3. DNA Replication – Essentials of Molecular Biology

3

DNA Replication

CONCEPT OUTLINE
  • Introduction
  • Chemistry of DNA Synthesis
  • Modes of DNA Replication
  • Semi-conservative Mode of Replication/Meselson and Stahl Experiment
  • Enzymes of Replication
    • Prokaryotic DNA polymerases
    • Helicases
    • Primases
    • Single-strand binding proteins
    • Ligases
    • Topoisomerases
  • Models of Replication
    • θReplication
    • Rolling circle replication
    • Bacteriophage ϕ ×174 replicates by rolling circle replication
    • D-loop replication
  • Prokaryotic Replication
    • Origin of replication
    • Initiation of DNA replication
    • Fidelity of DNA replication
  • Eukaryotic Replication
    • Cell cycle
    • Cell cycle control of DNA replication
    • Eukaryotic replication origins
    • Initiation of DNA replication
    • Elongation
    • Licensing of DNA replication
    • Termination of replication
    • Telomeres and telomerases
  • Inhibitors of Replication
    • Inhibitors of nucleotide biosynthesis
    • Inhibitors of purine biosynthesis
    • Analogues of purine and pyrimidine bases
    • Inhibitors of folate synthesis
    • Inhibitors of deoxynucleotide synthesis
    • Catabolite analogs
    • Inhibitors that modify DNA
    • Inhibitors that affects enzyme of replication
  • Summary
  • References
INTRODUCTION

DNA, the molecular chip of a cell, occupies a unique place among all the biological macromolecules, because it is considered the blueprint of an organism. The nucleotide sequence of DNA carries all the necessary information for forming the primary structures of all cellular RNAs and proteins. By encoding the enzymes, DNA indirectly affects the synthesis of all other cellular constituents. The information stored in DNA in the form of nucleotide sequence is transmitted from one generation of cells to the next without any error and in an uncorrupted state. The formulation of the structure of DNA by Watson and Crick in 1953 was accompanied by a proposal for its self-duplication. The process of DNA duplication is called ‘replication’, a biological process that occurs in all living organisms prior to cell division. The process of replication results in the production of two identical daughter DNAs originally copied from the parent DNA. Each strand of DNA double helix can serve as template for the synthesis of a new strand to form duplicated DNA. Watson and Crick proposed that the replication of DNA involves breakdown of weak hydrogen bonds that hold the duplex together and the process is followed by rotation and separation of both the polynucleotide strands. Each separated strand acts as a template as it contains the information required for the synthesis of the other strand. Each base of the template strand attracts the complementary nucleotide available within the cell and thus produces the replicas of the double helical molecule.

CHEMISTRY OF DNA SYNTHESIS

Copying of a DNA template strand into a complementary strand is a common feature of DNA replication. The polymerization of the new DNA strand is brought about by an enzyme called DNA polymerase. The building blocks added to a growing daughter strand are individual nucleotides. The substrates for DNA syn thesis are 2′-deoxyribonucleotides.

 

 

The addition of the nucleotides occurs via a nuclephilic attack. The 3′-OH at the growing DNA chain attacks the alpha phosphate group of an incoming nucleotide. This results in the formation of the phosphodiester bond between the growing daughter strand and the next nucleotide. The 3′-OH of the newly added nucleotide is now exposed on the end of the growing chain and can attack the next nucleotide in the same way (Figure 3.1).

 

Figure 3.1 Addition of nucleotides to a growing daughter strand. Schematic representation of growing polynucleotide chain. The lines represent the ribose sugar with 3′-OH; ‘P’ represents a phosphate group

 

The high-energy bond between the β- and γ-phosphates of the incoming nucleotide breaks and provides the necessary energy for its addition to the nucleic acid chain. The subsequent hydrolysis of the pyrophosphate drives the reaction in the forward direction.

MODES OF DNA REPLICATION

The biological information stored in the DNA must be exactly replicated and transmitted to the daughter cells. There are three modes of accomplishing this task.

They are conservative, dispersive and semi-conservative mode of replication (Figure 3.2).

 

Figure 3.2 Possible modes of DNA replication

 

  • Conservative replication: The original DNA molecule remains intact and a completely new daughter DNA molecule is generated.
  • Dispersive replication: It produces two DNA molecules with sections of both old and new DNAs interspersed along each strand.
  • Semi-conservative replication: Each strand of the parent DNA acts as the template for the synthesis of complementary daughter DNA. Thus, this mode of replication produces molecules with both old and new DNAs composed of one old strand and one newly synthesized strand.
SEMI-CONSERVATIVE MODE Of REPLICATION/MESELSON AND STAHL EXPERIMENT

According to the proposal made by Watson and Crick for DNA duplication, once DNA replication is initiated, both the old strands of the duplex serve as templates that direct the synthesis of new complementary strand. Thus, each daughter DNA retains half of the parental DNA, i.e., the replication is semi-conservative.

 

Figure 3.3 Semi-conservative replication-Messelson and Stahl experiment (a) Bacteria grown in 15N and 14N medium (b) Semi conservative replication

 

In 1958, Matthew Meselson and Franklin Stahl of California Institute of Technology confirmed the semi-conservative nature of DNA replication in bacteria using radioisotopes through a series of experiments. They grew E. coli cells for many generations in media containing 15N-ammonium chloride as the sole source of nitrogen. As the result, the nitrogen bases of the DNA of these bacterial cultures contained only the heavy nitrogen isotope. The cells were then transferred to a new medium containing the normal lighter isotope of nitrogen 14N. At various time after the transfer, bacterial cells were collected. The DNA was then extracted from the bacterial cells. The extracted DNA was loaded on a caesium chloride density gradient and subjected to equilibrium density gradient centrifugation. The DNA molecules moved on the gradient until their density matched with that of the gradient. The DNA bands were visualized under UV light and photographed. DNA containing 14N sank to a position determined by its density. DNA containing 15N is denser than 14N hence sank to a lower position in caesium chloride. After one generation in 14N medium, the bacteria yielded a single band of DNA with the density between that of 14N and 15N DNA, indicating that only one strand of the duplex contained 15N (Figure 3.3).

After two generations in 14N medium, two bands were obtained, one of intermediate density (in which one strand contained 15N) and one band of low density (in which neither strand contained 15N). Meselson and Stahl concluded that DNA replication involves building new molecules by separating parental strands and then adding new nucleotides to form the complementary strands on these templates.

ENZYMES Of REPLICATION

Prokaryotic DNA Polymerases

DNA polymerases are the enzymes responsible for the synthesis of DNA during replication.

There are three types of prokaryotic DNA polymerases; namely, DNA polymerase I (DNA Pol-I), DNA polymerase II (DNA Pol-II) and DNA polymerase III (DNA Pol-III).

