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Course : PGPathshala-Biophysics

Paper 11 : Cellular and Molecular Biophysics

Module 29 : Dna Replication Cell Cycle And Mechanism Of Cell Division Content Writer: Dr. Subhradip karmakar, AIIMS, NEW DELHI

ABSTRACT

DNA is replicated during the "S" stage of the cell cycle. DNA replication is necessary for both Mitosis and meiosis and signifies the production of identical DNA helices from a single double-stranded DNA molecule. Each molecule consists of a strand from the original molecule and a newly formed strand. Prior to replication, the DNA uncoils and strands separate. The three steps in the process of DNA replication are initiation, elongation and termination. A replication fork is formed which serves as a template for replication. Primers bind to the DNA and DNA polymerases add new nucleotide sequences in the 5′ to 3′ direction. This addition is continuous in the leading strand and fragmented in the lagging strand. Once elongation of the DNA strands is complete, the strands are checked for errors, repairs are made, and telomere sequences are added to the ends of the DNA.

1. OBJECTIVE

The genetic material Structure of DNA Details of replication Replication Enzymes Prokaryotic replication Eukaryotic replication

2. INTRODUCTION

DNA was the material responsible for genetic inheritance came into existence much later, previous thoughts support that proteins may have controlled the genetics of living things since there were so many different expressed traits. DNA is a very simple molecule in its structure, so how could something so simple be responsible for so many differences in life on Earth? It was only logical to conclude that it were the numerous proteins that controlled genetics.

It was not always known that deoxyribonucleic acid (DNA) was the genetic material for all living things. In fact, this is a fairly recent discovery in the history of science. It was not until the mid-1900s that enough about DNA was known to build its structure.

The first question arises, whether the DNA duplication was semi-conservative or conservative; the evidences were based on

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2.1 The Meselson and Stahl experiment,

2.2 Semi-conservative-one strand from parent in each new strand 2.3 Conservative-both strands from parent and other is all new strands

The complimentary base pairing produces semi-conservative replication process states that

2.4 Double helix unwinds

2.5 Each strand acts as a template

2.6 complementary base pairing ensures that T signals addition of A on the new strand and G signals addition of C and

Two daughter helices are produced after replication

The DNA replication can be described by three possible models Semi-conservative replication-Watson and crick model Conservative replication

-The parent double helix remains intact

-Both strands of daughter double helix are newly synthesized Dispersive replication

-At completion, both strands of double helices contain both original and newly synthesized material.

Fig1 The three possible models of DNA replication

The Meselson-Stahl experiments confirms the semi-conservative replication When bacteria grown in 15N were transferred to normal 14N containing

medium,

-The newly synthesized DNA strand had the 14N while the parental

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strand had 15N.

They checked the composition of the resulting DNA molecules by density gradient

centrifugation,

 found an intermediate band,

 indicating a hybrid molecule

 containing both 14N and 15N DNA.

Fig 2 The Meselson-Stahl experiment(adapted from The McGraw Hill

Companies Inc.)

3.1 DNA IS THE GENETIC MATERIAL

In 1952, Alfred Hershey and Martha Chase conducted a series of experiments that finally proved that it was DNA and not proteins that controlled inheritance of traits.

The experiment used DNA with radioactively labelled phosphorus and proteins with radioactively labelled sulphur. A bacteriophage was used to infect regular bacteria and through the process of transformation incorporate its genetic material into the bacteria. The phosphorus tracer was found in the transformed bacteria, but there was no sulphur tracer found. Therefore, it was concluded that DNA had to be the genetic material, and not proteins.

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3.2 FINDING THE STRUCTURE OF DNA

Once DNA was proven to be the genetic material, the race was on to be the first to figure out its structure. Even though DNA had been known to exist since the mid- 1800s, it was not extensively researched because of its relatively simplicity.

However, once Hershey and Chase published the results of their experiments, scientists across the globe turned their attention to figuring out more about the mysterious simple molecule that could create so much diversity in life on Earth.

Erwin Chargaff was the first to figure out the rules of nitrogen base pairing that helped lead to the discovery of the double helix structure. DNA has a total of four, and only four, nitrogen bases. These four bases are named Adenine (A), Cytosine (C), Guanine (G), and Thymine (T). The structure of Adenine and Guanine are similar in that they both have two rings of carbons. These were designated as purines. Likewise, Thymine and Cytosine are alike in that they have only one ring of carbon. These bases were called pyrimidines.

