Eukaryotic DNA Replication Mechanism:
Eukaryotic cells are endowed with a very complicated but sophisticated regulation of cell division. Embryonic cells go through first seven to eight cell divisions very fast at an interval of 30- 40 minutes, but differentiated cells remain in resting stage i.e. Go stage. Onion root tip cells divide once in every 12 hrs, but mammalian cultured cells take 24 hr for one cell cycle. Resting cells, whatever may the cell type, initiate cell division when there is loss of cells in the tissue or by mitotic signals. In order to replace the lost number, existing cells divide and redivide till the number is restored.
Cells in cell division mode go through certain number of sequential molecular expressions and we call them as stages such as, Interphase and Mitotic stages (M). Interphase is further divided into G1, S and G2 stages in succession; each event is independent and interdependent. An event does not enter to the next till the previous one is completed. The mitotic phase the M phase consists of Prophase, Metaphase, Anaphase and Telophase, all put together requires only one hour or so. In 24hrs cycle the longest phase is G1 about 10-12 hrs., S phase requires about 7-8 hrs and G2 takes 6-8 hrs; this is an approximation of 24 hrs cell cycle.
Activation of DNA replication is initiated at the end of G1 phase and replication initiation takes place at the very early S-phase (DNA replication or synthesis stage), till the replication completes at G2 (G stands for Gaps in interphase) cells don’t enter into M phase. Replication initiation occurs only once in a cell cycle and second round of replication is prevented in the same cell cycle. In S.cerevisiae and S.pombe, gene products of Cdc6 and Cdt1 respectively required for transition from G1 to S phase. They are similar to Xenopus’ p34 kinase. There are different forms of P34 CdKs (Kinases). One of them is cell cycle dependent CdK34, which is activated when specific S-cyclin binds to CDK. The protein complex has now been purified; it consists of p34 kinase and cyclin (or CDK2-CyclinB), called Mitosis-Promoting Factor (MPF). There are S-cyclins and M-cyclins, each have specific roles in S and M (Synthesis and Mitotic phases) phases. A START point or Restriction point is the stage at which commitment to initiation for S stage occurs at G1, when a whole battery of genes for DNA synthesis (DNA replication) is activated. The regulatory pathway consists of a cascade of cyclin dependent phosphorylation and dephosphorylation events. Until every bit of DNA sequence is replicated, this includes telomeric DNA synthesis, the cell doesn’t enter into M phase.
Eukaryotic DNA is linear and contains several Ori sites may be 1000 or more distributed all along the length of each chromosomal DNA. Taking SV40 DNA as a model, replication initiation starts with the binding of T-antigens as hexamers, to specific sites. T-antigen also acts as a helicase (motor protein). They bind to specific sequences called ORE (Origin Recognition Elements) and induce the opening of DNA in DUE (DNA unwinding Element). Location of origin in different system differs, but initiation always takes place by the binding of specific factors such as origin recognition complexes (ORC) to specific sequences that initiate the process.