DNA Pol-I

It is the first known DNA polymerase, which was discovered by Arthur Kornberg in 1956 and hence also called Kornberg enzyme. It is a monomeric 928-residue polypeptide. It couples dNTPs on DNA template in a reaction that occurs through the nucleophilic attack of the growing DNA chains 3′-OH on the α-phosphoryl group of the incoming nucleotide. The reaction is driven by the elimination of the pyrophosphate and its subsequent hydrolysis. The error rate of polymerase I in copying the template is very low about one wrong base pair per 10 million. Polymerase I is a ‘processive’ enzyme, i.e., it catalyses a series of successive polymerization steps without releasing from the template. Polymerase I selects an incoming nucleotide according to its ability to form Watson–Crick-shaped pair with the template (Figure 3.4).

Polymerase I has three important functions; namely:

  • 5′ → 3′ Polymerization activity,
  • 3′ → 5′ Exonuclease activity and
  • 5′ → 3′ Exonuclease activity.

If polymerase I erroneously incorporates a wrong nucleotide at the end of a growing chain, the polymerase activity is inhibited and 3′ → 5′ exonuclease activity excises the incorrect nucleotide. This is called ‘proofreading activity’.

 

Figure 3.4 Replication by DNA polymerase

Catalytic mechanism

Active site of DNA Pol-I has a shape complementary to the Watson–Crick base pairs. Although double-stranded DNA is mainly in the B conformation, the three base pairs near the active site assume the A conformation. The resulting wider and shallower minor groove permits protein side chains to form hydrogen bonds with the otherwise inaccessible N3 atoms of the purine bases and O2 atoms of the pyrimidine bases. DNA polymerases share a common catalytic mechanism for nucleotidyl transfer. Their active sites contain two metal ions usually Mg2+, which are ligated by two aspartate side chains. Metal ion B is liganded by all three phosphate groups of the bound dNTP, whereas metal ion A bridges the α-phosphate group of this dNTP and the primers 3′-OH. Metal ion A presumably activates the primer’s 3′-OH for a nucleophilic attack on the 5′-phosphate. Metal ion B function to orient its bound triphosphate group and to elctrostatically shield their negative charges as well as the additional negative charges on the transition state leading to the release of PPi. Polymerization rate 16–20 nucleotides/s. The processivity of the enzyme is 3–200.

The 5′ → 3′ exonuclease activity of DNA Pol-I is located in a distinct structural domain of the enzyme and can be separated from the enzyme by mild protease treatment. When the 5′ → 3′ exo-nuclease activity is removed, the remaining fragment retains the polymerization activity and is called the large or ‘Klenow fragment’.

Functions of DNA Pol-I

  1. Physiologically the enzyme functions to repair DNA. Damaged DNA is endonucleolytically cleaved on the 5′ side of the lesion thereby activating the polymerase 5′ → 3′ exonuclease activity. While excising this damaged DNA, polymerase I simultaneously fills the resulting single-strand gap through its polymerase activity.
  2. Polymerase I catalyses ‘nick translation’. The polymerase I’s combined 5′ → 3′ exonuclease activity and polymerase activity can replace the nucleotides on the 5′ side of a singlestrand nick. These reactions in effect translate (move) the nick towards the DNA strands 3′-end without otherwise changing the DNA molecule. This nick translation is synthetically employed to prepare radiolabelled DNA.
  3. 3. Polymerase 5′ → 3′ exonuclease activity removes the RNA primer at the 5′-end of newly synthesized DNA while its polymerase activity fills in the resulting gap.

 

DNA Pol-II

The enzyme has a mass of 90 kDa.

  • It has 5′ → 3′ polymerization activity.
  • It has 3′ → 5′ exonuclease activity.
  • Polymerization rate is about 40 nucleotides/s. This polymerase also fills the gaps and appears to facilitate DNA synthesis directed by damaged templates. Polymerase II has a low error rate but it is much too slow to be of any use in normal DNA synthesis. Polymerase II differs from polymerase I in that it lacks a 5′→3′ exonuclease activity (Table 3.1).

 

Table 3.1 Comparison of DNA polymerase of E. coli

DNA Pol-III

This enzyme is E. coli ‘DNA replicase’. The holoenzyme is a very large (>600 kDa) and highly complex protein composed of 10 different subunits. The core polymerase is composed of three subunits. The α-, ε- and the θ-subunits. The α-subunit contains the active site for nucleotide addition. The ε-subunit has 3′ → 5′ exonuclease activity and removes the incorrectly added nucleotides from the growing end. The θ is an accessory protein that stimulates the function of ε. Polymerase III holoenzyme consists of 10 subunits, which are α-, ε-, θ-, τ-, γ-, δ-, δ′-, χ-, ψ- and β-subunits. The β-subunit forms a donut-shaped dimer around the duplex DNA and holds the catalytic core polymerase near the 3′-end of the growing strand. Once tightly associated with the DNA, the β-subunit dimer functions like a ‘clamp’ that can slide freely along the DNA. In this way, the active site of core polymerase remains near the growing fork and the processivity of the enzyme is maximized. The five subunits of the enzyme; namely, γ, δ δ′,χ and ψ form the so-called γ complex that mediates two essential tasks: (Figure 3.5)

 

Figure 3.5 Bacterial DNA Polymerase III

  1. Loading of the β-subunit clamp on the duplex DNA—primer substrate, in a reaction that requires the hydrolysis of ATP.
  2. Unloading of the β-subunit clamp after the synthesis is completed. The τ-subunit acts to dimerize two core polymerases and is essential for co-ordinating the synthesis of the leading and lagging strands at each growing fork.

DNA Pol-V

Error prone synthesis occurs in E. coli. Functions involved in this pathway are identified by mutations in the genes umuD and umuC, which abolish UV-induced mutagenesis. This implies that umuD and umuC cause mutation to occur after UV irradiation. The genes constitute the umuDC operon and their expression is induced by DNA damage. Their products form a complex umuD′ 2C, consisting of two subunits of truncated umuD protein and one subunit of umuC. umuD′ 2C complex has DNA polymerase activity and is called DNA Pol-V and is responsible for synthesizing new DNA to replace sequences that have been damaged by UV. This is the only enzyme in E. coli that can bypass the pyrimidine dimers produced by UV.

Eukaryotic DNA Polymerases

There are many types of eukaryotic DNA polymerases; namely, DNA polymerases α, δ, ε, γ, β, ζ and η.

DNA polymerase α

It is a multi-subunit enzyme. It belongs to the A-family of DNA polymerases. It has primase activity. The larger subunit of the enzyme has polymerase activity. It does not have the proofreading activity. It is, therefore, unsuitable for high fidelity. DNA polymerase α is believed to function only in the synthesis of short primers. It has moderate processivity of about approximately 100 nucleotides.

Polymerase α/primase functions to synthesize 7–10-nucleotide-long RNA primers, which extend by an addition of approximately 15 nucleotides of DNA. Then, in a process called ‘polymerase switching’, replication factor C (RFC) displaces polymerase α and loads PCNA (proliferating cell nuclear antigen) on the template near the primer strand, following which polymerase δ binds to the PCNA and the processively extends the DNA.

