1.6. In Eukaryotes
DNA replication takes place in three steps, Initiation, elongation and termination in eukaryotes. Single eukaryotic chromosome has many replicons. In case of eukaryote DNA replication, formation of pre-replicative complex takes place in G1 stage of cell cycle. Entry from G1 stage to stage is mediated by regulatory checkpoint to ensure the presence of requirement of replication is completed like amount of RNA, protein, lipids, and carbohydrate and there should be not any kind of damage in DNA. Although individual replicons have characteristic time to get activated during S phase but replicons near one another are activated at the same time known by Regional activation patterns. Once replication get completed, no other activation of any origin takes place until next round of replication starts after cell division.
Only some of these replicons are engaged in replication at any point in S phase. A crucial difference between DNA replication of bacteria and eukaryotes is bacteria replication is occurring on DNA but in eukaryotes replication is occurring on chromatin and nucleosomes.
In eukaryotes, many origin of replication are present. Each origin of replication called as autonomously replicating sequence (ARS). ARS up to 100 to 200 base pair in eukaryotes. In yeast the ARS is 100 base pair. Long sequence ARS contain four consensus sequences.
(1)-A domain also called core box it is a 14 base pair in which first box 11 A-T base pairs sequence are highly conserve between all ARS these II base pain sequence are known as ACS. Known as ARS consensus sequence (ACS).
All present in cascade manner A-B1-B2-B3 and all of them comes around ~50 base pair in ARS.
Mutation in A the replication initiation get inhibited completely. Mutation in other three affect initiation of replication but not significantly. The order of inhibition of replication initiation is in decreasing order. A > B1 > B2 > B3 mutation in B1 effect more than B2 and B3, mutation in B2 effect more than B3 and mutation in B3 least effect the initiation of replication. It also reveal that an origin can function effectively in the presence of functional A along with any of two B elements.
Origin recognition complex (OR-C) is composed of six proteins act as initiator and binds to AT rich A and B1with ATP dependent manner. ORC2-5 binds strongly but ORC-6 binds weakly. ORC-6 has nuclear localization sequence. In G1 stage the ORC-6 is phosphorylated by CDK-2 and cyclin E. This phosphorylation causes transport of ORC-6 to nucleus and it binds with remaining ORC-2-5. After that transcription factor ABF I (ARS binding factor) binds to the B3 element. This directs initiation by affecting chromatin structure at the A and B1 elements. After binding ORC recruit helicase loader Cdc6 with Cdt1 protein Cdt1 enhance the activity of Cdc6. Cdc6 recruit helicase known as MCM. The loading of MCM on ARS also facilitate by Cdt1 along with ORC’s ATP hydrolysis. The complex of ORC, Cdt-1, Cdt-6 and MCM is called as pre-replicative complex (pre-RCs).
Now in S phase pre-replicative complex trigger in active state by phosphorylation of Cdc6 and Cdt1 through kinases name Cdk and Ddk. Phosphorylation of Cdc6 leads to its degradation and not available for further initiation the replication. Now the MCM recruit CdC-45 and GINS. This complex is called as CMG (CdC-45, MCM-GINS) complex. CDt-45 and GIN-45 enhance the helicase activity of MCM and MCM causes unwinding of Ds DNA. After that polymerase a get recruited by helicase and make RNA primer nucleotide. The polymerase a makes 10 nucleotide long RNA sequence which work as primer for DNA syubnthesis. A 20 base pair DNA also form by polymerase a called as initiator DNA, or “iDNA”. After forming the iDNA the polymerase a get dissociated from DNA template. Replication factor C (RFC), a clamp loader then binds to the iDNA and loads PCNA (clamp) on iDNA in ATP dependent manner with the same mechanism mention in prokaryotic replication, DNA polymerase ε for leading strand and DNA polymerase δ on lagging strand also comes there with help of its clamp. Further mechanism similar to prokaryote rather than primer removal which carried out when leading strand and lagging strand synthesis get completed. This marks the transition from initiation to elongation stage.
1.6.1. Primer removal
The RNA primers are removed by an exonuclease (MF1) in yeast and the gaps are filled by the DNA polymerase δ in yeast and nicks are sealed by DNA ligase in gene. In human flap endonuclease 1 (FEN1) break the phosphodiester bond between primer and DNA, then RNase H remove the RNA primer. RNA primers of Okazaki fragments are removed by exonuclease FEN I. DNA polymerase δ makes complex with FEN 1 to direct nick translation as in E. coli DNA polymerase I. after that nicks are sealed by DNA ligase. DNA polymerase δ responsible for displacing the 5′ end of the primer into a single-stranded RNA flap and cleavage of the short RNA flaps or for long flaps, get coatted by single-stranded DNA binding protein replication protein A (RPA) and then sequential cleavage by Dna2 nuclease and FEN1.
