammalian nuclei in Xenopus egg extracts declines with quiescence, but the residual DNA synthesis is independent of Xenopus MCM proteins.

In eukaryotes, the initiation of DNA synthesis requires the assembly of a pre-replicative complex (pre-RC) at origins of replication. This involves the sequential binding of ORC (origin-recognition-complex), Cdc6 and MCM proteins, a process referred to as licensing. After origin firing, the Cdc6 and MCM proteins dissociate from the chromatin, and do not rebind until after the completion of mitosis, thereby restricting replication to a single round in each cell cycle. Although nuclei normally become licensed for replication as they enter G(1), the extent to which the license is retained when cells enter the quiescent state (G(0)) is controversial. Here we show that the replication capacity of nuclei from Swiss 3T3 cells, in Xenopus egg extracts, is not lost abruptly with the onset of quiescence, but instead declines gradually. The decline in replication capacity, which affects both the number of nuclei induced to replicate and their subsequent rate of DNA synthesis, is accompanied by a fall in the level of chromatin-bound MCM2. When quiescent nuclei are incubated in egg extracts, they do not bind further MCMs unless the nuclei are first permeabilized. The residual replication capacity of intact nuclei must therefore be dependent on the remaining endogenous MCMs. Although high levels of Cdk activity are known to block MCM binding, we show that the failure of intact nuclei in egg extracts to increase their bound MCMs is not due to their uptake and accumulation of Cdk complexes. Instead, the failure of binding must be due to exclusion of some other binding factor from the nucleus, or to the presence within nuclei of an inhibitor of binding other than Cdk activity. In contrast to the situation in Xenopus egg extracts, following serum stimulation of intact quiescent cells, the level of bound MCMs does increase before the cells reach S phase, without any disruption of the nuclear envelope.

Sequential MCM/P1 subcomplex assembly is required to form a heterohexamer with replication licensing activity.

Replication licensing factor (RLF) is a multiprotein complex involved in ensuring that chromosomal DNA replicates only once in a single cell cycle. It comprises two components, termed RLF-M and RLF-B. Purified RLF-M consists of a mixture of complexes containing all six members of the MCM/P1 family of minichromosome maintenance proteins. The precise composition of these different complexes and their contribution to RLF-M activity has been unclear. Here we show that in Xenopus extracts, MCM/P1 proteins mainly form heterohexamers containing each of the six proteins. This heterohexamer is readily split into subcomplexes, whose interactions and subunit composition we characterize in detail. We show for the first time an ordered multistep assembly pathway by which the heterohexamer can be reformed from the subcomplexes. Importantly, this novel pathway is essential for DNA replication, since only the full heterohexamer can bind productively to chromatin and provide RLF-M activity.

The Cdc7/Dbf4 protein kinase: target of the S phase checkpoint?

Cdc7/Dbf4 is a protein kinase that is required for the initiation of DNA replication in eukaryotes. Recent work has provided new clues to the role that Cdc7/Dbf4 plays in this process. A range of other observations suggest that Cdc7/Dbf4 also plays another, less well characterized, role in checkpoint function and in the maintenance of genomic integrity. In this review we attempt to bring together new information to explain how Cdc7/Dbf4 may perform these two distinct functions.

Nucleoplasmin-mediated chromatin remodelling is required for Xenopus sperm nuclei to become licensed for DNA replication.

During late mitosis and early G(1), a series of proteins are assembled onto replication origins, resulting in them becoming 'licensed' for replication in the subsequent S phase. Four factors have so far been identified that are required for chromatin to become functionally licensed: ORC (the origin recognition complex) and Cdc6, plus the two components of the replication licensing system RLF-M and RLF-B. Here we describe the first steps of a systematic fractionation of Xenopus egg extracts to identify all the components necessary for the assembly of licensed replication origins on Xenopus sperm nuclei (the physiological DNA substrate in this system). We have purified a new activity essential for this reaction, and have shown that it is nucleoplasmin, a previously known chromatin remodelling protein. Nucleoplasmin decondenses the sperm chromatin by removing protamines, and is required at the earliest known step in origin assembly to allow ORC to bind to the DNA. Sperm nuclei can be licensed by a combination of nucleoplasmin, RLF-M and a partially purified fraction that contains ORC, Cdc6 and RLF-B. This suggests that we are likely to have identified most of the proteins required for this assembly reaction.