DNA polymerase δ

It belongs to the B-family enzyme. It lacks primase and exhibits proofreading activity. It is a highly processive enzyme, but only when it is associated with PCNA. Polymerase δ, in association with PCNA, is required for both leading- and lagging-strand synthesis.

DNA polymerase ε

This also belongs to the B-family of nuclear enzymes. It superficially resembles polymerase δ. It has 3′ → 5′ exonuclease activity. The enzyme is highly processive even in the absence of PCNA.

DNA polymerase γ

It belongs to the A-family of enzyme. It occurs exclusively in the mitochondria. It replicates mitochondrial DNA. The chloroplast also contain similar enzyme.

DNA polymerase β

It belongs to the X-family of enzymes. It is involved in the base excision repair and the low-fidelity repair.

DNA polymerase ζ

It is involved in thymine dimer repair.

DNA polymerase η

It is involved in base damage repair (Table 3.2).

 

Table 3.2 Eukaryotic DNA polymerases

Helicases

Helicases are the enzymes that unwind nucleic acid molecules. There are DNA and RNA helicases. DNA helicases are essential during DNA replication, because they separate double-stranded DNA into single strands allowing each strand to be copied. As helicase unwinds the DNA, it forms the replication fork. The process of breaking the hydrogen bonds between the nucleotide bases pairs in double-stranded DNA requires energy from ATP or GTP hydrolysis. A helicase is generally multimeric. A common form of the helicase is a hexamer. This typically translocates along the DNA by using its multimeric structure to provide multiple DNA binding sites. Helicases are likely to have one conformation that binds the duplex DNA and another conformation that binds the single-stranded DNA. Alternation between the conformations drives the motor that melts the duplex and require ATP hydrolysis. Typically, one ATP is hydrolysed for each base pair that is unwound. Helicases may function with a particular polarity that is they can be either 3′→5′ or 5′→ 3′ helicase.

Helicases have been classified into five super families:

Superfamily I: It includes the helicases such as UvrD (E. coli, DNA repair), Rep (E. coli, DNA replication), PcrA (Staphylococcus aureus, recombination), Dda (bacteriophage T4, replication initiation) and RecD (E. coli, recombinational repair). RNA helicases also belong to this family. They play a role during viral RNA replication.

Superfamily II: It includes the enzymes such as rec Q (E. coli DNA repair).

Superfamily III: It consists of the helicases that are encoded mainly by the small DNA viruses and some large nucleocytoplasmic DNA viruses.

Superfamily IV, DnaB-like family: It includes the enzymes such as DnaB (E. coli, replication), gp41 (bacteriophage T4, DNA replication) and T7gp4 (bacteriophage T7, DNA replication).

Superfamily V, Rho-like family: It includes the enzymes such as Rho protein (E. coli, transcription termination).

Primases

Primase catalyses the formation of RNA primers required to initiate DNA replication. Primers are short RNA segments complementary to single-stranded DNA templates. Primase is of key importance in DNA replication because no known DNA polymerases can initiate the synthesis of a DNA strand without an initial RNA or DNA primer. The gene locus DnaG codes for the enzyme primase, an RNA polymerase in prokaryotes. In eukaryotes, the polymerase α has a primase subunit that synthesizes the primer.

Single-Strand Binding Proteins

Single-strand binding (SSB) binds the unwinded single strands of DNA, protecting it and preventing it from rewinding. E. coli SSB is a tetrameric molecule while eukaryotic SSB is trimer. Eukaryotic SSB is also called RPA. SSB binds the DNA co-operatively in which the binding of one monomer favours the binding of the other.

Ligases

DNA ligases close nicks in the phosphodiester backbone of DNA. Biologically, the DNA ligases are essential for the joining of Okazaki fragments formed during replication and for completing the DNA synthesis occurring in DNA repair process. There are two classes of DNA ligases. The first uses NAD+as a cofactor and only found in bacteria. The second uses ATP as a cofactor and found in eukaryotes, viruses and bacteriophages. The RNA primers in the lagging strand are removed by the 5′ → 3′ exonuclease activity of DNA Pol-I and replaced with DNA by the same enzyme. The resulting nick is sealed by DNA ligase (Figure 3.6).

 

Figure 3.6 Mechanism of action of DNA ligase

DNA ligase mechanism

The reaction occurs in three stages in all DNA ligases:

  1. Formation of a covalent enzyme-AMP intermediate linked to a lysine side-chain in the enzyme.
  2. Transfer of the AMP nucleotide to the 5′ -phosphate of the nicked DNA strand.
  3. Attack on the AMP-DNA bond by the 3′ -OH of the nicked DNA sealing the phosphate backbone and resealing AMP.

Figure 3.6 illustrates the three reaction stages:

When the energy source is ATP as in viruses and eukaryotes during adenylation of the enzyme, DNA ligase pyrophosphate is released. When the energy source is NAD+ as in prokaryotes, NMN is released.

Topoisomerases

In the cell, DNA is not free to rotate on its own axis. In E. coli, the closed circular chromosome clamps the DNA. In eukaryotes, DNA is arranged in fixed loops and is attached to several proteins; therefore, free rotation becomes impossible. However, the separation of DNA strand demands the rotation of the DNA duplex. This causes over winding, creating positive supercoils ahead of the replication fork and as the helix tightens further separation is resisted. If unrelieved, this tension halts strand separation and DNA replication. Topoisomerases catalyse the removal of the positive supercoils formed ahead of the replication fork. Topoisomerase change the topology of the DNA (discussed in chapter 1).

MODELS of REPLICATION

θ Replication

Autoradiography of replicating DNA was obtained by John Cairns. This indicates how DNA replicates. Autoradiogram of circular chromosome grown in medium containing 3H thymidine shows the presence of ‘replication’ ‘eyes’ or ‘bubbles’. These are called ‘Θ structures’, because of their resemblance to Greek letter theta. This indicates that double-stranded DNA replicates by the progressive separation of its two parental strands accompanied by the synthesis of their complementary strands to yield two semi-conservatively replicated duplex daughter strands. DNA replication involving Θ structures is known as ‘Θ replication’.

Rolling Circle Replication

It occurs during viral DNA replication in E. coli during mating. According to this model, by some initiation events, a nick is made in the duplex circle and this has 3′-OH and 5′-PO4 termini. Under the influence of a helicase and SSB protein, a replication fork is generated. The synthesis of primer is unnecessary because of the 3′-OH group. So that, leading-strand synthesis proceeds by elongation from this terminus. At the same time, the parental template for lagging-strand synthesis is displaced. Polymerase used for this synthesis is polymerase III. The displaced parental strand is replicated in the usual way by means of precursor fragments. The result of this mode of replication is a circle with a linear branch. It resembles the Greek letter sigma and is also called as sigma replication.