1.7. End replication problem
In eukaryotes linear DNA is present thus eukaryotes face a difficulty at the end of DNA replication because the first primer on each strand is removed, there is no way to fill the gaps because no 3’-end is upstream direction and DNA polymerase cannot be extended in the 3’→5' direction. In prokaryote there is no problem in filling all the gaps because 3’-end of another DNA is always upstream to serve as primer as the DNA is circular in prokaryotes. DNA would grow shorter, If nothing was done to overcome this problem.
1.7.1. Telomere protection in germ cell, stem cell, cancerous cell
End of eukaryotic chromosome known as telomeres. Telomeres possess long array of multiple short tandem repeats of six bases TTAGGG up to 20 to several 100 (vertebrates including humans), which are rich in G-T bases. The telomere sequence of telrahymena is TTGGGG. Telomerase possess a small segment of RNA, complementary to the six-base-pair telomere repeat due to this region it recognize the telomeres and reminds it what sequence to add. The end replication problem is solved by telomerase enzyme. Telomerase in a ribonucleo protein. It is madeup of RNA and protein. The protein has reverse transcinptase activity and RNA is complementary to telomere sequence present at the end of DNA. Telomerase enzyme is a reverse transcriptase which is hold by two protein p43 and p123 responsible for catalytic activity of the enzyme thus called as TERT (telomerase reverse transcriptase). The human teloheric sequence is TTAGGG.
The G-rich strand of a telomere is added at the 3’-ends of DNA strands by telomerase. The RNA component of telomerase or serves as a template for addition of new DNA repeats at 3’ end. The C-terminal part of the TERT protein contains the reverse transcriptase activity. RNA appears to be tethered to the N terminal part of TERT. This allows the RNA perform its template role by moving with respect to the active site of the enzyme as each nucleotide is added to the growing telomere. When 3’-ends get elongated by telomerase after that the complementary strand can be filled by normal RNA priming followed by elongation by DNA polymerase d and joining by ligase. This process of protection available in germ cell, stem cell and cancerous cell because all of them possess telomerase enzyme.
Telomere repeat sequences have been conserved during evolution even though some variation is seen. Several monocotyledonous plants – TTAGGG, Aspergillus nidulans –TTAGGG, Aspergillus oryzae – TTAGGGTCAACA, Paramecium – TTGGGG, Arabidopsis – TTTAGGG, Many insects – TTAGG, Trypanosoma – TAGGG, Tetrahymena - TTGGGG. Drosophila having exception to this general pattern because in fruit fly telomeres consisting of tandem sequences generated by successive transposition of two retrotransposons (HeT-A and TART) instead of being synthesized by telomerase.
1.7.2. Telomere protection in somatic cell
Telomerase protection in somatic cell is done with the help of a group of protein complex known as Shelterin complex and protein in this group known as shelterin protein because those provide shelters to telomere. In mammals, these protein known as TRF1, TRF2, TIN2, POT1, TPP1, and RAP1. Shelterin act in specific way to remodel the telomere into a loop called a t-loop (for “telomere-loop”) and in t-loop each protein protects telomere in particular manner. TTAGGG repeat binding factor-1 (TRF1) binds to double-stranded telomere DNA with TTAGGG repeats. TRF2 paralog of TRF1 binds to the double-stranded parts of telomeres. Single-stranded telomeric DNA protected from endonucleases through binding of POT1 (protection of telomeres -1) to single-stranded 3’-tails of telomeres at just 2 nucleotides away from the 5’-end of the other strand in turn double-stranded telomeric DNA at 5’end of other strand also get protected from 5’-end exonucleases. TPP1 is a POT1-binding protein and present as partner of POT1 in a heterodimer. TIN2 (TRF1-interacting factor-2) plays an organizing role in shelterin complex, It connects TRF1 and TRF2 to TPP1/POT1 dimer. RAP1 (repressor activator protein-1) interact with TRF2. Shelterin proteins specifically present only at telomeres throughout the cell cycle and nowhere else.
1.7.3. Telomere structure and telomere-binding proteins in lower eukaryotes
Yeasts also have telomere-binding protein which protect the telomere ends by forming D-loop in which single strands not get hide and those protein bear a resemblance to mammalian shelterin proteins like Taz1 binds double-stranded telomere same as TRF. Taz1 with the help of Rap1 and Poz1 binds to a dimer of Tpz1 and Pot1 same asTPP1-POT1 dimer and protect single-stranded telomeric DNA. All these protein bond the telomere DNA up to 180 degrees by protein : protein interactions proteins bound to the double stranded telomere and also bound to its single-stranded tail.
Yeast Rap1 binds directly to double-stranded DNA show evolutionary relationship to mammalian Rap1. Rif1 and Rif2 are two partners of RAP1in yeast. Single-stranded telomeric tail binds by other protein complex Cdc13, Stn1, and Ten1. Ciliated protozoan Oxytricha in which telomere-binding proteins were firstly discovered having name TEBPa and TEBPb, which are evolutionarily related to POT1 and TPP1 in mammals binds with single-stranded 39-end of the organism’s telomeres and protect them from degradation. In schizosaccharomyces probe, Pot1 responsible for maintaining their integrity instead of limiting the growth of telomeres as mammalian POT1 does. Indeed loss of Pot1 can cause the loss of telomeres from this organism.