Through some biochemical analyses of the DNA molecule, Chargaff determined that Adenine was present at nearly the same percentages as Thymine. He also noticed that the percentage of Guanine was extremely similar to the percentage of Cytosine.

From this data, he created what would become known as the base pairing rules. A = T and C = G. He also noted that the percentages of each of the nitrogen bases differed from species to species. This explained how there could be so much diversity in life on Earth even though the DNA molecule was so simple.

James Watson and Francis Crick first proposed that DNA was shaped much like a ladder. The backbone of the DNA was the five carbon sugar called deoxyribose. This would make up the sides of the ladder. They also knew there were phosphate groups coming off of the backbone. The "rungs" of the ladder, they hypothesized, were made up of pairs of the nitrogen bases, either a rung composed of Adenine and Thymine or Cytosine and Guanine. It was very important that the DNA matched up one pyrimidine with one purine to keep the distance between the deoxyribose backbones constant at a total of three carbon rings between them.

However, the discovery of the structure of DNA did not end there. Watson and Crick collaborated with Rosalind Franklin who had been taking X-ray photographs of DNA molecules. Her pictures showed a sort of spiral shape to the molecule. Watson and Crick amended their model to be a sort of "twisted" ladder or even spiral staircase.

This came to be known as the DNA double helix.

3.3 WHY REPLICATE DNA?

DNA is the genetic material that defines every cell. Before a cell duplicates and is divided into new daughter cells through either mitosis or meiosis, biomolecules and organelles must be copied to be distributed among the cells. DNA, found within the nucleus, must be replicated in order to ensure that each new cell receives the correct number of chromosomes. The process of DNA duplication is called DNA replication. Replication follows several steps that involve multiple proteins called replication enzymes and RNA. In eukaryotic cells, such as animal cells and plant cells, DNA replication occurs in the S phase of interphase during the cell cycle. The

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process of DNA replication is vital for cell growth, repair, and reproduction in organisms.

3.4 IMPORTANCE OF REPLICATION

If DNA never replicated, meiosis and mitosis would slowly halve the size of the genome until each cell would die, which probably would not take long. Therefore, it is important that DNA doubles itself to account for the cells splitting during mitosis/meiosis. DNA replication is similar to RNA transcription.

3.5 HOW FAST DO DNA REPLICATE?

Eukaryotic human DNA replicates at a rate of 50 nucleotides per second. In both cases, replication occurs so quickly because multiple polymerases can synthesize two new strands at the same time by using each unwound strand from the original DNA double helix as a template.

3.6 DNA STRUCTURE

DNA or deoxyribonucleic acid is a type of molecule known as a nucleic acid. It consists of a 5-carbon deoxyribose sugar, a phosphate, and a nitrogenous base.

Double-stranded DNA consists of two spiral nucleic acid chains that are twisted into a double helix shape. This twisting allows DNA to be more compact. In order to fit within the nucleus, DNA is packed into tightly coiled structures called chromatin.

Chromatin condenses to form chromosomes during cell division. Prior to DNA replication, the chromatin loosens giving cell replication machinery access to the DNA strands.

Fig. 3 a)Structure of DNA( cartoon representation) b) Unlinked DNA under Electron Microscope

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3.8 PREPARATION FOR REPLICATION

DNA replication is copying genetic information for transmission to the next generation. replication occurs in S phase of cell cycle and signifies the process of DNA duplicating itself. this begins with unwinding of the double helix to expose the bases in each strand of DNA. Each unpaired nucleotide attracts a complementary nucleotide from the medium and forms base pairing via hydrogen bonding. the crucial role is played by the enzymes, which link the aligned nucleotides by phosphodiester bonds to form a continuous strand. The mechanism of DNA replication is a very tightly controlled process and occurs at specific S phase, involves the interplay of proteins and enzymes and requires energy in the form of ATP. the basic components are the template and the primer.

Fig.4 The overview of replication in different phases of cell cycle ( adapted from clinical tools Inc.)