Bacteriophage ϕ×174 Replicates by Rolling Circle Replication

The virus carries a single-stranded DNA known as its (+) strand. On infecting an E. coli this strand directs the synthesis of its complementary strand or (−) strand to form the circular duplex replicative form (RF).

Synthesis of (−) strand

  1. The (+) strand is coated with SSB, except for a 44-nucleotide-long hairpin known as primosome assembly site (PAS). This is recognized and bound by PriA, PriB and PriC proteins.
  2. DnaB and DnaC proteins in the form of DnaB6 and DNAC6 complex add to the DNA with the help of DnaT protein in an ATP-requiring process. DnaC protein is then released yielding the pre-primosome. The pre-primosome in turn binds primase yielding the primosome.
  3. Primosome is propelled in the 5′ → 3′ direction along the (+) strand by PriA and DnaB helicase at the expense of ATP.
  4. Primase synthesizes an RNA primer.
  5. Polymerase III extends the primer to form the Okazaki fragments.
  6. Polymerase I excises the primer and replaces them by DNA. The fragments are then joined by DNA ligase and supercoiled by DNA gyrase to form ϕ174, (−) DNA.

Synthesis of (+) strand by looped rolling circle replication

  1. (+) Strand synthesis begins with the primosome-aided binding of the gene A protein to its 30-bp recognition site.
  2. Gene A protein cleaves a specific phosphodiester bond on the (+) strand nucleotide by forming a covalent bond between a Tyr residue and the DNA’s 5′-phosphoryl group, thereby conserving the cleaved bond’s energy.
  3. Rep helicase subsequently attaches to the (−) strand at the gene A protein and with the aid of the primosome still associated with the (+) strand, commences the unwinding of the duplex DNA from (+) strand’s 5′-end.
  4. The displaced (+) strand is coated with SSB that prevents from reannealing to the (−) strand. E. coli polymerase III holoenzyme extends the (+) strand from its free 3′-OH group.
  5. 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 replication fork.
  6. It is thought that as the old (+) strand is peeled off the RF, the primosome synthesizes the primers required for the later generation of new (−) strand.
  7. When it has come full circle around the (−) strand, the gene A protein again makes a specific cut at the replication origin so as to form a covalent linkage with the new (+) strand 5′-end. Simultaneously, the newly formed 3′-OH group of the old and looped out (+) strand nucleophilically attacks its 5′-phosphoryl attachment to the gene A protein thereby liberating covalently closed (+) strand.

 

Synthesis of ϕ X174 (−) strand on a (+) strand template

 

The synthesis of ϕ X174 (+) strand by rolling circle replication

D-loop Replication

Mitochondrial and chloroplast DNA is replicated by a process in which leading-strand synthesis precedes lagging-strand synthesis. Leading strand, therefore, displaces the lagging-strand template to form a displacement or D-loop. The 15-Kb circular mitochondrial chromosome normally contains a single 500–600-nucleotide D-loop that undergoes frequent cycles of degradation and resynthesis. During replication, the D-loop is extended. When it has reached a point approximately two thirds of the way around the chromosome, the lagging-strand origin is exposed and its synthesis proceeds in the opposite direction around the chromosome. Lagging-strand synthesis is, therefore, only about one-third complete when the leading-strand synthesis terminates.

 

 

PROKARYOTIC REPLICATION

Whether a cell has only one chromosome or has many chromosomes, the entire genome must be replicated precisely once for ssevery cell division. Initiation of DNA replication commits the cell to further division. Once replication started, it continues until the entire genome has been duplicated. The unit of DNA in which an individual act of replication occurs is called the ‘replicon’. Each replicon fires only once in each cell cycle. The replicon is defined by its possession of the control elements needed for replication. It has an origin at which replication is initiated. It also has a terminus at which replication halts. The origin is a ‘cis -acting molecule’, i.e., it is able to affect only that molecule of DNA on which it resides. Bacteria and archaebacteria may contain additional genetic information in the form of ‘plasmids’. A plasmid is an autonomously circular DNA that constitutes a separate replicon.

The E. coli genome contains 4.7 × 106 nucleotide pairs. DNA replication proceeds at about 1,000 nucleotides/s and thus is done in not more than 40 min. One incorrect nucleotide is added for every 109 nucleotides inserted.

Origin of Replication

The replication starts at an origin by the separation of the two strands of the DNA duplex following a semi-conservative mode. When viewed under the electron microscope, the replicated region appears as a replication bubble. The point at which replication occurs is called replication fork also known as the growing point. A replication fork moves sequentially along the DNA from the origin. Replication may start either unidirectionally or bidirectionally from the origin. In E. coli, the origin is called oriC.

Initiation of DNA Replication

Events at the origin

E. coli oriC is a 240-bp DNA segment. oriC has a repetitive four 9-bp sequence and three AT-rich, 13-bp sequence referred to as ‘9-mers’ and ‘13-mers’ respectively. The 9-mer consensus sequence is 5′-TTATCCACA-3′. DnaA protein interacts with oriC to initiate replication. DnaA protein binds to the four 9-mers and causes it to become negatively supercoiled. Following this, the 13-mer sequences are melted. The 13-mer consensus sequence is 5′-GATCTNTTNTTTT-3′. Melting of the 13-mer sequences requires ATP that is hydrolysed by DnaA. Following melting, DnaA recruits hexameric helicase (six DnaB proteins) to opposite ends of the melted DNA. Recruitment of helicase requires six DnaC proteins, each of which is attached to one subunit of helicase. Once this complex is formed, an additional five DnaA proteins bind to the original five DnaA proteins to form five DnaA dimers. DnaC is then released and the ‘preprinting complex’ is complete. SSB protein is needed to prevent the single strands of DNA from forming any secondary structures and to prevent them from reannealing, and DNA gyrase is needed to relieve the stress (by creating negative supercoils) created by the action of DnaB helicase. The unwinding of DNA by DnaB helicase allows primase (DnaG) an RNA polymerase to prime each DNA template, so that DNA synthesis can begin. The DNA region at which all of the proteins come together to carry out the synthesis of daughter strand is called the replication fork, or ‘growing fork’. As replication proceeds, the growing fork and associated proteins move away from the origin. In order for DNA polymerase to move along and copy a duplex DNA, helicase must sequentially unwind the duplex and topoisomerase must remove the supercoils that form.

 

Regulation of replication initiation

In E. coli, DNA replication is regulated through several mechanisms, including the hemimethylation and sequestering of the origin sequence, the ratio of ATP to ADP and the levels of DnaA. All these control the process of initiator proteins binding to the origin sequences. E. coli methylates GATC DNA sequences. This results in the hemimethylated sequences that are recognized by the protein Seq A, which binds and sequesters the origin sequence. In addition, DnaA binds less to hemimethylated DNA. As the result, newly replicated origins are prevented from immediately initiating another round of replication. Elongation is a complex process involving many proteins.

Leading-and lagging-trand syntheses

A major complication in the operation of a DNA replication fork arises from two properties.

