Plant MCM proteins: role in DNA replication and beyond

Mini-chromosome maintenance (MCM) proteins form heterohexameric complex (MCM2–7) to serve as licensing factor for DNA replication to make sure that genomic DNA is replicated completely and accurately once during S phase in a single cell cycle. MCMs were initially identified in yeast for their role in plasmid replication or cell cycle progression. Each of six MCM contains highly conserved sequence called “MCM box”, which contains two ATPase consensus Walker A and Walker B motifs. Studies on MCM proteins showed that (a) the replication origins are licensed by stable binding of MCM2–7 to form pre-RC (pre-replicative complex) during G1 phase of the cell cycle, (b) the activation of MCM proteins by CDKs (cyclin-dependent kinases) and DDKs (Dbf4-dependent kinases) and their helicase activity are important for pre-RC to initiate the DNA replication, and (c) the release of MCMs from chromatin renders the origins “unlicensed”. DNA replication licensing in plant is, in general, less characterized. The MCMs have been reported from Arabidopsis, maize, tobacco, pea and rice, where they are found to be highly expressed in dividing tissues such as shoot apex and root tips, localized in nucleus and cytosol and play important role in DNA replication, megagametophyte and embryo development. The identification of six MCM coding genes from pea and Arabidopsis suggest six distinct classes of MCM protein in higher plant, and the conserved function right across the eukaryotes. This overview of MCMs contains an emphasis on MCMs from plants and the novel role of MCM6 in abiotic stress tolerance.     

Genome based identification and analysis of the pre-replicative complex of Arabidopsis thaliana

Eukaryotic DNA replication requires an ordered and regulated machinery to control G1/S transition. The formation of the pre-replicative complex (pre-RC) is a key step involved in licensing DNA for replication. Here, we identify all putative components of the full pre-RC in the genome of the model plant Arabidopsis thaliana. Different from the other eukaryotes, Arabidopsis houses in its genome two putative homologs of ORC1, CDC6 and CDT1. Two mRNA variants of AtORC4 subunit, with different temporal expression patterns, were also identified. Two-hybrid binary interaction assays suggest a primary architectural organization of the Arabidopsis ORC, in which AtORC3 plays a central role in maintaining the complex associations. Expression profiles differ among pre-RC components suggesting the existence of various forms of the complex, possibly playing different roles during development. In addition, the expression of the putative pre-RC genes in non-proliferating plant tissues suggests that they might have roles in processes other than DNA replication licensing.     

植物着丝粒结构和功能的研究进展

着丝粒是真核生物有丝分裂和减数分裂染色体正确分离和传递所必需的染色体区域。十多年来, 已对包括拟南芥、水稻、玉米在内的一些植物的着丝粒进行了较深入的分子生物学研究。在不同的植物间, 着丝粒DNA的保守性很低, 呈现快速进化, 但着丝粒的DNA序列类型和组织方式基本相似, 一般是由夹杂排列着的卫星DNA串联重复阵列和着丝粒专一的反转录转座子构成。与着丝粒DNA相反, 着丝粒/着丝点的结构性和瞬时蛋白质在包括植物在内的真核生物中保守。与其他真核生物的情况一样, 拥有含着丝粒组蛋白H3(CENH3)的核小体是植物功能着丝粒染色质最基本的特征, CENH3在着丝粒染色质的识别和保持中起着关键作用。     

Progress in understanding DNA replication control

Completion of genome duplication during the S-phase of the cell cycle is crucial for the maintenance of genomic integrity. In eukaryotes, chromosomal DNA replication is accomplished by the activity of multiple origins of DNA replication scattered across the genome. Origin specification, selection and activity as well as the availability of replication factors and the regulation of DNA replication licensing, have unique and common features among eukaryotes. Although the initial studies on the semiconservative nature of chromosome duplication were carried out in the mid 1950s in Vicia faba, since then plant DNA replication studies have been scarce. However, they have received an unprecedented drive in the last decade after the completion of sequencing the Arabidopsis thaliana genome, and more recently of other plant genomes. In particular, the past year has witnessed major advances with the use of genomic approaches to study chromosomal replication timing, DNA replication origins and licensing control mechanisms. In this minireview article we discuss these recent discoveries in plants in the context of what is known at the genomic level in other eukaryotes. These studies constitute the basis for addressing in the future key questions about replication origin specification and function that will be of relevance not only for plants but also for the rest of multicellular organisms.     

