1.1. Meselson and Stahl experiment (1957)
If E. coli cells grown in a medium with the sole nitrogen source (NH4Cl) contained N15, the “heavy” isotope of nitrogen. Density of DNA isolated from N15 containing cells is greater than that of normal N14 containing cell. The mixture of DNA of N14 and N15 can be separated by cesium chloride density gradient (Cscl) centrifugation. This technique was the basic of Meselson and Stahl experiment.
Esheria Coli cells were grown in a medium containing solo heavy nitrogen (N15), thus all the nitrogen in their DNA was (N15) then cellular DNA isolated and subjected to centrifuged in a CsCl density gradient. The band of N15 appear at the Bottom of falcon (tube). After that the cells had been transferred to light nitrogen (N14) containing medium, cellular DNA isolated after one generation from cell grown in N14 containing medium after that subjected to centrifuged in a CsCl density gradient and found to be equilibrated at a higher position in centrifugation falcon. The bybrid band (N15 – N14) is appear in the mid of falcon. To eliminate the confusion that DNA replication is semi conservative or not. Esheria coli cells were again allowed to double in number in the N14 medium for one more generation, thus Esheria coli. cell grown in two generation within in N14 containing medium after that subjected to centrifuged in a CsCl density gradient and equilibrated which show two type of bands, one a higher position in centrifugation falcon then N15 - N14 with a density equal to N14 DNA and another one share the same position like hybrid (N15-N14) DNA. The disappearance of N15 – N15 band indicate that the replication in semi conservative in nature. When the cell was allowed to grow in N14 medium for some more generation the N15 – N15 duplex never observe again.
1.2. DNA structure
DNA is a double stranded helical structure. It is made up of four types of nucleotide. Nucleotides have deoxyribose sugar, a phosphate and nitrogenous base. The four types of bases are adenine (A') guanine (G), Cytosine (C) and thymine (T). DNA is a polymer of these four nucleotides a phosphodiester bond link the adjacent nucleotide in DNA. One strand of DNA binds with other strand of DNA through Hydrogen bonding between complementary base pairs.
Directionality in DNA
In eukaryotes the DNA is linear. Linear DNA has two types of ends 5' phosphate end and 3' on end. In dsDNA, the strands are anti parallel in orientation. Directionality has consequences in DNA synthesis, because DNA polymerase synthesize DNA in 5' – 3' direction.
1.3. ORI (Origin of Replication)
DNA replication starts from unique point called origin of replication (Ori). This is proof by denaturation mapping experiment carried out by Ross Inman and colleagues on bacteriophage scientist proof that initiation of replication loop always start at unique point, which is rich in AT base pair known as ORI (origin of replication).
1.4. Enzymes of DNA replication:
1.4.1. DNA polymerase
DNA polymerase catalyzes the polymerization of deoxy nucleotide tri phosphate (dNTP) and synthesizing a new DNA strand on a template strand. DNA polymerase add new nucleotide at 3' OH of existing nucleotide. Thus addition of new nucleotide at 3' end make the growth of chain in 5' – 3' direction. This 5’- 3’ polymerization activity, is present in all DNA polymerase. DNA polymerase also have some other function like 5’ – 3’ exonuclease activity responsible for removal of nucleotide from polynucleotide strand and 3’ – 5’ exonuclease activity responsible for proofreading to check correct complementary nucleotide addition on newly synthesized strand. All these three activities are not universal in all DNA polymerases. DNA polymerase also known as “Replicases”.
Structure of DNA polymerase with its mechanism of polymerization
All DNA polymerase use common catalytic mechanism to polymerize nucleotide. DNA polymerase structure resemble to right hand of human. It has three domain which resemble like thumb, finger and palm. Catalytic active site is presents in the conserved motifs of palm region. This catalytic active site is composed of β sheet. DNA polymerase binds with two divalent metal ion (Mg+2 and Zn+2) in this region. The mg+2 is helps in the binding between asparate residue and phosphate (PO4–2) of incoming nucleotide. Mg+2 and Zn+2 stabilise the interaction as these ions forms salt bridges.
DNA polymerase exists in open and closed conformation. These conformations are due to change in conformation of O helix of finger domain. When DNA polymerase is not associated with incoming dNTP, polymerase present in open conformation. After reading the template DNA the DNA polymerase recruit its complementary nucleotide. This complementary binding between template DNA and incoming nucleotide allows the finger to close. This is called closed confrmation of polymerase.