The two strands of parental DNA duplex are antiparallel and DNA polymerase can add nucleotides to the growing new strands only in the 5′ → 3′ direction.

At the replication fork, the 3′ → 5′ parental DNA strand is copied by continuous synthesis initiated by RNA primer and proceeds in the direction of movement of the replication fork. Since this daughter DNA is continuously synthesized, it is called as the ‘leading strand’.

The 5′ → 3′ side of the parental strand is copied in the direction opposite to the movement of the replication fork by discontinuous synthesis. A cell accomplishes this by synthesizing a new primer every few hundred bases or so. Each of these primers base paired to the template is elongated in the 5′ → 3′ direction forming discontinuous segments called ‘Okazaki fragments’ after the discoverer Reiji Okazaki. The RNA primer of each Okazaki fragment is removed and replaced by DNA chain growth from the neighbouring Okazaki fragment. Finally, the enzyme DNA ligase joins the fragments. This discontinuously synthesized daughter strand is called the ‘lagging strand’.

DnaG primase binds to the DnaB helicase at the replication fork. DnaG primase initiates primer synthesis on the lagging strand followed by the binding of polymerase III. β-clamp is loaded by γ ‘clamp loader’ complex subunit onto the primer template junction. The polymerase III holoenzyme transits from the previously completed Okazaki fragment to the new primer terminus. DnaG primase is released and polymerase III synthesizes the new fragment. The τ processivity switch stops polymerase III when the end of previously synthesized Okazaki fragment reaches the active site of the enzyme.

The leading and lagging strands are synthesized concurrently

The co-ordinated synthesis of both leading and lagging DNA strands is thought to involve a dimeric DNA polymerase and a looping of the lagging strand, so that both strands can be synthesized in the same direction. A single molecule of polymerase III holoenzyme acts at the replication fork catalysing concurrently both leading- and lagging-strand syntheses. In prokaryotic systems, the directionality problem seen in lagging-strand synthesis is solved by the formation of a loop in the lagging strand at the replication fork to reorient the lagging-strand DNA polymerase, so that it advances in parallel with the leading-strand polymerase.

The two core-polymerase molecules at the fork are linked together by a τ-subunit dimer. The core polymerase synthesizing the leading strand moves, together with its β-subunit clamp, along its template in the direction of the movement of the fork, elongating the leading strand. The other core polymerase molecule that elongates the lagging strand moves with its β-subunit clamp in the direction opposite to that of the fork movement. As elongation of lagging strand proceeds, the size of the DNA loop between the core polymerase and the fork increases. The replication loop grows and shrinks during each cycle of the Okazaki fragment synthesis.

 

 

Double-stranded and newly synthesized DNA will be pushed into this loop. Eventually, the core polymerase synthesizing the lagging strand will complete an Okazaki fragment. It then dissociates from the DNA template, but the τ-subunit dimer continues to tether it to the fork protein complex. Simultaneously, DnaG primase binds adjacent to DnaB helicase on the lagging-strand template and initiates the synthesis of another RNA primer. This is followed by the binding of the P-clamp and the rebinding of the core polymerase. This polymerase molecule then proceeds to elongate the RNA primer to form another Okazaki fragment. As each Okazaki fragment nears completion, the RNA primer of the previous fragment is removed by the 5′ → 3′ exonuclease activity of DNA Pol-I. This enzyme also fills in the gaps between the lagging-strand fragments, which then are ligated together by DNA ligase. The E. coli enzyme uses NAD as a cofactor, while T4 DNA ligase (and other phage and eukaryotic ligases) uses ATP as a cofactor. Although the two core polymerase molecules are linked by the τ-subunit dimer, they are oriented in the opposite directions. Thus, the 3′ growing ends of both the leading and lagging strands are close together but offset from each other. The two core polymerases can add deoxyribonucleotides to the growing strands at the same time and rate, so that leading- and lagging-strand syntheses occur concurrently.

Topoisomerase, helicase and SSB are required continually throughout elongation to relive torsional stress, unwind the replication fork and to keep DNA single-stranded respectively.

 

Figure 3.7 Termination sites of DNA replication in E. coli

Termination

Replication must be terminated properly both to dis-tangle the two daughter chromosomes and to regulate and co-ordinate replication with cell division.

The terminus of replication in E. coli is a large region of about 350 kb flanked by 10 nearly identical 23-bp terminator sites. These sites are non-palindromic and they are oriented differently on either side of the terminus. The Ter A, Ter D, Ter E, Ter I and Ter H are oriented in one direction, while Ter B, Ter C, Ter F, Ter G and Ter J are oriented in the other direction (Figure 3.7). They function in conjunction with a protein, Tus—terminator utilization substance, a 36-kDa protein, which depending on the orientation, permits a replisome to pass in one direction but not in the other. Tus interacts with DnaB and blocks its helicase activity, terminating replication (Figure 3.8).

 

Figure 3.8 Tus-Ter complex

 

Newly replicated daughter chromosomes are prone to recombination reactions that may result in the production of circular dimers. These must be resolved into monomers before the segregation of the new DNA into two daughter cells. In E. coli, a specific locus called dif is required for resolution. dif is a 28-bp sequence located in the centre of the replication terminus region. Two recombination proteins, xer C and xer D, bind to the dif locus and catalyse a site-specific recombination reaction that generates two monomeric DNA. Another possible problem that arises at the termination of replication is the formation of catenated chromosomes. These are resolved by the concerted action of DNA gyrase and topoisomerase IV.

Fidelity of DNA Replication

The process of DNA replication is remarkably accurate. Error occurs once every 109–1,010 nucleotides incorporated. DNA polymerase, however, are not so accurate. They make mistakes once every 104–105 nucleotides incorporated. The proofreading activity of DNA polymerase improves the overall error rate by 102–103. However, this still leaves a difference of 102–103 in the error rates between DNA synthesis and replication. This difference is accommodated by mismatch repair systems that quickly fix any errors made during replication.

EUKARYOTIC REPLICATION

The chromosomes in eukaryotes have much complex structure than prokaryotic chromosomes. The duplication of the chromosomes of eukaryotes involves not only the replication of their DNA but also the synthesis of the associated histone and non-histone chromosomal proteins. However, at the molecular level, the replication of DNA in eukaryotes is quite similar to prokaryotes regardless of the complexity of its genome. Eukaryotic replication is semi-discontinuous and semi-conservative. Like DNA replication in E. coli, eukaryotic DNA replication occurs bidirectionally from RNA primers made by a primase; the synthesis of the leading strand is continuous, while the synthesis of the lagging strand is discontinuous. In contrast to the situation in E. coli, however, two distinct DNA polymerases, α and either δ or ε, function at the eukaryotic growing fork.

Cell Cycle

The cell cycle represents the general sequence of events that occur during the lifetime of a eukaryotic cell. The cell cycle comprises the following phases (Figure 3.9).