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Fig. 5 Computer artwork of a DNA (deoxyribonucleic acid) molecule during replication. DNA is composed of two strands. Each strand consists of a sugar- phosphate backbone (gray) attached to nucleotide bases. During replication the two strands unwind and separate, forming a replication bubble that enlarges to form a Y- shaped molecule termed a replication fork. It is here that daughter strands form a the parent DNA acts as a template for the construction of a new matching strand In this way the sequence of bases (or genetic information) along the DNA molecule is replicated.(Adapted from EQUINOX GRAPHICS/Science Photo Library)

Step 1: Replication Fork Formation

Before DNA can be replicated, the double stranded molecule must be “unzipped”

into two single strands. DNA has four bases called adenine (A), thymine (T), cytosine (C) and guanine (G) that form pairs between the two strands. Adenine only pairs with thymine and cytosine only binds with guanine. In order to unwind DNA, these interactions between base pairs must be broken. This is performed by an enzyme known as DNA helicase. DNA helicase disrupts the hydrogen bonding between base pairs to separate the strands into a Y shape known as the replication fork. This area will be the template for replication to begin.

DNA is directional in both strands, signified by a 5' and 3' end. This notation signifies which side group is attached the DNA backbone. The 5' end has a phosphate (P) group attached, while the 3' end has a hydroxyl (OH) group attached. This directionality is important for replication as it only progresses in the 5' to 3' direction.

However, the replication fork is bi-directional; one strand is oriented in the 3' to 5' direction (leading strand) while the other is oriented 5' to 3' (lagging strand). The two sides are therefore replicated with two different processes to accommodate the directional difference.

Step 2: Primer Binding

The replication begins, the leading strand is the simplest to replicate. Once the DNA strands have been separated, a short piece of RNA called a primer binds to the 3' end of the strand. The primer always binds as the starting point for replication.

Primers are generated by the enzyme DNA primase

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Fig 6. DNA polymerases (blue) attach themselves to the DNA and elongate the new strands by adding nucleotide bases.(Adapted from BSIP/UIG)

Step 3: Elongation

Enzymes known as DNA polymerases are responsible creating the new strand by a process called elongation. There are five different known types of DNA polymerases in bacteria and human cells. In bacteria such as E. coli, polymerase III is the main replication enzyme, while polymerase I, II, IV and V are responsible for error checking and repair. DNA polymerase III binds to the strand at the site of the primer and begins adding new base pairs complementary to the strand during replication.

In eukaryotic cells, polymerases alpha, delta, and epsilon are the primary polymerases involved in DNA replication. Because replication proceeds in the 5' to 3' direction on the leading strand, the newly formed strand is continuous

Fig. 7. This is an illustration of a DNA polymerase molecule. DNA polymerase is an enzyme that synthesizes DNA. Callista Image/Cultura

The lagging strand begins replication by binding with multiple primers. Each primer is only several bases apart. DNA polymerase then adds pieces of DNA, called Okazaki fragments, to the strand between primers. This process of replication is discontinuous as the newly created fragments are disjointed.

Step 4: Termination

Once both the continuous and discontinuous strands are formed, an enzyme called exonuclease removes all RNA primers from the original strands. These primers are then replaced with appropriate bases. Another exonuclease “proofreads”

the newly formed DNA to check, remove and replace any errors. Another enzyme called DNA ligase joins Okazaki fragments together forming a single unified strand.

The ends of the linear DNA present a problem as DNA polymerase can only add nucleotides in the 5′ to 3′ direction. The ends of the parent strands consist of repeated DNA sequences called telomeres. Telomeres act as protective caps at the end of chromosomes to prevent nearby chromosomes from fusing. A special type of DNA polymerase enzyme called telomerase catalyzes the synthesis of telomere sequences at the ends of the DNA. Once completed, the parent strand and its complementary DNA strand coils into the familiar double helix shape. In the end,

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replication produces two DNA molecules, each with one strand from the parent molecule and one new strand.

4. REPLICATION ENZYMES

DNA replication would not occur without enzymes that catalyze various steps in the process.the multienzyme complex is called Replisome. Enzymes that participate in the eukaryotic DNA replication process include:

DNA helicase - unwinds and separates double stranded DNA as it moves along the DNA. It forms the replication fork by breaking hydrogen bonds between nucleotide pairs in DNA.

DNA primase - a type of RNA polymerase that generates RNA primers.

Primers are short RNA molecules that act as templates for the starting point of DNA replication.