Actually "fingers" are also responsible for accurate placing of template at the active site inducing 90° band in backbone of template just after the active site, thus only first template base expose after primer. Metal ion mg+2 binds to 3’OH and get separate the oxygen to hydrogen O to H by reducing their affinity and create 3’O-, thus it acts as nucleophili and attack on α phosphate of incoming dNTP. Metal ion Zn+2 make interaction with triphosphate of dNTP and it is responsible to neutralize negative charge of dNTP phosphates and stabilize pyrophosphate (β and γ phosphate). The newly form DNA when exit the enzyme binds with thumb region and thumb region play important role in possessive reaction, for accurate positioning of primer and active site and strong union between polymerase and its substrate.
The domain palm interact with the minor groove of recently added base pair by hydrogen bonding. This interaction occur only if correct nucleotide is added. If wrong nucleotide added then it causes a distortion of the helix and result in slow catalysis is reduced. The rate of phosphodiester bond formation siqniticantly when wrong nucleotide is added. This wrong nucleotide move towards proofreading site and incorrect nucleotide is removed by 3’- 5’ exonuclease activity. The exonuclease activity is located in an independent domain with its own catalytic site.
Processivity is a tendency of DNA polymerase to remain associate with DNA template rather than dissociate and reassociate. High processivity of DNA polymerase make it more reliable. The speed of DNA replication depends upon processivity. The β-clamp enhance the processivity of DNA polymerase III. The DNA polymerase III has less tendency to get dissociate from template DNA strand due to β–clamp. β–clamp is a clamp like structure, which allows the binding between DNA template and DNA polymerase III.
220.127.116.11. Fidelity (reliability/ accuracy) of DNA Replication
DNA replication is very accurate proves. How ever the accuracy is not 100%. Now LET'S TALK about the addition of one wrong nucleotide. Different event and how the accuracy increases due to different mechanism responsible for correct addition of nucleotide at the fidelily of DNA replication has three steps.
- Due to thermodynamics of base pairing one wrong nucleotide added per 102
- Base selection by polymerase one wrong nucleotide added per 105.
- Proofreading by polymerase one wrong nucleotide added per 107
- Post-replicative mismatch repair one wrong nucleotide added per 1010 nucleotide. Mismatch repair last event to check and correct the addition of wrong nucleotide and due to this event accuracy increase enormously. The difference in free energy between correct (G-C and A-T) base pairing and incorrect (G-T for example) predicts 1/100 mistakes.
18.104.22.168. Family of DNA polymerase
There are seven different family of DNA polymerase A, B, C, D, X, Y, and RT.
22.214.171.124. DNApolymerase in Prokaryotes
There are five DNA polymerase. Palm is conserve in all families but finger and thumb present as similar secondary structure elements from different sequences.
DNA polymerase I:
It was isolated by Arthur Kornberg in 1956 from E.Coli. It has single subunit and encoded by polA gene. It is the first DNA polymerase to be identified. Polymerase I has three activities.
- 5’ –3’ exonuclease activity
- 3’-5’ exonuclease and
- 5’ –3’ polymerase activities.
When klenow treated the pol I with proteases. Pol I is cleaved by proteases such as subtilisin or trypsin it gives two fragments:
A larger C- terminal or Klenow fragment (residues 324–928 and mw 68 KD), has both the polymerase and the 3’-5’ exonuclease activities.
N-terminal fragment (residues 1–323 and mw 35 KD) smaller one, has the 5’–3’ exonuclease activity. Thus Pol I contains three active sites on a single polypeptide chain. It has 5 '-3' exonucleolytic activity coordinated with the synthetic/proofreading activity. Polymerization rate of Pol I is 10 to 20 nucleotides per second. Role of DNA polymerase in recombination, nick translation, excise RNA Primers and repair.
Nick translation during DNA repair and in vitro is mediated by 5' - 3' exonuclease activity. This is also called as forward exonuclease activity. DNA polymerase I is able to start replication in vitro at a nick in DNA. Nick is a breakage of phosphodiester bond. Nick produces two types of ends 5' phosphate and 3' OH ends. The enzyme extends the 3–OH end at a point where a phosphodiester bond has been broken in a double-stranded DNA and the new segment of DNA is synthesized by move the existing homologous strand in the duplex. Thus one strand of a duplex DNA can be degraded and change by resynthesis of new material also used to introduce radioactively labeled nucleotide into DNA in vitro.