 

Figure 3.9 Cell cycle

 

Mitosis and cell division occur during the relatively brief M-phase.

Followed by G1-phase, the longest part of the cell cycle. G1-phase gives way to the S-phase the only period in the cell cycle when DNA is synthesized.

During G2-phase, the new tetraploid cell prepares for mitosis.

Cell cycle for cells in culture occupies 16-24 h period. Cell cycle time varies for different types of cells of multicellular organism and may vary from as little as 8 h to ξ100 days. Most of this variation occurs in the G1-phase. Many terminally differentiated cells such as neurons or muscle cells never divide. They assume quiescent state known as G0-phase. Cells irreversible decision to proliferate is made during G1-phase. Quiescence is maintained, for example, if nutrients are in short supply or the cell is in contact with other cells (contact inhibition). DNA synthesis may be induced by various agents such as carcinogens or tumour viruses or by proteins known as mitogens, which bind to the cell receptor and induce cell division. The progression of the cell through the cell cycle is regulated by proteins known as cyclins and cyclin-dependent protein kinase.

Cell Cycle Control of DNA Replication

In cycling somatic cells, cells synthesize RNA and proteins during the G1-phase, preparing for DNA synthesis and chromosome replication during the S-phase. After progression through the G2-phase, cells begin the complicated process of mitosis. The concentrations of cyclins the regulatory subunits of heterodimeric protein kinases that control cell cycle events, increase and decrease as cells progress through the cell cycle. Cyclin kinases have no kinase activity unless they are associated with cyclin. Each cyclin-dependant kinase (CDK) can associate with different cyclins and the type of associated cyclin determines which proteins are phosphorylated by a particular cyclin–CDK complex.

There are three major classes of cyclin–CDK complex; namely, G1 cyclin–CDK complex, S cyclin–CDK complex and mitotic cyclin–CDK complex. When cells are stimulated to replicate, G1 cyclin–CDK complex are expressed first. These prepare the cell for the S-phase by activating transcription factors that promote the transcription of genes encoding enzymes required for DNA synthesis and the genes encoding S-phase cyclin–CDKs. The three G1 cyclins associate with CDK to form S-phase-promoting factor (SPF). SPF phosphorylates and regulates proteins required for DNA synthesis. The degradation of S-phase inhibitor triggers DNA replication.

The S-phase cyclin–CDK complexes, Clb 5-CDk and Clb 6-CDK begin to accumulate in G1; however, they are inactivated by the binding of an inhibitor, called Sic1, that is expressed late in mitosis and early G1. Sic1 specifically inhibit B-type cyclin–CDK complexes, but has no effect on G1 cyclin CDK complexes, i.e., it functions as an S-phase inhibitor.

Sic 1 inhibitor is degraded following its polyubiquitination by a ubiquitin ligase called SCF. Once Sic1 is degraded, the S-phase cyclin–CDK complex induces replication by phosphorylating multiple proteins bound to replication origins (Figure 3.10).

 

Figure 3.10 Cyclins regulating initiation of DNA replication

Eukaryotic Replication Origins

Eukaryotic chromosomes are replicated from multiple origins (Figure 3.11). Some of these initiate replication early in the S-phase, some later and still others towards the end. However, no eukaryotic origin initiate more than once per S-phase. Moreover, S-phase continues until replication from all origins along the length of each chromosome results in the replication of the chromosomal DNA in its entirety. These two factors ensure that the correct gene copy number is maintained as cell proliferates.

 

Figure 3.11 Eukaryotic origins of replication

Initiation of DNA Replication

Yeast replication origins contain 11 bp conserved sequence to which is bound a hexameric protein, the origin recognition complex (ORC). ORC remains associated with origin during all phases of the cell cycle. Several replication factors are required for the initiation of DNA replication such as Cdc 6, Cdt 1, MCM 10 and MCM hexamer a complex of six additional closely related MCM proteins. These proteins associate with origins during G1 or M. During G1, the various initiation factors assemble with ORC into a pre-replication complex at each origin.

The initiation of replication requires an active S-phase cyclin–CDK complex, a heterodimeric protein kinase Dbf 4-Cdc 7, which is expressed in G1. Dbf 4-dependent kinase, Cdc 7, is often called DDK. DDK phosphorylates the components of pre-replication complex. This leads to the binding of Cdc 45 which is followed by the activation of MCM helicase that unwinds the parental DNA strand and the release of the phosphorylated Cdc 6 and Cdt 1 initiation factors. RPA binds to the resulting single-stranded DNA. A complex of DNA polymerase a and primase initiates the synthesis of the daughter strands. ORC remains bound to the origin sequence in the daughter double-stranded DNA but the phosphorylated initiation factors cannot assemble the pre-replication complex on it. B-type cyclin CDK complexes maintain the initiation factors in a phosphorylated state through the remainder of S-phase, G2-phase and early anaphase. The initiation factors cannot assemble pre-replication complex until B-type cyclins are degraded following their polyubiquitination in the late anaphase (Figure 3.12).

 

Figure 3.12 Eukaryotic replication-initiation and elongation

Elongation

The DNA polymerase α and primase initiate the synthesis of the daughter strands, however, DNA polymerase α is not capable of a lengthy DNA synthesis, as it has low processivity. This is followed by PCNA binding that aids the binding of DNA polymerase δ, which carries out the rest of the synthesis.

Flap endonuclease, FEN1 (previously called MF1), is associated with DNA polymerase 8 complex at the 3′-end of an Okazaki fragment in order to degrade the primer from the 5′-end of the adjacent fragment. FEN1 cannot initiate the primer degradation because the ribonucleotide at the extreme 5′-end of the RNA primer carries a triphosphate that blocks FEN1 activity. The alternative models to circumvent this problem have been proposed (Figure 3.13).

The first possibility is that a helicase breaks the base pairs holding the primer to the template strand, enabling the primer to be pushed aside by DNA polymerase δ, as it extends the adjacent Okazaki fragment into the region thus exposed. The flap that results can be cut off by FEN1. Alternatively, most of the RNA component of the primer could be removed by RNase H, which can degrade the RNA part, i.e., the primer. However, this enzyme cannot cleave the phosphodiester bond between the last ribonucleotide and the first deoxyribonucleotide. This ribonucleotide will carry a 5′-monophosphate than a triphosphate and hence can be removed by FEN1.

 

Figure 3.13 DNA elongation assisted by FEN1

Licensing of DNA Replication

Various chromosomal regions are not replicated simultaneously. Clusters of 20–80 adjacent replicons are activated. Replicons are activated throughout the S-phase until the entire chromosome has been replicated. During this process, the replicons that have already been replicated are distinguished from those that are not, i.e., cells chromosomal DNA is replicated only once per cell cycle. A pre-replication complex is assembled at the origin only during the G1-phase of the cell cycle. G1 is the only period during which pre-replication complex can form. Hence, this process is known as licensing. However, licensed pre-replication complex cannot initiate the replication until it is activated during the S-phase.