The ORC (Origin Replication Complex) is a protein binding complex, made up of 6 subunits (ORC p1, ORC p2, ORC p3, ORC p4, ORC p5, ORC p6) that has a binding specificity to DNA, more specifically to the origins of replication, in the presence of ATP, in the genomes of the eukaryotes. Theoretically this binding complex’s function is the initiation and/or regulation of the DNA replication in eukaryotes. More specifically, the ORC bound to the origins of replication act as the foundation for the assembly of the pre-replication complex (pre-RC), which is a protein complex essential for the initiation of the replication, in other words, the pre-RCs act as licensing factor of the chromosomes. That’s why ORCs do not work directly in DNA replication initiation but indirectly instead. In addition to this, the ORC is target of protein kinases which will phosphorylate some of it’s subunits in order to regulate DNA replication initiation and to block the reinitiation of the G2/M phases.(Blogspot)
We the authors mentioned below report that a highly purified human origin recognition complex (HsORC) has intrinsic DNA-binding activity, and that this activity is modestly stimulated by ATP. HsORC binds preferentially to synthetic AT-rich polydeoxynucleotides, but does not effectively discriminate between natural DNA fragments that contain known human origins and control fragments. The complex fully restores DNA replication to ORC-depleted Xenopus egg extracts, providing strong evidence for its initiator function. Strikingly, HsORC stimulates initiation from any DNA sequence, and it does not preferentially replicate DNA containing human origin sequences. These data provide a biochemical explanation for the observation that in metazoans, initiation of DNA replication often occurs in a seemingly random pattern, and they have important implications for the nature of human origins of DNA replication, Sanjay Vashee1,4, Christin Cvetic2,4, Wenyan Lu3, Pamela Simancek3, Thomas J. Kelly3,6, and Johannes C. Walter2,5
a | The origin recognition complex (ORC) is first recruited to the replication origin. b | ORC recruits Cdc6 and Cdt1. c | ORC, Cdc6 and Cdt1 act together to load multiple minichromosome maintenance (Mcm)2–7 protein hexamers onto the origin, which licenses the DNA for replication. d | Initiation-competent complexes are probably formed by the back-to-back assembly of two Mcm2–7 complexes. As the ORC is asymmetrical, this might require deposition of a second ORC molecule to load Mcm2–7 in the opposite orientation; J. Julian Blow & Anindya Dutta; Nature-2005
A very important event, of the most significance, happens in yeast. It is the role of licensing factors, which also control cell division via initiation of replication.
In yeast certain proteins like OBFs (Origin Binding Factors) or origin recognition complex (ORC) bind to ORE (Origin Recognition Elements) region, they act as positional factors, that facilitates the binding of other factors to induce melting of the DNA in that region, which is A/T rich region (DUE). The binding of accessory factors or auxiliary factors to AUX-I AUX-II regions can further facilitate or augment melting of the DNA.
In yeast CD6 and Cdt1 activate MCM to load on to the ORC (hexamer), which is already bound to origin site. Yeast, autonomously replicating plasmid origin site, has been very well elucidated.
The binding of MCM2-7 and phosphorylation of the same initiates opening of the DNA into Replication bubble; but it requires other factors such a Cdc7/DbF1. Phosphorylation of MCM2 by Cdc7/DbF1 activates its helicase activity to unwind the dsDNA. Among the six MCMs, MCM 3, 5 and 6 are essential and others’ role is not clear. For initiation of replication Cdc45 is required and the same is loaded on to fork joints. At this point ORC, Cdc6 and Cdt1 are unloaded from the origin complex, the same are subjected ubiquitin- mediated degradation. The unused MCMs are also degraded. Recruitment of MCM dimer induces the MCM complex switch from ds DNA binding to ssDNA strands.
The ORC is a six-protein machine with a slightly twisted half-ring structure (yellow). ORC is proposed to wrap around and bend approximately 70 base pairs of double stranded DNA. (The MCM complex consists of Cdc46p, Cdc47p, Cdc54p, Mcm2p, Mcm3p, and Mcm6p), Mcm4p (Cdc54p), 6p and 7p (Cdc47p) are responsible for helicase activity during elongation (You et al., 1999; Labib and Diffley, 2001),Mcm4p (Cdc54p), 6p and 7p (Cdc47p) are responsible for helicase activity during elongation (You et al., 1999; Labib and Diffley, 2001),
The initiation of chromosomal DNA replication in eukaryotes can be divided into two general steps. The first occurs during the G1 phase of the cell cycle and involves the formation of a pre-replication complex (pre-RC) containing the origin recognition complex (ORC), Cdc6, Cdt1 and MCM. At the G1/S transition, cyclin-dependent kinase (Cdks) and the Cdc7-Dbf4 kinase convert the pre-RC into an active replication fork by a currently unknown mechanism. There is evidence that the substrate of Cdc7-Dbf4 may be the MCM complex (?), whereas the Cdk substrates are not known. To investigate whether PP2A is involved in the replication of chromosomal DNA, authors used Xenopus cell-free replication system and found that removal of PP2A from egg extract caused complete inhibition of DNA replication, and that PP2A is required for initiation but not elongation of DNA replication. (Stephen Kearsey, Regulation of eukaryotic DNA replication). Mcm10 binds to the complex depending on assembly of the CMG components. Loading of Mcm10 does not cause release of Sld3, Cut5 or Drc1. (iv) Mcm10 forms homo-multimers and promotes conversion of the Mcm2-7 complex from the dsDNA-bound double hexamer into two ssDNA-bound single hexamers through its interactions with multiple subunits of Mcm2-7. Once single stranded DNA templates are made available RPA bind to maintain the ssDNA strands till the new strand is synthesized.