Divergent functions of multiple eukaryote-like Orc1/Cdc6 proteins on modulating the loading of the MCM helicase onto the origins of the hyperthermophilic archaeon Sulfolobus solfataricus P2

The Cdc6 protein has been suggested as a loader for the eukaryotic MCM helicase. Archaeal replication machinery represents a core version of that in eukaryotes. In the current work, three eukaryotic Orc1/Cdc6 homologs (SsoCdc6-1, -2, and -3) from crenarchaeon Sulfolobus solfataricus were shown to have totally different effects on the interactions with SsoMCM helicase. SsoCdc6-2 stimulates the binding of the SsoMCM onto the origin DNA, but SsoCdc6-1 and SsoCdc6-3 significantly inhibit the loading activities, and these inhibitive effects can not be reversed by the stimulation of SsoCdc6-2. Using pull-down assays, we showed that three SsoCdc6 proteins interacted physically with the SsoMCM. Furthermore, the C-terminal domains of SsoCdc6 proteins were shown to physically and functionally affect the interactions with SsoMCM. This is the first report on the divergent functions of multiple eukaryote-like Orc1/Cdc6 proteins on regulating the loading of the MCM helicase onto the origins in the archaeon.     

Hexameric ring structure of the full-length archaeal MCM protein complex

In eukaryotes, a family of six homologous minichromosome maintenance (MCM) proteins has a key function in ensuring that DNA replication occurs only once before cell division. Whereas all eukaryotes have six paralogues, in some Archaea a single protein forms a homomeric assembly. The complex is likely to function as a helicase during DNA replication. We have used electron microscopy to obtain a three-dimensional reconstruction of the full-length MCM from Methanobacterium thermoautotrophicum. Six monomers are arranged around a sixfold axis, generating a ring-shaped molecule with a large central cavity and lateral holes. The channel running through the molecule can easily accommodate double-stranded DNA. The crystal structure of the amino-terminal fragment of MCM and a model for the AAA+ hexamer have been docked into the map, whereas additional electron density suggests that the carboxy-terminal domain is located at the interface between the two domains. The structure suggests that the MCM complex is likely to act in a different manner to traditional hexameric helicases and is likely to bear more similarity to the SV40 large T antigen or to double-stranded DNA translocases.     

Control of DNA replication: regulation and activation of eukaryotic replicative helicase, MCM

DNA replication is a key event of cell proliferation and the final target of signal transduction induced by growth factor stimulation. It is also strictly regulated during the ongoing cell cycle so that it occurs only once during S phase and that all the genetic materials are faithfully duplicated. DNA replication may be arrested or temporally inhibited due to a varieties of internal and external causes. Cells have developed intricate mechanisms to cope with the arrested replication forks to minimize the adversary effect on the stable maintenance of genetic materials. Helicases play a central role in DNA replication. In eukaryotes, MCM (minichromosome maintenance) protein complex plays essential roles as a replicative helicase. MCM4-6-7 complex possesses intrinsic DNA helicase activity which translocates on single-stranded DNA form 3' to 5'. Mammalian MCM4-6-7 helicase and ATPase activities are specifically stimulated by the presence of thymine-rich single-stranded DNA sequences onto which it is loaded. The activation appears to depend on the thymine content of this single-strand, and sequences derived from human replication origins can serve as potent activators of the MCM helicase. MCM is a prime target of Cdc7 kinase, known to be essential for activation of replication origins. We will discuss how the MCM may be activated at the replication origins by template DNA, phosphorylation, and interaction with other replicative proteins, and will present a model of how activation of MCM helicase by specific sequences may contribute to selection of replication initiation sites in higher eukaryotes.     

Ancient diversification of eukaryotic MCM DNA replication proteins

Background  

Yeast and animal cells require six mini-chromosome maintenance proteins (Mcm2-7) for pre-replication complex formation, DNA replication initiation and DNA synthesis. These six individual MCM proteins form distinct heterogeneous subunits within a hexamer which is believed to form the replicative helicase and which associates with the essential but non-homologous Mcm10 protein during DNA replication. In contrast Archaea generally only possess one MCM homologue which forms a homohexameric MCM helicase. In some eukaryotes Mcm8 and Mcm9 paralogues also appear to be involved in DNA replication although their exact roles are unclear.     