Termination of Replication

Eukaryotic DNAs are linear. The ends of linear chromosome cannot be replicated easily. This is because the RNA primers at the 5′-end of a completed lagging strand cannot be replaced with DNA, as the polymerase required to do this would have no place to bind. At the extreme end of a chromosome, there is no way to synthesize this region when the last primer is removed. Therefore, the lagging strand is always shorter than its template by at least the length of the primer. This is the so-called ‘end-replication problem ′. For this reason, eukaryotic chromosomes have linear DNA sequences at the ends called telomeres.

Telomeres and Telomerases

Telomeric DNA has an unusual sequence. It consists of up to several thousand tandem repeats of a simple, species-dependent and G-rich sequence concluding the 3′ terminus of each chromosomal terminus. The enzyme that synthesizes the G-rich strand of telomeric DNA is named telomerase. Telomerase adds tandem repeats of the telomeric sequence TTTGGG to the 3′-end of the lagging-strand template.

 

Figure 3.14 Replicating ends of eukaryotic DNA by telomerases

 

Telomerase is a ribonucleoprotein. Its RNA component contains a segment that is complementary to the telomeric sequence. This sequence acts as a template in a kind of reverse transcriptase reaction that synthesizes the telomeric sequence, translocates to the DNA’s new 3′-end and repeats the process (Figure 3.14).

Without the action of telomerase, a chromosome would be shortened by 50–1,000 nucleotides with every cycle of DNA replication and cell division. The loss of telomerase function in somatic cells is the basis for aging in multicellular organisms. Cancer cells have active telomerases.

 

 

Exposed telomeric DNA would result in the end-to-end fusion of chromosomes, which is a process that will lead to chromosome instability and eventually cell death. This is prevented by capping the DNA. Telomeric DNA is specifically bound by proteins that sequester the DNA ends forming T-loops.

INHIBITORS of REPLICATION
  • Inhibitors of DNA replication are used as prime drugs for suppressing proliferative, viral, bacterial and auto-immune diseases. The inhibitors of nucleic acid synthesis have various effects.
  • They block the synthesis of nucleotide precursors or their polymerization.
  • Some inhibitors are incorporated as nucleotide analogues.
  • Other inhibitors interfere with template function by binding, modifying or degrading DNA. Some inhibitors bind and inactivate the replication proteins.
  • Physical agents such as UV and X-ray inhibit the DNA replication by damaging the DNA.
  • Some inhibitors act indirectly. The inhibitors of mitosis profoundly affect the DNA synthesis by preventing its initiation (Figure 3.15) (Table 3.3).

Inhibitors of Nucleotide Biosynthesis

Reducing the supply of precursors limits the rate of nucleic acid biosynthesis and leads to mutations in DNA template. The inhibitor can function as:

  • an antimetabolite that is blocking the enzymatic use of the substrate,
  • an alternative substance incorporated into the DNA and
  • an analogue of feedback inhibition of the enzyme that is responsible for its biosynthetic regulation.

Inhibitors of Purine Biosynthesis

Azaserine and 6-diazo-5-oxo-L-norleucine (DON)—the analogues of glutamine—inhibit three reactions in purine biosynthesis. Their activities in inhibiting cell division primarily arise from the inhibition of formylglycinamide ribonucleotide amidotransferase. They form covalent bond with the cysteine residue in the active site of the enzyme. They also inhibit cytidine triphosphate synthase.

 

 

Figure 3.15 Inhibitors of DNA replication

 

Table 3.3 Inhibitors of DNA replication

 

Analogue chain terminators Incorporated into DNA or RNA Inhibition
2′3′ Dideoxy NTPs
AZT—azidothymidine DNA Chain growth 3′→5′ Exonuclease activity of polymerase.
Arabinose NTP DNA
Acyclovir NTP (analogue of G) DNA Chain growth
Cordycepin DNA, RNA Chain growth
Defective nucleic acid
Uracil dNTP (analogue of T) DNA DNA integrity, excision leads to chain breakage
5-Bromouracil DNA Fidelity of replication
5-Iodouracil DNA Mutation
Allopurinol DNA Xanthine oxidase
5-Azacytidine DNA Chain growth
Thioguanine DNA Fidelity of DNA replication

6-Diazo-5-oxo-L-norleucine (DON)DON is a cytotoxic inhibitor of many enzymes of nucleotide synthesis. DON treatment leads to apoptosis, the programmed cell death.

Analogues of Purine and Pyrimidine Bases

A few examples of the analogues of purine and pyrimidine bases are 6-mercaptopurine and 6-thioguanine.

6-Mercaptopurine, brand name Purinethol, is an immunosuppressive drug, which is used in the treatment of leukaemia. It interferes with nucleotide interconversion and glycol protein synthesis.

6-Thioguanine is also used in the treatment of cancer. It is guanine analogue. After incorporation into DNA, the thiocarbonyl group of thioguanine has the tendency to be methylated. This produces a base similar to 6-o-methylguanine. During second round of replication, the mismatch repair system will recognize the mismatch between the methylated base and cytosine. This results in persistent single-strand breaks in the DNA. The genotoxic stress triggers cell death.

 

Inhibitors of Folate Synthesis

The conversion of folate to tetrahydrofolate is required for its coenzymic function in all cells. The inhibition of dihydrofolate reductase (DHFR) depletes the coenzyme thereby blocking the synthesis of purine nucleotides; for example, trimethoprin for bacterial enzyme and methotrexate/aminopterin for animal cell enzymes. Sulpha drugs also block the microbial biosynthesis of folate. Folate analogues such as Lecoverin (5-formyl-tetrahydrofolate) can also inhibit the action of DHFR.

 

Inhibitors of Deoxynucleotide Synthesis

Hydroxyl urea destroys free radicals of E. coli ribonucleotide reductase and is a potent reversible inhibitor of the mammalian reductase. Its action blocks the production of all deoxyribonucleotides and hence blocks the DNA replication. 5-Flurouracil in the form of deoxynucleotide covalently binds and inactivates thymidylate synthase and inhibits the biosynthesis of thymidylate.

 

Catabolite Analogs

Anti-tumour activity of allopurinol, the structural analogue of hypoxanthine, Nis due the inhibition of xanthine oxidase enzyme. Adenosine analogues inhibitadenosine deaminase and also inhibit replication.

 

Inhibitors that Modify DNA

Although DNA is relatively unreactive chemically, the need to preserve its conformation and continuity makes it vulnerable to agents that bind it covalently or non-covalently. For example, acridine, ethidium and propidium dyes. The intercalation of these agents into the DNA does not allow strand separation and hence inhibits replication. Mitomycin and porfiromycin inhibit DNA synthesis by interstrand cross-linking of DNA. Aureomycin introduces single-strand breaks in linear duplex, a superhelical DNA by damaging the deoxyribose to release free base. cis -Diamminedichloridoplatinum (II) (CDDP) is a platinum-based chemotherapy drug used to treat various types of cancers, including sarcomas, some carcinomas (e.g., small cell lung cancer and ovarian cancer), lymphomas and germ cell tumours. It was the first member of its class, which now also includes carboplatin and oxaliplatin. Platinum complexes are formed in cells, which bind and cause cross-linking of DNA ultimately triggering apoptosis, or programmed cell death.

