Topoisomerase is able to introduced nick in single strand and double strand by cleaving the phosphodiester bond in single strand or both the strand of double strand DNA thus it is a type of nuclease. There are two main classes of topoisomerase. In Escheria coli four individual topoisomerase present and get classified in above mention two classes.
Type I Topoisomerase:
It is able to produce break in one DNA strand and cause the linking number to be increase by one. DNA binds with in cleft of enzyme thus get placed near active tyrosine. Active-site Tyr tysosine attacks as nucleophile and break a phosphodiester bond in one DNA strand, cleaving it and generating a covalent 5’-phosphotyrosyl protein- DNA linkage and free 3’ OH cause the formation of open conformation of enzyme, this result in a gap in cleaved strand and the gap bridge by enzyme. After that uncut strand pass through the gap cause the formation of closed conformation of enzyme as a result liberated 3’-OH of cleave strand attacks the 5’-phosphotyrosyl protein-DNA linkage to religate the cleaved DNA strand. Type I Topoisomerase not use ATP and responsible for catenane / decatenated, relaxation of supercoiling and knotting / unknotting. E.g. Type I Topoisomerase which relax negative supercoiling in E.coli, Type III Topoisomerase of E.coli. and Type I Topoisomerase of calf thymus which will relax negative and positive supercoiling.
Type I divide in two subclass; Type I which form covalent intermediate at 5’ end of DNA with changing the linking number by one integer and Type IB which form covalent intermediate at 3’ end of DNA with changing the linking number by two integer.
Type II Topoisomerase:
It is dimeric or in some cases tetrameric, able to produce break in two DNA strand and cause the linking number to be increase by two. It requires energy released by ATP hydrolysis to facilitate conformational change in enzyme not for cleave and rejoin DNA and responsible for catenation / decatenated, relaxation / induction of supercoiling and knotting / unknotting. The general mechanism features the passage of one intact duplex DNA segment through a transient double-strand break in another segment. The DNA segment enters and leaves the topoisomerase through gated cavities above and below the bound DNA, which are called the N gate and the C gate, enter through N gate and release through C gate. Two tyrosine present in each active site subunit and each tyrosine form 5’-phosphotyrosyl protein- DNA linkage with one DNA strand with 3’-OH in other site of cleave strand, two ATP require in over all process. The broken DNA is religated, and the second DNA segment is released.
E.g. Type II Topoisomerase in E.coli known as DNA gyrase which is tetramer of two Gyr A subunit and two GyrB subunit, can introduce negative supercoils and decrease Lk (Linking Number) through the utilization of ATP. DNA strand cutting and rejoining done by GyrA which is inhibited by nalidixic a quinolone antibiotic and its fluorinated derivatives norfloxacin and ciprofloxacin. ATP hydrolysis done by GyrB which is inhibited by novobiocin. DNA gyrase uses in initiaton of replication and transcription.
Other example include Type IV Topoisomerase of E.coli. Type II topoisomerases of eukaryotes can relax both positive and negative supercoils but cannot unwind DNA or introduce negative supercoils in DNA. In vertebrates Type II Topoisomeras has two isoform IIα and IIβ . In Archaea DNA gyrase belongs to type IIA and topoisomerase VI belongs to IIB family of Type II Topoisomerase.
1.5. Process of DNA replication in prokaryotes
Replicon is a unit of DNA replication which possess origin of replication. If the replication fork proceed in both the direction the replication is said to be bidirectional and when it proceed in one direction it is called unidirection replication. Replication proceeds generally bidirectionaly. Replicon should posses complete molecule of DNA.
In prokaryote only one replicon is present. Thus entire chromosome act as a single replicon but in eukaryote each chromosome contain multiple origin of replication in turn divided each chromosome in multiple replicon.
According to replicon model, control of replication depends on two component: First, replicator define as entire set of cis acting DNA sequence that is enough themselves to carries out DNA replication, infact origin of replication is the part of replicator, but in eukaryotes origin of replication act as replicator themselves because DNA sequence within origin of replication sufficient to direct replication. Second initiator, a protein that recognizes and binds to specific DNA element in replicator to activate initiation of replication.
In Prokaryotes DNA replication takes places in three steps-Initiation, elongation and termination.
In prokaryotes, eg. Escheria coli the ori C is a 245 bp long sequence. Ori C contains a number of repeat of two conserve or consensus sequence, first 13 mer consensus sequence with three repeat and second 9 mer sequence with five repeat. 13 mer which is rich in AT base pair is present upstream to 9 mer. The GATC sequence present at ori C is methylated at the adenine base. Dam enzyme methylates the 6th position of adenine base. Only fully methylated DNA can undergo DNA replication in pro karyotes. DNA-A-ATP bends with R1, R2 and R4 boxes of 9 mer sequence. These boxes are comparatively conserve than the R3 9bp box. The bending of DNA-A-ATP complex displace the Fis protein present at these boxex. The Fis interacts with 1 HF protein which causes binding at 9 bp sequence. This bending allows loading of DNA-A-ATP complex to 13 bp sequences. The DNA C now binds at 13 bp sequences. DNA C is helicase loader. It recruits the DNA B (helicase) at 13 bp sequence. Initiator protein DNA A make complex with ATP and DNA A-ATP binds to 9 mer sequence repeats on fully methylated chromosome. Which stimulates cooperative binding of DNA A.