DNA Clamp- A protein which prevents elongating DNA polymerases from dissociating from the DNA parent strand

SINGLE-STRAND BINDING (SSB) PROTEINS- Bind to ssDNA and prevent the DNA double helix from re-annealing after DNA helicase unwinds it, thus maintaining the strand separation, and facilitating the synthesis of the nascent strand.

DNA Ligase- Re-anneals the semi-conservative strands and joins Okazaki Fragments of the lagging strand.

Primase- Provides a starting point of RNA (or DNA) for DNA polymerase to begin synthesis of the new DNA strand.

DNA polymerases - synthesize new DNA molecules by adding nucleotides to leading and lagging DNA strands.

Topoisomerase or DNA Gyrase - unwinds and rewinds DNA strands to prevent the DNA from becoming tangled or supercoiled.

Exonucleases - group of enzymes that remove nucleotide bases from the end of a DNA chain.

DNA ligase - joins DNA fragments together by forming phosphodiester bonds between nucleotides

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Fig 8. The functional details of Replisome

5. DIFFERENCE IN EUKARYOTIC AND PROKARYOTIC DNA REPLICATION

Fig 9. The salient differences in the DNA replication in Eukaryotes and Prokaryotes

5.1 REPLICATION IN PROKARYOTES

EUKARYOTES PROKARYOTES

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DNA replication in prokaryotes is semi-conservative, accurate and fast. Replication is an enzymatic process in which synthesis of a daughter or progeny duplex DNA molecule, identical to the parental duplex DNA occurs. Rate of replication in E.Coli (prokaryotic cell) is 1500 nucleotides per second. To complete replication of whole E.Coli genome it takes 40 minutes. Rate of replication in eukaryotes is about 10 - 100 nucleotides per second. To complete replication of simple eukaryotic genome 6 - 8 hours required. In prokaryotic circular DNA only one replication fork is present but in eukaryotic DNA several replication forks are present. Space between two- replication forks in eukaryotes is about 20kbps apart.

Fig 9. The DNA replication in Prokaryotes is semi-conservative (adapted from Molecular Biology of the cell; 4th Edition)

5.2 The Replication Of Circular DNA In E.Coli (Prokaryotic Duplex DNA Replication)

The synthesis or replication of DNA molecule can be divided into three stages;

Initiation (Formation of Replisome), Elongation (Initiation of synthesis and elongation) and Termination

Initiation

The replication begins at a specific initiation point called OriC point or replicon.

(Replicon: It is a unit of the genome in which DNA is replicated; it contains an origin for initiation of replication) It is the point of DNA open up and form open complex leading to the formation of pre-priming complex to initiate replication process.

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Fig 10. The OriC site is situated at 74" minute near the ilv gene. The OriC site consists of 245 basepairs, of which three of 13 basepair sequence are highly conserved in many bacteria and forms the consensus sequences (GATCTNTTNTTTT). Close to OriC site, there are four of 9 basepair sequences each (TTATCCACA).

The sequence of reactions in the initiation process is as follows:

DNA protein recognizes and binds up to four 9bp repeats in OriC to form a complex of negatively supercoiled OriC DNA wrapped around a central core of DNA A protein monomers. This process requires the presence of the histone like HU or 1 HC proteins to facilitate DNA bending.

DNA A protein subunits then successively melt three tandemly repeated 13bp segments in the presence of ATP at >=22*C (open complex).

The DNA A protein then guides a DNA B - DNA C complex into the melted region to form a so called prepriming complex. The DNA C is subsequently released. DNA B further unwinds open complex to form prepriming complex.

DNA gyrase, single stranded binding protein (SSB), Rep protein and Helicase - II are bound to prepriming complex and now complex is called as priming complex.

In the presence of gyrase and SSB, helicases further unwinds the DNA in both directions so as to permit entry of primase and RNA polymerase. Then RNA polymerase forms primer for leading strand synthesis while primase in the form of primosome synthesis primer for lagging strand synthesis.

To the above complex, DNA polymerase - III will bind and forms replisome

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Fig. 11.The initiation of priming and replication

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Fig. 12.The interplay of enzymes in Replisome

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Replisome

It is the multiprotein structure that assembles at the bacterial replicating fork to undertake synthesis of DNA. It contains DNA polymerase and other enzymes.