Inhibitors that Affects Enzyme of Replication

  1. Acridines, anthracyclins, ellipticines and epipodophyllotoxins inhibit all four categories of eukaryotic type II topoisomerases. Nalidixic acid and Fluoroquinolones are antibiotics used to inhibit bacterial topoisomerases.
  2. Aphidicolin inhibits eukaryotic polymerase a and polymerase δ.
  3. Rifamycin and Rifampicin inhibit RNA polymerase.
  • The process of DNA duplication is called ‘replication’, a biological process that occurs in all living organisms prior to cell division. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule. Each strand of DNA double helix can serve as template for the synthesis of a new strand to form duplicated DNA.
  • Once the DNA replication is initiated, both the old strands of the duplex serve as templates that direct the synthesis of new complementary strand. Thus, each daughter DNA retains half of the parental DNA, i.e., the replication is ‘semi-conservative’.
  • There are three types of prokaryotic DNA polymerases; namely, DNA Pol-I, Pol-II and Pol-III.
  • Eukaryotic DNA polymerases are of different types; namely, DNA polymerase α, ζ, ε, γ, β, ζ, η,
  • If polymerase I erroneously incorporates a wrong nucleotide at the end of a growing chain, the polymerase activity is inhibited and 3′ → 5′ exonuclease activity excises the incorrect nucleotide. This is called ‘proofreading activity’.
  • The 5′ → 3′ exonuclease activity of DNA Pol-I is located in a distinct structural domain of the enzyme and be separated from the enzyme by mild protease treatment. When the 5′ → 3′ exonuclease activity is removed, the remaining fragment retains the polymerization activity and is called the large or ‘Klenow fragment’.
  • Polymerase I catalyses ‘nick translation’. The polymerase I’s combined 5′ → 3′ exonuclease activity and polymerase activity can replace the nucleotides on the 5′ side of a single-strand nick. These reactions in effect translate (move) the nick towards the DNA strands 3′-end without otherwise changing the DNA molecule. This nick translation is synthetically employed to prepare radioactive DNA.
  • ‘DNA Pol-III’ is E. coli ‘DNA replicase’;.
  • Polymerase α/primase functions to synthesize 7–10-nucleotide-long RNA primers, which extends by an addition of approximately 15 nucleotides of DNA. Then, in a process called ‘polymerase switching’, replication factor C (RFC) displaces polymerase α and loads PCNA (proliferating cell nuclear antigen) on the template near the primer strand, following which polymerase δ binds to the PCNA and the processively extends the DNA.
  • Autoradiogram of a circular chromosome that is grown in a medium containing 3H thymidine shows the presence of ‘replication’; ‘eyes’ or ‘bubbles’. These are called ‘Θ structures’, because of their resemblance to Greek letter theta. This indicates that double-stranded DNA replicates by the progressive separation of its two parental strands accompanied by the synthesis of their complementary strands to yield two semi-conservatively replicated duplex daughter strands. DNA replication involving Θ structures is known as ‘Θ replication’.
  • Mitochondrial and chloroplast DNAs are replicated by a process in which the leading-strand synthesis precedes the lagging-strand synthesis. Leading strand, therefore, displaces the lagging-strand template to form a displacement or D-loop.
  • At the replication fork, the 5′ → 3′ parental DNA strand is copied by a continuous synthesis initiated by RNA primer and proceeds in the direction of the movement of the replication fork. Since this daughter DNA is continuously synthesized, it is called as the ‘leading strand’.
  • The 5′ → 3′ side of the parental strand is copied in the direction opposite to the movement of the replication fork by discontinuous synthesis. A cell accomplishes this by synthesizing a new primer every few hundred bases or so. Each of these primers base paired to the template is elongated in the 5′ → 3′ direction forming discontinuous segments called ‘Okazaki fragments’ after the discoverer Reiji Okazaki. This discontinuously synthesized daughter strand is called ‘lagging strand’.
  • Eukaryotic chromosomes have linear DNA sequences at the ends called telomeres. Telomeric DNA has an unusual sequence. It consists of up to several thousand tandem repeats of a simple, species-dependent and G-rich sequence concluding the 3′ terminus of each chromosomal terminus. The enzyme that synthesizes the G-rich strand of telomeric DNA is named telomerase.
  1. What are the modes of DNA replication?

  2. Explain in detail - Meselson and Stahl’s experiment with illustrations.

  3. Mention the role of DNA polymerases in replication with its types and mechanism.

  4. What are the different types of Eukaryotic DNA polymerase. Explain any one in detail.

  5. Differentiate Eukaryotic and Prokaryotic DNA polymerase.

  6. Define helicases and primases.

  7. Write short notes on DNA ligase.

  8. What are different models of replication?

  9. Discuss in brief the mechanism of rolling circle replication.

  10. Differentiate between prokaryotic and eukaryotic replication.

  11. Explain the functions and significance of Inhibitors of Nucleotide Biosynthesis.

  12. What are Okazaki fragments.

MULTIPLE-CHOICE QUESTIONS
  1. Helicases have been classified into ———superfamilies.

    1. 1
    2. 2
    3. 3
    4. 4
    5. 5
  2. Helicases like UvrD belong to superfamily ———.

    1. I
    2. II
    3. III
    4. IV
    5. V
  3. DNA polymerase ——— is highly processive even in the absence of PCNA.

    1. β
    2. α
    3. ε
    4. δ
  4. A ——— is an autonomously circular DNA that constitutes a separate replicon.

    1. Z-DNA
    2. DNA primer
    3. plasmid
    4. none of the above
  5. ——— phase is the longest part of the cell cycle.

    1. M
    2. G2
    3. G1
    4. S
  6. When the 5′-------→3′ exonuclease activity is removed, the remaining fragment retains the polymerization activity and is called the ———.

    1. Klenow fragment
    2. Okazaki fragment
    3. replication bubble
    4. leading strand
  7. The rate of polymerization rate is about ——— nucleotides/sec.

    1. 30
    2. 40
    3. 50
    4. 60
  8. Superfamily III consists of helicases encoded mainly by ———.

    1. DNA small viruses
    2. Rho protein encoding enzymes
    3. Enzymes like rec Q
    4. Enzymes like UvrD, PcrA etc.
  9. ——— catalyze the formation of RNA primers required to initiate DNA replication.

    1. DNA polymerases
    2. restriction endonucleases
    3. Ligases
    4. Primases
  10. During ——— phase the new tetraploid cell prepares for mitosis.

    1. G1
    2. G2
    3. M
    4. S

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