DNA replication in eukaryotic organisms
Eukaryotic DNA replication is a conserved mechanism that restricts DNA replication to once per cell cycle. Eukaryotic DNA replication of chromosomal DNA is central for the duplication of a cell and is necessary for the maintenance of the eukaryotic genome.
DNA replication is the action of DNA polymerases synthesizing a DNA strand complementary to the original template strand. To synthesize DNA, the double-stranded DNA is unwound by DNA helicases ahead of polymerases, forming a replication fork containing two single-stranded templates. Replication processes permit copying a single DNA double helix into two DNA helices, which are divided into the daughter cells at mitosis. The major enzymatic functions carried out at the replication fork are well conserved from prokaryotes to eukaryotes, but the replication machinery in eukaryotic DNA replication is a much larger complex, coordinating many proteins at the site of replication, forming the replisome.
The replisome is responsible for copying the entirety of genomic DNA in each proliferative cell. This process allows for the high-fidelity passage of hereditary/genetic information from parental cell to daughter cell and is thus essential to all organisms. Much of the cell cycle is built around ensuring that DNA replication occurs without errors.
In G1 phase of the cell cycle, many of the DNA replication regulatory processes are initiated. In eukaryotes, the vast majority of DNA synthesis occurs during S phase of the cell cycle, and the entire genome must be unwound and duplicated to form two daughter copies. During G2, any damaged DNA or replication errors are corrected. Finally, one copy of the genomes is segregated into each daughter cell at the mitosis or M phase. These daughter copies each contains one strand from the parental duplex DNA and one nascent antiparallel strand.
This mechanism is conserved from prokaryotes to eukaryotes and is known as semiconservative DNA replication. The process of semiconservative replication for the site of DNA replication is a fork-like DNA structure, the replication fork, where the DNA helix is open, or unwound, exposing unpaired DNA nucleotides for recognition and base pairing for the incorporation
of free nucleotides into double-stranded DNA.
Initiation.
Initiation of eukaryotic DNA replication is the first stage of DNA synthesis where the DNA double helix is unwound and an initial priming event by DNA polymerase α occurs on the leading strand. The priming event on the lagging strand establishes a replication fork. Priming of the DNA helix consists of the synthesis of an RNA primer to allow DNA synthesis by DNA polymerase α. Priming occurs once at the origin on the leading strand and at the start of each Okazaki fragment on the lagging strand.
Origin of replication.
Replication starts at origins of replication. DNA sequences containing these sites were initially isolated in the late 1970s on the basis of their ability to support replication of plasmids, hence the designation of autonomously replicating sequences (ARS). Origins vary widely in their efficiency, with some being used in almost every cell cycle while others may be used in only one in one thousand S phases. The total number of yeast ARSs is at least 1600, but may be more than 5000 if less active sites are counted, that is, there may be an ARS every 2000 to 8000 base pairs.
Pre-replicative complex.
Multiple replicative proteins assemble on and dissociate from these replicative origins to initiate DNA replication. with the formation of the pre-replication complex (pre-RC) being a key intermediate in the replication initiation process.
Association of the origin recognition complex (ORC) with a replication origin recruits the cell division cycle 6 protein (Cdc6) to form a platform for the loading of the minichromosome maintenance (Mcm 2–7) complex proteins, facilitated by the chromatin licensing and DNA replication factor 1 protein (Cdt1). The ORC, Cdc6, and Cdt1 together are required for the stable association of the Mcm2-7 complex with replicative origins during the G1 phase of the cell cycle.