Replication initiation steps.
Initiation requires a stepwise association of proteins with replication origins before DNA synthesis can begin. The origin recognition complex (ORC) binds to DNA and provides a site on the chromosome where additional replication factors can associate. An early step leading to initiation, called licensing or pre-replicative complex formation, involves the association of Mcm2-7 complex with DNA at ORC, in a process requiring Cdt1 and Cdc6. Mcm2-7 proteins provide helicase activity for DNA synthesis and loading of these proteins confers competence on the origin to fire in S phase. Onset of DNA synthesis requires the action of two protein kinases (cyclin dependent kinase (CDK) and Cdc7), which trigger the association of additional proteins with the origin, such as Cdc45 and GINS. During the process of initiation, DNA polymerases are also recruited and DNA synthesis starts. During replication, Mcm2-7 proteins move away from the origin and further assembly of pre-replicative complexes is blocked. This ensures that origins can only fire a single time per cell cycle. For further details see www.dnareplication.net.
The role of Ctf4 in promoting replication fork stability
Ctf4 is the budding yeast And1 homolog and links polymerase alpha to the RF, specifically the helicase. Gambus et al. (2009) demonstrate in this paper that Ctf4 associates with helicase components (MCM proteins and also the GINS complex). They also show that cells lacking both Ctf4 and Mrc1 (delta-ctf4 and delta-mrc1 mutants) cannot survive, demonstrating how important the FPC is to cell health. If Mrc1 is not present (delta-mrc1), the Ctf4 component of the FPC becomes critical to holding the RF together. © 2009 Nature Publishing Group Gambus, A. et al. A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase α within the eukaryotic replisome. The EMBO Journal 28, 2992–3004 (2009) doi:10.1038/emboj.2009.226. All rights reserved.
Replication fork components; The RF is a multiprotein complex with helicase and DNA synthesis activities. It is called a fork because the structure resembles a two-pronged fork. The helicase activities unwind DNA in front of the fork to create regions of singled-stranded DNA (ssDNA). The helicase components shown are the minichromosome maintenance (MCM) helicase 2-7 hexamer, CDC45, and associated GINS complex (simplified as a single entity here). The ssDNA is coated in RPA (yellow circles) to keep strands from reannealing. Fork protection complex (FPC) components shown are Timeless (TIM), Tipin (TIPIN), Claspin (CLASPIN), and And1 (AND1). Claspin (MRC1 in yeast) helps connect the leading-strand polymerase epsilon (light blue circle) to the helicase. And1 connects the lagging-strand polymerase alpha (tan circle) to the helicase. Pol-alpha is part of the primase complex, which synthesizes primers (thick tan line) on the lagging strand. These primers allow the polymerase of the main lagging-strand (polymerase delta, light green circle) to start synthesis. The direction of DNA synthesis and RF movement into DNA is indicated by arrows.© 2010 Nature Education All rights reserved
The melting and stabilization of melted DNA is also facilitated by upstream transcriptional initiation factors like Sp1 (GC binding factors in SV 40) and other TFs. The RNA Pol II Holozyme is involved in this process. Probably enhancers too contribute to this event.
Chromosomal DNA is bound by histones and nonhistones. The replication factors have to find their Ori sites to bind; it means the chromatin in the region should be remodeled to DNA free from chromatin proteins; Tomas Aparicio etal
Replication proceeds in bi-directional manner, so each of the replication forks meet from opposite directions. Though replication is initiated in most of the origins simultaneously, replication of heterochromatin segments is delayed in comparison to euchromatin DNA. The last region that replicates is telomeric DNA.
As in the case of SV 40, yeast and other Eukaryotes, replication initiation is controlled by what is called licensing factors RFM and RFL. The activity of these factors in regulating replication initiation events takes place only once per cell cycle.