Elongation

The initiation of synthesis and the elongation to proceed. But this occurs in two mechanistically different pathways in the 5'-->3' template strand and 3'-->5' template strand.

6. Initiation of synthesis and Elongation on the 5'-->3' template (synthesis of leading strand) (If replication fork moves in 3'-->5' direction)

The DNA daughter strand that is synthesized continuously on 5'-->3' template is called leading strand. DNA pol-III synthesizes DNA by adding 5'-P of deoxynucleotide to 3'-OH group of the already presenting fragment. Thus chain grows in 5'-->3' direction. The reaction catalyzed by DNA pol-III is very fast. The enzyme is much more active than DNA pol - I and can add 9000 nucleotides per minute at 370C. The RNA primer that was initially added by RNA polymerase is degraded by RNase.

Fig. 13. Synthesis and Elongation on the 5'-->3' template (synthesis of leading strand)

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7. Initiation of synthesis and Elongation on 3'-->5' template when fork moves in 3'-->5' direction (Synthesis of lagging strand)

The daughter DNA strand which is synthesized in discontinuous complex fashion on the 3'-->5' template is called lagging strand. It occurs in the following steps:

Synthesis of Okazaki fragment- To the RNA primer synthesized by primosome, 1000-2000 nucleotides are added by DNA pol-III to synthesis Okazaki fragments.

Excision of RNA primer- When the Okazaki fragment synthesis was completed up to RNA primer, then RNA primer was removed by DNA pol - I using its 5'-->3' exonuclease activity.

Filling the gap (Nick translation)- The gap created by the removal of primer, is filled up by DNA pol - I using the 3'-OH of nearby Okazaki fragment by its polymerizing activity.

Joining of Okazaki fragment: (Nick sealing)- Finally, the nick existing between the fragments are sealed by DNA ligase which catalyze the formation of phosphodiester bond between a 3'-OH at the end of one strand and a 5' - phosphate at the other end of another fragment. The enzyme requires NAD for during this reaction.

Fig 14. Synthesis of lagging strand

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Fig 14. Ligation of two DNA fragments 8. Termination

Termination occurs when the two replicating forks meet each other on the opposite side of circular E.Coli DNA. Termination sites like A, B, C, D, E and F are found to present in DNA. Of these sites, Ter A terminates the counter clockwise moving fork while ter C terminates the clockwise moving forks. The other sites are backup sites. Termination at these sites are possible because, at these sites tus protein (Termination utilizing substance) will bound to DNA B protein and inhibits its helicase activity. And DNA B protein released and termination result.

After the complete synthesis, two duplex DNA are found to be catenated (knotted).

This catenation removed by the action of topoisomerase. Finally, from single parental duplex DNA, two progeny duplex DNA synthesized.

Fig 15. Terminator sequences in E.coli genome

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Fig 16. Synthesis of progeny duplex DNA

9. REGULATION OF PROKARYOTIC REPLICATION

Especially initiation of replication is regulated. DNA A protein when available in high concentration then ratio of DNA to cell mass is quiet high but at low DNA A concentration, the ratio found to be low. This shows that DNA protein regulates the initiation of replication.

The sequence most commonly methylated in E.Coli is GATC including in three of 13mer sequence. Thus, the observation that E.Coli defective in the GATC methylation enzyme are very inefficiently replicated, suggests that the DNA replication trigger also responds to the level of OriC methylation.

10. OTHER MODELS FOR CIRCULAR DNA REPLICATION

There are two such models are available namely Rolling Circle and D-loop models.

10.1. Rolling circle model OR -replication

In the rolling circle model of replication, a nick is made in one of the strands of the circular DNA, resulting in replication of circle and a tail. This form of replication occurs in the F plasmid or E.Coli Hfr chromosome during conjugation. The F+ or Hfr cell retains the circular daughter while passing the linear tail into the F- cell, where replication of tail takes place. This method is also used in several phages (Viruses), which fill their heads with linear DNA replicated form a circular parent molecule. In some cases, because there is no termination point, synthesis often continues beyond a single circle unit, producing concatamers i.e., a series of linked chains, of several circle lengths, which are then processed by recombination to yield normal length circles.