Changes in association of the Xenopus origin recognition complex with chromatin on licensing of replication origins.

During late mitosis and early G1, a series of proteins are assembled onto replication origins that results in them becoming 'licensed' for replication in the subsequent S phase. In Xenopus this first involves the assembly onto chromatin of the Xenopus origin recognition complex XORC, and then XCdc6, and finally the RLF-M component of the replication licensing system. In this paper we examine changes in the way that XORC associates with chromatin in the Xenopus cell-free system as origins become licensed. Restricting the quantity of XORC on chromatin reduced the extent of replication as expected if a single molecule of XORC is sufficient to specify a single replication origin. During metaphase, XOrc1 associated only weakly with chromatin. In early interphase, XOrc1 formed a strong complex with chromatin, as evidenced by its resistance to elution by 200 mM salt, and this state persisted when XCdc6 was assembled onto the chromatin. As a consequence of origins becoming licensed the association of XOrc1 and XCdc6 with chromatin was destabilised, and XOrc1 became susceptible to removal from chromatin by exposure to either high salt or high Cdk levels. At this stage the essential function for XORC and XCdc6 in DNA replication had already been fulfilled. Since high Cdk levels are required for the initiation of DNA replication, this 'licensing-dependent origin inactivation' may contribute to mechanisms that prevent re-licensing of replication origins once S phase has started.

The regulation of replication origin activation.

At the start of the cell-division programme, proteins must be assembled onto replication origins to establish competence for initiation of DNA synthesis. At the correct moment, other effectors must then coordinate appropriate firing of the various origins to control entry into and progress through S phase. These processes are key targets of cell-cycle control, and understanding their regulation will provide a deeper knowledge of the mechanisms controlling cell proliferation.

The RLF-B component of the replication licensing system is distinct from Cdc6 and functions after Cdc6 binds to chromatin.

Replication licensing factor (RLF) is an essential initiation factor that can prevent re-replication of DNA in a single cell cycle [1] [2]. It is required for the initiation of DNA replication, binds to chromatin early in the cell cycle, is removed from chromatin as DNA replicates and is unable to re-bind replicated chromatin until the following mitosis. Chromatography of RLF from Xenopus extracts has shown that it consists of two components termed RLF-B and RLF-M [3]. The RLF-M component consists of complexes of all six Xenopus minichromosome maintenance (MCM/P1) proteins (XMcm2-7), which bind to chromatin in late mitosis and are removed as replication occurs [3] [4] [5] [6] [7]. The identity of RLF-B is currently unknown. At least two factors must be present on chromatin before licensing can occur: the Xenopus origin recognition complex (XORC) [8] [9] and Xenopus Cdc6 (XCdc6) [10]. XORC saturates Xenopus sperm chromatin at approximately one copy per replication origin whereas XCdc6 binds to chromatin only if XORC is bound first [9] [10] [11]. Although XORC has been shown to be a distinct activity from RLF-B [9], the relationship between XCdc6 and RLF-B is currently unclear. Here, we show that active XCdc6 is loaded onto chromatin in extracts with defective RLF, and that both RLF-M and RLF-B are still required for the licensing of XCdc6-containing chromatin. Furthermore, RLF-B can be separated from XCdc6 by immunoprecipitation and standard chromatography. These experiments demonstrate that RLF-B is both functionally and physically distinct from XCdc6, and that XCdc6 is loaded onto chromatin before RLF-B function is executed.

The replication licensing system.

The replication licensing system acts to ensure that no section of the genome is replicated more than once in a single cell cycle. Experiments using Xenopus egg extracts have revealed that the licensing system consists of two components, named RLF-M and RLF-B. Whereas the function of RLF-B is still unclear, RLF-M has been shown to consist of all six members of the MCM/P1 family proteins, which appear to be the structural component of the licensing system. The origin recognition complex (ORC) and Cdc6/Cdc18 are needed on chromatin before the licensing reaction can take place, although they are not themselves components of the licensing system. Cell cycle events and cyclin-dependent protein kinases (Cdks) also seem to be involved in controlling the licensing system to ensure once per cell cycle DNA replication. The subject of this review is to detail our current understanding of the licensing system and the way that it interacts with other components of the cell cycle machinery.