Eukaryotic origins of replication control the formation of several protein complexes that lead to the assembly of two bidirectional DNA replication forks. These events are initiated by the formation of the pre-replication complex (pre-RC) at the origins of replication. This process takes place in the G1 stage of the cell cycle. The pre-RC formation involves the ordered assembly of many replication factors including the origin recognition complex (ORC), Cdc6 protein, Cdt1 protein, and minichromosome maintenance proteins (Mcm2-7). Once the pre-RC is formed, activation of the complex is triggered by two kinases, cyclin-dependent kinase 2 (CDK) and Dbf4-dependent kinase (DDK) that help transition the pre-RC to the initiation complex before the initiation of DNA replication. This transition involves the ordered assembly of additional replication factors to unwind the DNA and accumulate the multiple eukaryotic DNA polymerases around the unwound DNA. Central to the question of how bidirectional replication forks are established at replication origins is the mechanism by which ORC recruits two head-to-head Mcm2-7 complexes to every replication origin to form the pre-replication complex.
Origin recognition complex.
The first step in the assembly of the pre-replication complex (pre-RC) is the binding of the origin recognition complex (ORC) to the replication origin. In late mitosis, the Cdc6 protein joins the bound ORC followed by binding the Cdt1-Mcm2-7 complex. ORC, Cdc6, and Cdt1 are all required to load the six protein minichromosome maintenance (Mcm 2–7) complex onto the DNA. The ORC is a six-subunit, Orc1p-6, protein complex that selects the replicative origin sites on DNA for initiation of replication and ORC binding to chromatin is regulated through the cell cycle. Generally, the function and size of the ORC subunits are conserved throughout many eukaryotic genomes with the difference being their diverged DNA binding sites.
The most widely studied origin recognition complex is that of "Saccharomyces cerevisiae" or yeast which is known to bind to the autonomously replicating sequence (ARS). The "S. cerevisiae" ORC interacts specifically with both the A and B1 elements of yeast origins of replication, spanning a region of 30 base pairs. The binding to these sequences requires ATP.
The atomic structure of the "S. cerevisiae" ORC bound to ARS DNA has been determined. Orc1, Orc2, Orc3, Orc4, and Orc5 encircle the A element by means of two types of interactions, base non-specific and base-specific, that bend the DNA at the A element. All five subunits contact the sugar phosphate backbone at multiple points of the A element to form a tight grip without base specificity. Orc1 and Orc2 contact the minor groove of the A element while a winged helix domain of Orc4 contacts the methyl groups of the invariant Ts in the major groove of the A element via an insertion helix (IH). The absence of this IH in metazoans explains the lack of sequence specificity in human ORC. Removing the IH from the "Sc"ORC causes it to lose its specificity for the A element, and to bind promiscuously and preferentially (83%) to promoter regions. The ARS DNA is also bent at the B1 element through interactions with Orc2, Orc5 and Orc6. The bending of origin DNA by ORC appears to be evolutionarily conserved suggesting that it may be required for the Mcm2-7 complex loading mechanism.
When the ORC binds to DNA at replication origins, it serves as a scaffold for the assembly of other key initiation factors of the pre-replicative complex. This pre-replicative complex assembly during the G1 stage of the cell cycle is required prior to the activation of DNA replication during the S phase. The removal of at least part of the complex (Orc1) from the chromosome at metaphase is part of the regulation of mammalian ORC to ensure that the pre-replicative complex formation prior to the completion of metaphase is eliminated.
Cdc6 protein.
Binding of the cell division cycle 6 (Cdc6) protein to the origin recognition complex (ORC) is an essential step in the assembly of the pre-replication complex (pre-RC) at the origins of replication. Cdc6 binds to the ORC on DNA in an ATP-dependent manner, which induces a change in the pattern of origin binding that requires Orc1 ATPase. Cdc6 requires ORC in order to associate with chromatin and is in turn required for the Cdt1-Mcm2-7 heptamer to bind to the chromatin. The ORC-Cdc6 complex forms a ring-shaped structure and is analogous to other ATP-dependent protein machines. The levels and activity of Cdc6 regulate the frequency with which the origins of replication are utilized during the cell cycle.
Cdt1 protein.