DNA polymerase II :
It has seven subunit and encoded by polB gene. DNA polymerase II play role in DNA repair, when replication fork progress is blocked due to damage in DNA.
DNA polymerase III:
It has nine subunit and encoded by pol C gene. It is a main replicating enzyme in E. coli with dimeric structure. DNA polymerase III core enzyme contain a catalytic core which contains alpha (α) subunit responsible for DNA polymerase activity, Epsilon (ε) subunit responsible for 3’- 5’ exonuclease proofreading activity and theta (θ) subunit which stimulates 5' – 3' activity of E submit. DNA polymerase III holoenzyme contain a dimerization subunit tall (τ) which link the two catalytic cores together, a processivity component (the β clamp) and core enzyme in its each monomeric unit. DNA polymerase III holo enzyme also contain a clamp loader which remain associated with its one monomeric subunit.
Two copies of clamp present in Pol III, also known as β sliding clamp which is responsible for holding each catalytic cores on to their template strands. β clamp is homodimer. b clamp is crucial for processivity. The γ clamp loader is require to load b clamp on DNA strand at the replication fork and it is a group of 5 proteins (δδ’χγΨ) or heteopentameric and function in ATP dependent manner. Firstly γ loader binds to 3’ end of DNA and then it open β sliding clamp to encircle DNA in ATP dependent manner. Polymerisation rate of Pol III 1000 nucleotides per second.
DNA polymerase IV and V:
Pol IV has one subunit and encoded by dnaB and Pol V has three subunit and encoded by UmuD'2C or UmuC, both comes from Y family of polymerase. Both of them has low fidelity on undamaged templates and they are able to replicate through damaged DNA. Thus members of this family known as translesion synthesis (TLS) polymerases or error-prone polymerases because allowing replication to bypass certain types of damage. Family of polymerase are error prone because 3' – 5' exonuclease activity is absent.
126.96.36.199. DNA polymerase in eukaryotes
Eukaryotic cells have approximately 15 types of DNA polymerases. However only three three DNA polymerases α alpha, δ delta, and ε epsilon required for nuclear DNA replication. DNA polymerase γ gamma require for mitochondrial DNA replication. Other DNA polymerase associated with repair and translession synthesis (TLS). For example DNA polymerase eta (Pol η) take part in the DNA repair by translesion DNA synthesis. DNA polymerase eta is also known as XPV. A mutation and loss of this gene causes the Xeroderma Pigmentosum. Xeroderma pigmentosum mutation in polymerase eta (h) causes loss of DNA repain, when the damage is caused by Ultra Violet light. When the eoisesmal cells of skin get exposed to Ultra Violet light blister appear on skin.
DNA polymerase Alpha (Pol α)
In prokaryotes the DNA replication is initiated by RNA polymerase however in eukaryotes the DNA replication is initiated by DNA polymerase a. It is responsible for the initiation of DNA replication at autonomously replicating sequence (ARS) both is leading and lagging strand of DNA. The okazaki fragment required DNA polymerase again and again.
Polymerases alpha has four subunit in which two subunit function as primase so that primase from outside not require and other two subunit act as DNA polymerase. DNA polymerases α has DNA binding domain. Pol α after synthesizing primer of 10 base, it also synthesized 20-30 nucleotide of DNA. The DNA synthesis by polymerase a is know as iDNA. α get replace by leading polymerase δ in lagging strand and poly e (epsilon) in leading strand. This phenomenon of pol replacement is called pol switching.
Proliferating Cell Nuclear Antigen (PCNA) :
It is a homotrimeric and initially recognized as an antigen which is expressed during the DNA synthesis phase or S phase of the cell cycle within the nuclei of cells thus called PCNA. PCNA act as processivity factor or DNA clamp for DNA polymerases in eukaryotes. Replication factor C (RFC), which is a heteropentameric (RFC 1–5) act as loader of PCNA. PCNA and RFC analogue of β sliding clamp and γ loader respectively. During DNA damage, PCNA is involved in the RAD6-dependent DNA repair pathway and get ubiquitinated. PCNA along with δ take part in repair especially post-replication repair.