Identification and characterization of a novel component of the human minichromosome maintenance complex

Minichromosome maintenance (MCM) complex replicative helicase complexes play essential roles in DNA replication in all eukaryotes. Using a tandem affinity purification-tagging approach in human cells, we discovered a form of the MCM complex that contains a previously unstudied protein, MCM binding protein (MCM-BP). MCM-BP is conserved in multicellular eukaryotes and shares limited homology with MCM proteins. MCM-BP formed a complex with MCM3 to MCM7, which excluded MCM2; and, conversely, hexameric complexes of MCM2 to MCM7 lacked MCM-BP, indicating that MCM-BP can replace MCM2 in the MCM complex. MCM-BP-containing complexes exhibited increased stability under experimental conditions relative to those containing MCM2. MCM-BP also formed a complex with the MCM4/6/7 core helicase in vitro, but, unlike MCM2, did not inhibit this helicase activity. A proportion of MCM-BP bound to cellular chromatin in a cell cycle-dependent manner typical of MCM proteins, and, like other MCM subunits, preferentially associated with a cellular origin in G(1) but not in S phase. In addition, down-regulation of MCM-BP decreased the association of MCM4 with chromatin, and the chromatin association of MCM-BP was at least partially dependent on MCM4 and cdc6. The results indicate that multicellular eukaryotes contain two types of hexameric MCM complexes with unique properties and functions.     

Putative subunits of the maize origin of replication recognition complex ZmORC1-ZmORC5

The finding in animal species of complexes homologous to the products of six Saccharomyces cerevisiae genes, origin of replication recognition complex (ORC), has suggested that ORC-related mechanisms have been conserved in all eukaryotes. In plants, however, the only cloned putative homologs of ORC subunits are the Arabidopsis ORC2 and the rice ORC1. Homologs of other subunits of plant origin have not been cloned and characterized. A striking observation was the absence from the Arabidopsis genome of an obvious candidate gene-homolog of ORC4. This fact raised compelling questions of whether plants, in general, and Arabidopsis, in particular, may have lost the ORC4 gene, whether ORC-homologous subunits function within a complex in plants, whether an ORC complex may form and function without an ORC4 subunit, whether a functional (but not sequence) protein homolog may have taken up the role of ORC4 in Arabidopsis, and whether lack of ORC4 is a plant feature, in general. Here, we report the first cloned and molecularly characterized five genes coding for the maize putative homologs of ORC subunits ZmORC1, ZmORC2, ZmORC3, ZmORC4 and ZmORC5. Their expression profiles in tissues with different cell-dividing activities are compatible with a role in DNA replication. Based on the potential of ORC-homologous maize proteins to bind each other in yeast, we propose a model for their possible assembly within a maize ORC. The isolation and molecular characterization of an ORC4-homologous gene from maize argues that, in its evolution, Arabidopsis may have lost the homologous ORC4 gene.     

Molecular machines in archaeal DNA replication

The archaeal DNA replication apparatus is a simplified version of that of eukaryotes and has attracted attention as a tractable model system for the orthologous, but significantly more complex eukaryal machinery. A variety of archaeal model organisms have been investigated with strong emphasis on structural and biochemical analyses of replication-associated proteins. In this review we will describe recent advances in understanding the properties of the replicative helicase, the MCM complex, and the role of the sliding clamp, PCNA, in mediating a range of protein-DNA transactions. Although both complexes form ring shaped assemblies, they play very distinct roles at the leading and trailing edges of the replication fork machinery respectively.     

A key role for the GINS complex at DNA replication forks

The GINS complex is the most recently identified component of the eukaryotic DNA replication machinery and is required both for the initiation of chromosome replication and also for the normal progression of DNA replication forks. Several recent studies suggest that GINS associates at replication forks with the MCM helicase that is responsible for unwinding the parental DNA duplex. Archaea also have an equivalent GINS complex that can interact with MCM and other replisome components. It seems likely that GINS couples MCM to other key proteins at forks, and we discuss here the current literature regarding this important late-comer to the DNA replication field.     