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Fig 17. Rolling circle model

10.2. Displacement loop or D-Loop model

Chloroplasts and mitochondria in eukaryotic cell have their own circular DNA molecules that appear to replicate by a slightly different mechanism than those described. The origin of replication is at a different point on each of the two parental template strands. Replication begins on one strand, displacing the other while forming a displacement loop or D-loop structure. Replication continues until the process passes the origin of replication on the other strand. The newly synthesized strand, known as leading strand. Replication is then initiated on the second strand in the opposite direction which is the lagging strand. When leading strand completely replicated, only 1/3 of lagging strand is replicated. Then finally the result is two circles

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Fig 18. Mitochondrial DNA replication starts at D loop

or Fi X174 BACTERIOPHAGE REPLICATION

X174 Bacteriophage contains a small single-stranded circular DNA. It occurs in two stages namely formation of Replicative form and formation of '+' strand.

11. Synthesis of '-' strand or Replicative form I (RFI) This occurs in six steps

To the (+) strand, PriA, PriB and PriC (formerly known as n', n, and n'' respectively) bound to the pas (Primosome assembly site) site. It contains 70 nucleotides hairpin. It is present between genes F and G.

DNA B and DNA C complex then bound to the DNA with the help of DNA T protein (formerly known as 'i') in an ATP - requiring process. DNA C then

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released and forming the preprimosome complex. When Primase bound to the preprimosome complex, it is referred as primosome.

Primosome complex moves in 5’-->3' direction on DNA with the help of ATP hydrolysis. Pri A and DNA B aid this function.

During the movement on the '+' DNA strand Primase synthesis RNA Primer.

This function also requires DNA B protein to maintain the DNA template conformation.

Then DNA pol III extends the primers to form Okazaki fragments.

DNA pol I then removes the primers and replaces them with DNA. The nicks are then joined by DNA ligase and supercoiled by DNA gyrase. Thus RF I form of The primosome remains complexed with '+' DNA even in RF I form.

Fig 19. Synthesis of '-' strand

11.1. Synthesis of '+' Strand or Looped Rolling Circle model or Model This occurs in four steps

Gene A protein bound to the 30bp region near the beginning of gene A in RF I with the primosome aiding function. Gene A cleaves the phosphodiester bond and forms a covalent bond between a Tyr residue and the DNA's 5’- phosphoryl group. This reaction is used to conserve the energy present in the phosphodiester bond.

Rep protein binds to (-) strand and commences unwinding the duplex DNA form the 5' end. The binding is aided by the primosome complex. The displaced (+) strand is coated with SSB protein which prevents the recoiling of (+) strand with (-) strand. Rep protein is essential for

unlike E.Coli replication. DNA pol - III then extends the (+) strand from 3' - end.

The extension process generates a looped rolling circle structure in which the 5'end of the old (+) strand remains linked to the gene A protein at the

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replication fork. Primosome found to be attached with the old (+) strand which may used for later generation of

After full circle of (-) strand replicated, Gene A protein make a cut in the (+) strand at the replication fork and joins the old (+) strands and new (+) strands. Because of this, the SSB bound old (+) strand released. At the end of this step, RF I form regenerated and one (+) strand DNA synthesized. RF I is used for the production of more (+) strand and viral proteins where as (+) strand utilized for the formation of new phages.

Fig.20. Synthesis of '+' strand

12. EUKARYOTIC DNA REPLICATION (Replication of Linear DNA)

Eukaryotic replication occurs during s-phase of cell cycle. Replication usually occurs only one time in a cell. Replication in eukaryotes occur in five stages namely,

Pre-initiation Initiation Elongation Termination

Telomerase function

12.1. Pre-initiation

During pre-initiation stage, replicator selection occurs. Replicator selection is the process of identifying the sequences that will direct the initiation of replication and occur in G1 phase(prior to S phase). This process leads to the assembly of a multi- protein complex at each replicator in the genome. Origin activation only occurs after cells enter S phase and triggers the Replicator - associated protein complex to

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initiate DNA unwinding and DNA polymerase recruitment. Replicator selection is mediated by the formation of pre-replicative complexes (pre-RCs). The first step in the formation of the pre-RC is the recognition of the replicator by the eukaryotic initiator, ORC (Origin recognition Complex). Once ORC is bound, it recruits two helicase loading proteins (Cdc6 and Cdtl). Together, ORC and the loading proteins recruit a protein that is thought to be the eukaryotic replication fork helicase (the Mem 2-7 complex). Formation of the pre-RC does not lead to the immediate unwinding of origin DNA or the recruitment of DNA polymerases. Instead the pre- RCs that are formed during Gl are only activated to initiate replication after cells pass from the G1 to the S phase of the cell cycle.