At this stage, Cdc4-6 and G1 Cyclins are inhibited by Sic1p, but soon the inhibitor gets phosphorylated and degraded by SPF (S phase Promoting Factor ) by ubiquitination process. In addition few more Cdk-cyclin inhibitors are also inactivated. Activated CDC2, 4/6-Cyclins phosphorylate Retino Blastoma (RB) (tumor suppressor proteins) and release E2F transcription factors. Similarly p107 and p130, that have sequestered few other E2Fs, are also released. The released E2Fs bind to their respective promoters and activate gene expression required factors for S-phase DNA replication events.
Cdc6 has a very short half-life say 2-5 minutes (in S.pombe, the fission yeast, it is called cdc18P). This is acquired only when the chromosomes are free from nuclear membrane i.e. between late M and G1. The presence of phosphorylated Cdc6-P is essential for loading MCM 2-7 proteins on to the replication fork, one on each side of the fork. The movement is like unwinding.
The Geminin by sequestering Cdc6/Cdt1 prevents reinitiation chromosomal DNA replication before the completion of Mitotic stage; later Geminin is degraded by APC1. Once the replication bubble is formed and stabilized by ssDNA binding protein RPA, other replication components join.
The replication components are RPA 1, DNA Pol-α , RFC1, PCNA DNA Pol δ- and ε-proteins, they assemble at replication fork one at each fork.
MCM proteins dissociate from DNA after replication but remain intact and stable, only Cdc6/Cdt1 proteins are destroyed in cell cycle dependent manner, that too by ubiquitination process.
As long as these factors are not available as a pre initiation components, DNA replication is not initiated, hence Cdc6 and Cdt1 are called as licensing factor B and MCM are called licensing factor M.
The licensing factors are synthesized only once in a cell cycle and they are acquired by the nucleus when the nuclear membrane reassembles. This happens only once in one cell cycle.
Interestingly, if Cdc6 is made available by ectopic expression at G2 stage, MCM complex doesn’t bind to the ORC; it means that there is another control point with another licensing factor?
As the replication of DNA completes Cdh1 activates Anaphase promoting complex (APC) by Cdc20. Then they in turn mark Cyclin-A and Cyclin-B for ubiquitinated degradation. They also mark the degradation of securins. That leads the release of a protease which degrades cohesin components thus the daughter DNA molecules are released from one another at centromere, which is essential for the anaphasic movement of chromosomes. Degradation of CyclinA and Cyclin-B prevent reinitiation of Mitosis before another round of DNA replication.
Opening of the DNA into ss DNA is stabilized by ss binding proteins RF-A (RPA). They also prevent reannealing the DNA strands. The replication bubbles provide fork joints at which helicases bind.
In SV 40, T-antigen, itself acts like a helicase (an hexamer) with 3’>>5’ directional movement.
In eukaryotic systems one MCM2-7 helicase complexes one each at fork found. As and when the replication bubble is formed, it is stabilized by SSB proteins, DNA pol-a, which has both 5’>>3’ polymerase and primase activity, recognizes single stranded DNA and using specific sequences, binds to ssDNA and lays RNA primers on both leading and lagging strands in opposite orientation. This means, at central position one primer in each strand is laid, one for leading strand replication and another set of primers for lagging strands; however orientation of the primers on each of the strands depends upon the orientation the replicating strand. Leading and lagging strands are differentiated based on the 5’-3’ and 3’ to 5’ orientation of the strands.
Multiple origins in the linear chromosomal DNA
The required proteins are abundant, and exceed the stoichiometry of origins by 100 fold. In the cartoon above, a diagram is presented of these three models; the origin recognition proteins are shown in green; the replication machinery (polymerases etc) is shown in red, and the MCM proteins are shown in blue. Note that these models are not mutually exclusive.