GINS, a central nexus in the archaeal DNA replication fork

In eukaryotes, the GINS complex is essential for DNA replication and has been implicated as having a role at the replication fork. This complex consists of four paralogous GINS subunits, Psf1, Psf2, Psf3 and Sld5. Here, we identify an archaeal GINS homologue as a direct interaction partner of the MCM helicase. The core archaeal GINS complex contains two subunits that are poorly conserved homologues of the eukaryotic GINS subunits, in complex with a protein containing a domain homologous to the DNA-binding domain of bacterial RecJ. Interaction studies show that archaeal GINS interacts directly with the heterodimeric core primase. Our data suggest that GINS is important in coordinating the architecture of the replication fork and provide a mechanism to couple progression of the MCM helicase on the leading strand with priming events on the lagging strand.     

Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and essential for DNA replication. By applying serial extractions to mammalian cells synchronized by release from quiescence, we reveal dynamic changes to the sub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix. The data distinguish 3 states that correspond to loose association with chromatin prior to DNA replication, transient highly stable binding to the nuclear-matrix coincident with initiation, and a post-initiation phase when MCM2 remains tightly associated with chromatin but not the nuclear-matrix. The data suggests that functional MCM complex loading takes place at the nuclear-matrix.     

Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and essential for DNA replication. By applying serial extractions to mammalian cells synchronized by release from quiescence, we reveal dynamic changes to the sub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix. The data distinguish 3 states that correspond to loose association with chromatin prior to DNA replication, transient highly stable binding to the nuclear-matrix coincident with initiation, and a post-initiation phase when MCM2 remains tightly associated with chromatin but not the nuclear-matrix. The data suggests that functional MCM complex loading takes place at the nuclear-matrix.     

Characterization of Leishmania donovani MCM4: expression patterns and interaction with PCNA

Events leading to origin firing and fork elongation in eukaryotes involve several proteins which are mostly conserved across the various eukaryotic species. Nuclear DNA replication in trypanosomatids has thus far remained a largely uninvestigated area. While several eukaryotic replication protein orthologs have been annotated, many are missing, suggesting that novel replication mechanisms may apply in this group of organisms. Here, we characterize the expression of Leishmania donovani MCM4, and find that while it broadly resembles other eukaryotes, noteworthy differences exist. MCM4 is constitutively nuclear, signifying that, unlike what is seen in S.cerevisiae, varying subcellular localization of MCM4 is not a mode of replication regulation in Leishmania. Overexpression of MCM4 in Leishmania promastigotes causes progress through S phase faster than usual, implicating a role for MCM4 in the modulation of cell cycle progression. We find for the first time in eukaryotes, an interaction between any of the proteins of the MCM2-7 (MCM4) and PCNA. MCM4 colocalizes with PCNA in S phase cells, in keeping with the MCM2-7 complex being involved not only in replication initiation, but fork elongation as well. Analysis of a LdMCM4 mutant indicates that MCM4 interacts with PCNA via the PIP box motif of MCM4--perhaps as an integral component of the MCM2-7 complex, although we have no direct evidence that MCM4 harboring a PIP box mutation can still functionally associate with the other members of the MCM2-7 complex- and the PIP box motif is important for cell survival and viability. In Leishmania, MCM4 may possibly help in recruiting PCNA to chromatin, a role assigned to MCM10 in other eukaryotes.     

Licensing of DNA replication by a multi-protein complex of MCM/P1 proteins in Xenopus eggs.

In eukaryotes, chromosomal DNA is licensed for a single round of replication in each cell cycle. Xenopus MCM3 protein has been implicated in the licensing of replication in egg extract. We have cloned cDNAs encoding five immunologically distinct proteins associated with Xenopus MCM3 as members of the MCM/P1 family. Six Xenopus MCM proteins formed a physical complex in the egg extract, bound to unreplicated chromatin before the formation of nuclei, and apparently displaced from replicated chromatin. The requirement of six XMCM proteins for the replication activity of the egg extract before nuclear formation suggests that their re-association with replicated chromatin at the end of the mitotic cell cycle is a key step for the licensing of replication.