DNA polymerase can not initiate the DNA replication done. It require a 3'oH to add deoxy ribohucleotide (dNtPs). That means to "prime" a reaction of polymerization of monomer, the DNA polymerase synthesize a short segment (sequence) of RNA that work as primer. Primase is RNA polymerase. Thus DNA polymerase themselves cannot initiate DNA synthesis de novo. Primase is encoded by dnaG gene in prokaryotes. In leading strand only one primer requires but each okazaki fragments has its own primer. In eukaryotes Pol α act as primase.
Helicases is hexameric ring shape protein which is mechanically separate the two strands of double stranded nucleic acid by translocation on one strand in ATP dependent manner. Helicase translocate on DNA strand in particular direction which is popularly known as polarity of helicase means either move in 5’ – 3’direction or 3’-5’ direction. Helicase functions in various event like DNA replication, recombination, repair, transcription termination, RNA splicing and RNA editing. Mechanism used by helicase to perform its function known as kinetic selectivity.
Super families of helicases
Helicase loader transfer the helicase to replication origin. These helicase loader break the ring of hexamer helicase and allow the DNA to pass. DnaC act as halicase loader in prokaryotic and Cdc6 act as halicase loader in eukaryote. These proteins load the helicase on DNA. In yeast, Cdc6 is a highly unstable protein and get degraded rapidly with a half-life of <5 minutes. Cdt 1 prevent its degredation. Cdt-1 is conserved from yeast to humans. Examples of orthologs of Cdt1 in S. pombe is cdt1 (cdc10-dependent transcript 1), in Drosophila melanogaster is 'double parked' or Dup, in Xenopus laevis is Cdt1. Licensing factor are protein which provide license for DNA replication of start (just like driving license to drive a motor). The origin of replication are required to start replication once per cell cycle. The start of replication is known as firing. This lead to development of an idea that licensing factor do exist in the cell. These factors are synthesised in cytoplasm and transported to nucleus.
The Cd C-6 and Cdt-1 are synthesized in G1 stage of cell cycle. These proteins bind with origin recognition complex (ORC) and fored pre-replication complex. This allows the binding of MCM.
In S phage the CDK-cyclin complex phosphorylates the Cd C-6. The phosphorylated CD C-6 is transported out of nucleus and degraded by Ubiquition proteasome pathway. Thus Cd t-1 and CD6-6 act as licensing factor
1.4.4. Single-strand binding protein
Helicase unwinds double stranded DNA two single stranded DNA and those single stranded regions of DNA get prevented from annealing by binding of single-strand binding protein, (SSB) SSB because separated strand having tendency to get reassociate and form duplex thus allowing the DNA replication machinery to perform its function. SSBP present in viruses to humans. In Escheria coli SSB present in homo tetrameric form.
In high salt concentration tetrameric form SSB65 binds to approximately 65 nucleotide of DNA, in contrast low salt concentration dimeric form (SSB)35 binds to 35 nt DNA. The SSB binds in a cooperative manner means binding of one monomer facilitate the binding of second monomer and further so no. In eukaryotes SSB present in trimeric form known as heterotrimeric RPA (Replication Protein A). SSBs function as monomers in many phage and virus.
1.4.5. DNA ligase
Once the RNA primer has been removed and replaced by DNA through Pol I, then with the help of DNA ligase phosphodiester bond is formed between the adjacent Okazaki fragments One okazaki fraginent has 3' oH end and other fregment of okazaki has 5' phosphate end. These two ends are seal by DNA ligase. Nick remains in leading strand sealed by DNA ligase. DNA ligase also requires energy to make phosphodiester bond. NAD (nicotinamide adenine dinucleotide) work as a cofactor and source of energy for DNA ligase in Escheria coli whereas ATP used by T4 DNA ligase. DNA ligases are present in both prokaryotes and eukaryotes.
Both enzymes undertake a two step reaction in first step DNA ligase interact with ATP and form an enzyme-AMP complex. The AMP in the enzyme-AMP complex becomes attached to the 5 ' phosphate of the nick, and then a phosphodiester bond is formed with the 3'-0H terminus of the nick, releasing the enzyme and the AMP.
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.