MCMs have homology to a wide class of putative DNA-dependent ATPases, and bind together as a hexamer. SV40 T antigen and bacterial DnaB protein are the helicases in their respective systems, and also have hexameric structure. However, in vitro helicase activity is only observed for a complex of Mcm4-6-7. Thus, there are some issues yet to be resolved. (1) All six MCMs are essential in eukaryotes, and evidence suggests that in the normal nucleus, they are present at 1:1:1:1:1:1 stoichiometry. But the in vitro data doesn't account for Mcm2, Mcm3, or Mcm5.
MCMs are amazingly abundant, far exceeding the stoichiometry of replication origins. Reducing the dose of MCMs has severe phenotypes, even though the cells can still synthesize DNA. Cytological data suggests that MCMs liberally decorate unreplicated chromatin, but do not co-localize with the "replication factories" that contain PCNA and other elongation factors.
Pair wise interactions in vitro suggest this organization of the MCM is in the form of heterohexamer. The P-loop (Walker A motif) and SRF motif (arginine finger) are proposed to act together as ATPase domains. In isolation, this complex does not have helicase activity in vitro (see the text).
A hypothetical rotary pump model showing two stages in the distribution and function of MCM hexameric ATPase complexes.
Figure: Activation of the eukaryotic replicative helicase. The isolated Mcm2-7 assembly exists in two forms, a planar, notched ring and a spiral state, with an opening between subunits 2 and 5. Binding of the Cdc45 and GINS activators to the side of the ring seal off the opening and stabilize the planar configuration. Nucleotide binding tightens the ring, giving rise to two topologically segregated conduits involved in tracking the leading and lagging strands at the replication fork, Alessandro Costa
First, in G1 phase, MCM hexamers move spirally along the helical grooves of unreplicated DNA, away from ORC, which is required for their loading and orientation. Second, in S phase, MCMs become immobilized, so that exactly the same rotary mechanism moves the DNA instead of the MCM proteins. This would result in translocation of DNA towards the replication forks. As DNA is twisted by fixed MCMs in S phase, it would become unwound at the distant replication fork, which is itself immobilized in fixed clusters. The direction of MCM is 3’ -5’, bound to single strand.
Step 1: Primer synthesis by DNA polymerase-α (Pol-α); step 2: replication factor C (RFC) displacement of DNA polymerase alpha and recruitment of proliferating cell nuclear antigen (PCNA); step 3: elongation by the newly recruited DNA polymerase-δ holoenzyme (Pol-δ); step 4: strand displacement
Schematic representation of the subunit interactions of Pol α.
Diagram of the formation of the pre-replicative complex transforming into an active replisome. Mcm 2-7 complex loads onto DNA at replication origins during G1 and unwinds DNA ahead of replicative polymerases. Cdc6 and Cdt1 bring Mcm complexes to replication origins. CDK/DDK-dependent phosphorylation of pre-replicative proteins leads to replisome assembly and origin firing. Cdc6 and Cdt1 are no longer required and are removed from the nucleus or degraded. Mcms and associated proteins, GINS and Cdc45, unwind DNA to expose template DNA. At this point replisome assembly is completed and replication in initiated. "P" represents phosphorylation-WIKI
Eukaryotic Replisome complex -WIKI
MCM proteins and checkpoint kinases get together at the fork
A schematic view of the signaling pathways inhibiting DNA replication. ssDNA-RPA intermediates and DSBs arise as a consequence of external insults (irradiation and polymerase inhibitors) or during normal replication. These aberrant DNA structures trigger the activation of ATR and ATM protein kinases. In turn, these protein kinases inhibit origin firing through the Chk1/Chk2-dependent down-regulation of Cdk2 and Cdc7 protein kinases. MCM7 interaction with ATRIP could tether ATR protein kinase to the replication fork, providing a way to locally regulate ATR response to ssDNA. In addition to activating the canonical checkpoint pathways leading to Cdk2 and Cdc7 down-regulation, ATM and ATR directly phosphorylate MCM2 and MCM3. Although the consequences of these modifications are not yet known, they could modulate the activity of MCM proteins and either halt fork progression, prevent re-replication, or participate in origin selection
Figure. Diagram of the replication fork generated by MCM helicase, which separates the two strands of DNA, and single stranded binding proteins (RPA), which protect the strands. One of the strands is replicated in a continuous strand by DNA polymerase ε complexed with the protein PCNA (green). The second strand is replicated in shorter fragments, each beginning with the RNA/DNA primer made by pol-prim and extended by a DNA polymerase δ (blue) complex. Here you see pol-prim (Pol α-Primase - Red) bound to the DNA strand ready to displace RPA as it synthesizes a new primer. The fragments are linked together by the FEN1/DNA ligase complex. Image reproduced with the permission of Professor Peter Burgers, http://biochem.wustl.edu/~burgersw3/Replication.htm.