Fig 21. The process of pre-initiation 12.2 Initiation

Pre-RCs are activated to initiate replication by two protein kinases namely Cdk (Cyclin Dependant Kinase) and Ddk (Ddt4 Dependant Kinase). Kinases are proteins that covalently attach phosphate groups to target proteins. Each of these kinases is inactive in Gl and is activated only when cells enter S phase. Once activated, these

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kinases target the pre-RC and other replication proteins. Phosphorylation of these pro-proteins results in the assembly of additional replication proteins at the origin and the initiation of replication.

These new proteins include the three eukaryotic DNA polymerases and a number of other proteins required for their recruitment. Interestingly, the polymerases assemble at the origin in a particular order. DNA Pol and associate first, followed by

DNA Pol DNA polymerases are present

at the origin prior to the synthesis of the first RNA primer (by DNA Pol Once present at the origin, DNA Pol

briefly extends it. Thus initiation of replication started.

Fig 22. The interplay of polymerases and the synthesis of RNA primer

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12.3. Elongation

The resulting primer-template junction is recognized by the eukaryotic sliding clamp loader (RF-C), which assembles a sliding clamp (PCNA) at these sites. Either DNA Pol or e recognizes this primer and begins leading strand synthesis. After a period of DNA unwinding, DNA

the initiation of lagging strand DNA synthesis by either DNA Pol

diagram, Pol was used for lagging strand

synthesis. DNA Pol possess activity to remove primer and fills the gap with DNA like DNA Pol I in prokaryotes. SSB like activity was played by replication protein A (RP A) which is denoted as accessory factors during replication.

12.4. Termination

When the replication forks meet each other, then termination occurs. It will result in the formation of two duplex DNA. Even though replication terminated, 5’ end of telomeric part of the newly synthesized DNA found to have shorter DNA strand than the template parent strand. This shortage corrected by the action of telomerase enzyme and then only the actual replication completed.

12.5. Telomerase Function

In Linear eukaryotic chromosome, once the first primer on each strand is remove, then it appears that there is no way to fill in the gaps, since DNA cannot be extended in the 3'->5' direction and there is no 3' end upstream available as there would be in a circle DNA. If this were actually the situation, the DNA strand would get shorter every time they replicated and genes would be lost forever.

Elizabeth Blackburn and her colleagues have provided the answer to fill up the gaps with the help of enzyme telomerase. So, that the genes at the ends, are conserved.

Telomerase is a ribonucleoprotein (RNP) i.e. it has RNA with repetitive sequence.

Repetitive sequence varies depending upon the species example Tetrahymena thermophilia RNA has AACCCC sequence and in Oxytrica it has AAAACCCC.

Telomerase otherwise known as modified Reverse Transcriptase. In human, the RNA template contains AAUCCC repeats. This enzyme was also known as telomere terminal transferase.

The 3'-end of the lagging strand template base pairs with a unique region of the telomerase associated RNA. Hybridization facilitated by the match between the sequence at the 3'-end of telomere and the sequence at the 3'-end of the RNA. The telomerase catalytic site then adds deoxy ribonucleotides using RNA molecule as a template, this reverse transcription proceeds to position 35 of the RNA template.

Telomerase then translocates to the new 3'-end by pairing with RNA template and it continues reverse transcription. When the G-rich strand sufficiently long, Primase can make an RNA primer, complementary to the 3'-end of the telomere's G-rich strand. DNA polymerase uses the newly made primer to prime synthesis of DNA to fill in the remaining gap on the progeny DNA. The primer is removed and the nick between fragments sealed by DNA ligase.

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Fig 22. Interplay of telomerase enzyme with other enzymes

13. REGULATION OF EUKARYOTIC REPLICATION

The tight connection between pre-RC function, Cdk levels, and the cell cycle ensures that the eukaryotic genome is replicated only once per cell cycle (Figure 8-32).