Gyrase or Type II topoisomerase work in front of replication fork responsible to induce negative supercoiling by which it causes removal of torsional strain (overwinding) in the DNA generate by helicase movement in 5’ – 3’ direction. SSB protein binds to single stranded DNA created by helicase to prevent it reassociation. The length of duplex DNA for initiation of replication is probably less than 60 bp. This complex of DNA-A, DNA-B and DNA-C is called as pre priming complex. Once a sufficient size of replication fork is being made by DNA-B helicase it recruits the primase enzyme. The primase causes release of DNA-C complex from the ori and bind to helicase. The helicase-primase complex is called as primosome.
After RNA primer synthesis the beta (β) clamp loaded on DNA with the help of γ clamp loader through the ATP hydrolysis. This result in the increase the affinity of clamp to core polymerase and the loading of core polymerase on DNA then τ binds to other component of DNA polymerase III and facilitated dimerization. This results in assembly of DNA polymerase III holoenzyme on DNA. After binding of polymerase this complex called replisome. As primer get synthesised, primase get replace by core polymerase on leading strand template, this replacement occur every time for each okazaki fragments.
DNA polymerase of leading strand move continuously along the template but the DNA polymerase of lagging strand can not move continuously thus creating a loop in the DNA. The single-stranded template must extend for the length of at least one Okazaki fragment before the lagging polymerase finish one fragment and is prepared to begin the next. DnaB need to be present on lagging strand template and moving in the same direction in which the replicating fork is moving. This attaches the primosome to the lagging strand template because it is needed for priming Okazaki fragment synthesis every time.
In elongation DNA polymerse polymerise the DNA strand by the addition of dNTP through same mechanism.
In prokaryote primer get remove by DNA polymerase I. DNA polymerase I binds at 3’ OH of previous okazaki tragment and cause the displacement of RNA base of primer by its 5’-3’ exonuclease activity. Polymerase I also add new dNTPs at the 3' on end of okazaki fragment by its 5’-3’ polymerisation activity. The last phosphodiester bond is bond by DNA ligase. In case of both leading and lagging strand and then synthesis of leading and lagging strand complete.
Tesmination of DNA replication in Escheria Coli. is completed through the use of termination sequences and Tus protein. Termination takes place within 350 bp region when both replication fork approach each other at termination sequence which are characterize by the presence of unidirectional Ter sites or Terminator sites (Ter A –Ter I and Ter K) of ~23 bp. These Ter site recognize by unidirectional Tus (terminus utilization substance). Ter site present in two clusters and positioned on 100 kb on either side of this termination region and each cluster of ter site specific for one direction of fork movement thus allow the movement of replication fork of one direction and inhibit the other. At the time of termination each side of ter cluster block the movement of opposite specification replication fork this arrangement generate a "replication fork trap”. Thus Tus is contra helicase in nature Tus prevent the replication fork from proceeding by directly interacting with helicase. Tus protein stop the movement of helicase (DnaB). In one cluster Ter A, Ter E, Ter D, Ter I, Ter K Ter present and in another cluster Ter C, Ter B, Ter F, Ter H, Ter G ter site present. Thus leaves the two daughter duplexes entangled. They must become disentangled before cell division occurs.
The two daughter duplexes remain in Interlink form due to circular nature of DNA in bacteria after termination, this form called as catenane. They must be unlinked, to move to the two daughter cells, unlinking process known as decatenated. If decatenation occurs before repair synthesis, a single nick will be sufficient to disentangle both the DNA and type I topoisomerase use to perform decatenation before repair synthesis and after repair type II topoisomerase required to disentangle. It is proved that Top IV is actually decatenating enzyme and is inhibited by norfloxacin.
1.5.1. Control of Initiation of Replication
Two protein DnaA and SeqA (sequestration protein) responsible for control of initiation of replication. It is maintaining one replication per cell division i.e. one initiation of replication per replication. [To regain full methylation state origin takes 10 to 15 minutes, so it is slow process. For prevention of reinitiation depends on above mention two protein].
On DnaA level: DnaA act as initiator and active when present with ATP. Once act as initiator convert in DnaA-ADP which is inactive initiator and to be convert in active form, it is time taken because its ADP get converted into ATP by membrane phospholipid which is acidic in nature, so it is slow reaction thus the replication prevented by reinitiation because newly synthesis DNA is hemimetylated and DnaA require fully methylated DNA to act as initiator. In chromosome dak locus present, which is another DnaA binding sites thus concentration of DnaA reduce according to binding site and not present in adequate active amount to initiate replication. DnaA has a weak intrinsic activity that converts the ATP to ADP, Had factor responsible for enhancement of intrinsic activity but it is recruited by 13 mer sequence when whole replication machinery assemble and origin get activated, at that time Had switch off DnaA thus preventing second round of replication because time is required is DnaA get on. The promoter of the dnaA gene also act like one other oriC , but due to new strand get synthesis and its Hemimethylated and at this situation dnaA promoter is repressed in turn reduction in the level of DnaA protein. Membrane-associated inhibitor binds to hemimethylated origin and get displaced by DnaA when origin becomes fully methylated. By all these reason DNA replication prevented.
On SeqA (sequestration protein): SeqA protein binds to GATC sequence at hemimetylated state, at this condition GATC site which subjected to methylation reduced, due to its binding in proximal oriC site inhibiting binding of DnaA to oriC.
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.