The chromatin licensing and DNA replication factor 1 (Cdt1) protein is required for the licensing of chromatin for DNA replication. In "S. cerevisiae", Cdt1 facilitates the loading of the Mcm2-7 complex one at a time onto the chromosome by stabilising the left-handed open-ring structure of the Mcm2-7 single hexamer. Cdt1 has been shown to associate with the C terminus of Cdc6 to cooperatively promote the association of Mcm proteins to the chromatin. The cryo-EM structure of the OCCM (ORC-Cdc6-Cdt1-MCM) complex shows that the Cdt1-CTD interacts with the Mcm6-WHD. In metazoans, Cdt1 activity during the cell cycle is tightly regulated by its association with the protein geminin, which both inhibits Cdt1 activity during S phase in order to prevent re-replication of DNA and prevents it from ubiquitination and subsequent proteolysis.
Minichromosome maintenance protein complex.
The minichromosome maintenance (Mcm) proteins were named after a genetic screen for DNA replication initiation mutants in "S. cerevisiae" that affect plasmid stability in an ARS-specific manner"." Mcm2, Mcm3, Mcm4, Mcm5, Mcm6 and Mcm7 form a hexameric complex that has an open-ring structure with a gap between Mcm2 and Mcm5. The assembly of the Mcm proteins onto chromatin requires the coordinated function of the origin recognition complex (ORC), Cdc6, and Cdt1. Once the Mcm proteins have been loaded onto the chromatin, ORC and Cdc6 can be removed from the chromatin without preventing subsequent DNA replication. This observation suggests that the primary role of the pre-replication complex is to correctly load the Mcm proteins.
The Mcm proteins on chromatin form a head-to-head double hexamer with the two rings slightly tilted, twisted and off-centred to create a kink in the central channel where the bound DNA is captured at the interface of the two rings. Each hexameric Mcm2-7 ring first serves as the scaffold for the assembly of the replisome and then as the core of the catalytic CMG (Cdc45-MCM-GINS) helicase, which is a main component of the replisome. Each Mcm protein is highly related to all others, but unique sequences distinguishing each of the subunit types are conserved across eukaryotes. All eukaryotes have exactly six Mcm protein analogs that each fall into one of the existing classes (Mcm2-7), indicating that each Mcm protein has a unique and important function.
Minichromosome maintenance proteins are required for DNA helicase activity. Inactivation of any of the six Mcm proteins during S phase irreversibly prevents further progression of the replication fork suggesting that the helicase cannot be recycled and must be assembled at replication origins. Along with the minichromosome maintenance protein complex helicase activity, the complex also has associated ATPase activity. Studies have shown that within the Mcm protein complex are specific catalytic pairs of Mcm proteins that function together to coordinate ATP hydrolysis. These studies, confirmed by cryo-EM structures of the Mcm2-7 complexes, showed that the Mcm complex is a hexamer with subunits arranged in a ring in the order of Mcm2-Mcm6-Mcm4-Mcm7-Mcm3-Mcm5-. Both members of each catalytic pair contribute to the conformation that allows ATP binding and hydrolysis and the mixture of active and inactive subunits presumably allows the Mcm hexameric complex to complete ATP binding and hydrolysis as a whole to create a coordinated ATPase activity.
The nuclear localization of the minichromosome maintenance proteins is regulated in budding yeast cells. The Mcm proteins are present in the nucleus in G1 stage and S phase of the cell cycle, but are exported to the cytoplasm during the G2 stage and M phase. A complete and intact six subunit Mcm complex is required to enter into the cell nucleus. In "S. cerevisiae", nuclear export is promoted by cyclin-dependent kinase (CDK) activity. Mcm proteins that are associated with chromatin are protected from CDK export machinery due to the lack of accessibility to CDK.
Initiation complex.
During the G1 stage of the cell cycle, the replication initiation factors, origin recognition complex (ORC), Cdc6, Cdt1, and minichromosome maintenance (Mcm) protein complex, bind sequentially to DNA to form a head-to-head dimer of the MCM ring complex, known as the pre-replication complex (pre-RC). While the yeast pre-RC forms a closed DNA complex, the human pre-RC forms an open complex.