Simplified cartoon model of a eukaryotic replication fork. Protein depictions are based on currently accepted subunit composition of S. cerevisiae proteins but are not meant to be accurate structure-based models. The assignment of pol to the leading strand is based on a recent report 120, but has not been definitively established for all replication. Pol is consequently assigned to the lagging strand, consistent with earlier reports 121, 122, 123. Helicase hexamer (magenta); replication protein A (RPA; light blue ovals); proliferating cellnuclear antigen (PCNA; purple torus); pol -primase complex (blue); RNA-DNA hybrid primer (red zig-zag and arrow); pol (red); pol (green); template strand DNA (black lines); newly synthesized DNA (gray lines). FEN1 is a n exonuclease which remove RNA promers and DNA pol delta fills the gap and DNA ligase ligates the ends
As the primers are laid, RF-C binds to POL-a; this leads to the extension of RNA primer into a short sequence of DNA called iDNA (initiator DNA). The same enzyme uses dNTPs and assembles DNA segment of about 100 ntds long. Now RF-C clamp loading factor similar to E.coli beta clamp loader (i.e. li E.c oli delta-gamma complex), loads the trimeric clamp called PCNA.
PCNA displaces the DNA Pol-a; and the RF-C now facilitates the loading of POL-E on to the iDNA. DNA Pol-ε loaded extend as iDNA on leading strand.
Similarly primers laid on lagging strands are extended by PCNA-Pol-d complex. RF-C performs the loading of PCNA and Pol-delta every time when they dissociate on lagging strand. The replication model is similar to that of prokaryote-‘Trombone model’.
Whether the Pol-e -PCNA complexes are found as dimers at each fork or not is not clear, but it is logical that a pair of Pol-d-PCNA complexes to be present at each fork. One of them synthesizes new strand on leading template and the other works on lagging strand in reverse orientation and produces DNA fragments in discontinuous manner.
DNA-pol a continues to assemble on lagging strand at frequent intervals and lays short primers, and RF-C activates the pol-a to extend it as I- DNA.
Unlike the primase of prokaryotes, where the primase translocates with the helicase, the pol-a seems not to bind to any helicase or any other translocation protein on DNA.
Similar to prokaryotes, primers in eukaryotes are removed by MF-1, FEN1 and RNase H proteins by their 3’à5’exonuclease and endonuclease activities. The gaps are filled by pol-α and d polymerase.
The gaps are sealed by DNA-ligases. Mechanism of Ligases is similar to that of prokaryotes.
The extended forks from opposite sides meet they end up without any problem for the helicases dissociate. Incomplete strands with 3’OH are extended and then ends are ligated. But the Telomeric ends replication requires Telomerase and its partner RNA for the extension of the ends. This is delt separately.
The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide, ("acceptor") with the 5' phosphate end of another ("donor"). ATP is required for the ligase reaction, which proceeds in three steps: (1) adenylation (addition of AMP) of a residue in the active center of the enzyme; pyrophosphate is released; (2) transfer of the AMP to the 5' phosphate of the so-called donor, formation of a pyrophosphate bond; (3) formation of a phosphodiester bond between the 5' phosphate of the donor and the 3' hydroxyl of the acceptor.  A pictorial example of how a ligase works (with sticky ends):
Ligase will also work with blunt ends, but rquires higher concentrations and different reaction conditions.
If the replication machinery makes any error, DNA repair enzymes become active; even p53 induces gene expression for repair enzymes, and repair is done. Till then the cell does not enter M-stage. If the DNA damage is beyond repair, the p53 and other components induce the cell to Apoptosis.
Note: Refer to Cell Cycle and Its Regulation for DNA replication regulation;