Active Cdk is absent during Gl, whereas elevated levels of Cdk are present during the remainder of the cell cycle (S, G2, and M phases). Thus, during each cell cycle

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there is only one opportunity for pre-RCs to form during Gl and only one opportunity for those pre-RCs to be activated during S, G2, and M phase, although in practice all pre-RCs are activated or disrupted by replication forks in S phase. Pre-RCs are disassembled after they are activated or after the DNA to which they are bound is replicated. These exposed replicators are then available for new pre-RC formation and rapidly bind to ORC. Despite the presence of the initiator at these sites, the elevated levels of Cdk activity in S, G2, and M phase cells prevents the association of the other members of the pre-RC complex with ORC. It is only when cells segregate their chromosomes and complete cell division that Cdk activity is eliminated and new pre-RC complexes are formed.

Inhibition of replication was achieved by antisense RNA. Once antisense RNA produced for particular gene, it bound with single strand DNA in open complex and inhibits the movement of replisome and thus replication. Antisense RNA synthesized in opposite direction to the normal RNA synthesis.

14. FIDELITY OF REPLICATION

It refers to the précised state or accuracy of replication. The rate of error occurring in E.Coli is one mispairing per 108 to 1010 base pairs replicated. This corresponds to one error per 1000 bacteria per generation. There are four main reasons for this high level of accuracy in replication.

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Cells maintain balanced level of dNTPs, thus each base added correctly to the binding site of the polymerase enzyme. But if one present at low levels then it is more likely to be replaced by the dNTPs present at higher levels.

The catalytic mechanism of action of polymerase itself responsible for high fidelity. This is because polymerization reaction occurs in two stages namely binding and catalysis. In the binding step, nucleotide binds to binding site in the enzyme and it forms Watson-crick base pair with the base in the template and then phosphodiester bond forms between the bases after it forms complementary base pairing. Binding process actually checks the binding of the correct base by checking the base pairing.

The 3'-->5' exonuclease activity of DNA POL I and DNA POL III also detect and eliminate the errors made by their polymerase function during replication.

Normal repair system present in all cells also plays a vital role in the maintenance of DNA.

In addition to the above four major reasons for the fidelity, the two minor reasons also present are Primer requirement and Lagging strand synthesis. Due to cooperative nature of base pairing, first few nucleotides might be added incorrectly.

But due to the presence of primers, this error does not occur because primer is replaced by the DNA later correctly. If DNA polymerase synthesizes DNA in 3'-->5' direction also, then when wrong base pair is added during proof reading function, then polymerase introduce the 5'-OH or 5' - Phosphate terminal region, and then the extension of the end is not possible without reactivation. Thus by forming lagging strand the end extension is possible and proof reading function also occurs normally so it prevents unusual base pairing during replication.

15. SV 40 VIRAL REPLICATION

Replication initiated by the binding of T antigen (Virus encoded protein). This multifunctional protein then unwinds DNA through its helicase activity. This reaction requires ATP. RF A (Replication Factor A), host cell protein then binds to single strands and stabilize it similar to SSB. To the open complex, pol - Primase complex binds and Primase forms RNA primer. This primer elongated by pol using deoxy nucleotides whose activity is stimulated by RF C. PCNA binds to the primer template terminus and displaces pol - Primase complex. Pol then binds to PCNA at the 3' - ends of other growing strand and continues the leading strand synthesis. PCNA was responsible for the processivity of pol As unwinding of the duplex DNA progresses further away from the origin, the Primase - pol complex associates with the unwound template strands downstream from the leading strand primers.

Synthesis of lagging strand then is carried out by primase - pol and RF C. Finally, topoisomerase probably play an important role in relieving tortional stress induced by growing fork movement and in separating the two daughter duplex DNA.

(29)

Fig 23. SV 40 viral replication

16. Summary

DNA replication is a semiconservative process in which each parental strand serves as a template for the synthesis of a new complementary daughter strand. The central enzyme involved is DNA polymerase, which catalyzes the joining of

(30)

deoxyribonucleoside 5′-triphosphates (dNTPs) to form the growing DNA chain.

However, DNA replication is much more complex than a single enzymatic reaction.

Other proteins are involved, and proofreading mechanisms are required to ensure that the accuracy of replication is compatible with the low frequency of errors that is needed for cell reproduction. Additional proteins and specific DNA sequences are also needed both to initiate replication and to copy the ends of eukaryotic chromosomes.

End of Module

References

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