1.7.4. Telomeric protection through G-quadruplexes
Telomeres are protein–DNA structures at the ends of eukaryotic chromosomes that protect chromosome ends from fusion and are vital in safeguarding genomic stability. In Tetrahymena tandem repeats of short G-rich sequences (TTGGGG) present at 3′ strand of telomeres that project as a single-stranded DNA overhang and forming G-quarters, which is form in a cyclic array through synchronization or coordination of four guanine residue and multiple layers of this G-quarters leads to the formation of G-quadruplexes. This arrangement takes place through non Watson-Crick pairing or Hoogsteen hydrogen-bonding with the help of centrally positioned cation.
1.8. The Hayflick Limit
Normal human cell can be grown in culture upto 50 generations (or cycles of subculturing) for a limited period, after they enter a period of senescence, in which they slow down and afterward stop dividing, and lastly they attain a crisis stage and get die. This upper limit on the life span of normal cells is known as the Hayflick limit, discovered by Leonard Hayflick in 1960s. But tumor cells do not follow any such limit. Tumor cell divides generation after generation, for an indefinite period because. Tumor cell contain telomerase enzyme which replicates the end of DNA.
1.9. Modes of Replication
There are various mode of DNA replication - (1) Theta Replication, (2) Rolling Circle, (3) Linear Replication. Differ to each other with respect to the initiation and progression of replication, but all produce new DNA molecules by semiconservative replication.
1.9.1. Rolling-circle replication
It involves a single stranded intermediate that seems to roll off the plasmid. Plasmid’s double stranded DNA has positive and negative strand. In rolling circle replication double stranded ori that place within stem-loop structure get nicked by Rep A protein at positive strand and free 3’ OH serve as a primer which get replicate by DNA pol III in leading strand manner and displacing the 5’ end which is non-template strand by strand displacement reaction. The ends of the displaced strand are ligated to make a single stranded circular DNA molecule. RNA polymerase makes a primer at the 5’ end containing strand, thus also capable to act as template for lagging strand synthesis. After synthesis of new strand primer get remove by DNA pol I. The ends of the displaced strand are ligated by ligase to make a second double- stranded plasmid. E.g. M13, Φ 174, F plasmid.
Most plasmids use one of two common mechanisms of replication, both of which depend on the host cell replicative machinery for replication (DNA polymerases, ligases, primases, helicases, etc. are usually supplied by the host cell).
1.9.2. Theta replication –
It is normal mode of DNA replication which resembles the greek letter theta (θ) during replication. The theta mode of DNA replication is bidirectional in nature.
1.9.3. Linear replication –
This the type of replication takes place in linear chromosome like in eukaryotes.
1.9.4. Replication of organelle DNA (Models for mtDNA replication)
DNA polymerase γ is used exclusively for mtDNA replication and proofreading. Two models have been proposed for the mode of mtDNA replication, 1) strand displacement model, appeal to continuous DNA replication and 2) coupled model, proposes semidiscontinous DNA replication.
Strand displacement model
The strand displacement model (also called the strand asynchronous model) for mammalian mtDNA replication is the most widely accepted, longest standing model. In strand displacement model, replication is unidirectional around the circle and there is one replication fork for each strand in which One strand is called the heavy strand (H) and other strand is called light strand (L). The designation of the strands as H and L due to their different buoyant densities in denaturing CsCl density gradients centrifugation. Two origins (OH and OL) because here one priming event per template strand and it start within a region called the displacement or “D” loop of 500–600 nucleotide. The RNA primer synthesis by mitochondrial RNA polymerase. Firstly replication start on H strand starting at OH. When DNA polymerase polymerise approximately two-thirds of the mtDNA as a result replication fork get passes the major origin of L strand synthesis, thus exposing the site in single-stranded form at which new H strand synthesis start from OL. Synthesis is continuous around the circle on both strands. Multifunction endoribonuclease, RNase MRP cleave RNA primer. Same mechanism for cpDNA (cytoplasmic DNA) replication.
Strand coupled model
In strand coupled model at multiple sites lagging strand replication (L strand) get initiated, showing discontinuous synthesis of short Okazaki fragments thus requiring multiple primers. So, in this model the coupled lagging strand and leading (H strand) synthesis represents a semidiscontinous, bidirectional mode of DNA replication. The endoribonuclease, RNase MRP (mitochondrial RNA processing) has at least two functions, 1) removal of RNA primers in mtDNA replication, 2) pre-ribosomal RNA’s nucleolar processing. Mutation in RNase MRP cause cartilage-hair hypoplasia a rare form of dwarfism and due to its consequences multiple phenotypic manifestations (pleiotropy) occur which included short limbs, short stature, fine sparse hair, impaired cellular immunity, anemia, and predisposition to several cancers.