Index

Chapter 1 Chapter 4
DNA replication Post Transcriptional Processing
1.1. Introduction 1 4.1 Introduction 79
1.2. Basic Concepts of Replication 1 4.2 RNA splicing 80
1.3. Proteins Involved In Replication 5 4.3 Self-splicing Introns 82
1.4. DNA Replication in Bacteria 14 4.4 Fidelity of RNA splicing 84
1.5. Nuclear Replication in Eukaryotes 18 4.5 Alternative Splicing 85
1.6. The End-Replication Problem 24 4.6 Trans-splicing 90
1.7. Chromatin Replication 28 4.7 Origin and Significance of Introns 90
1.8. Extra-Chromosomal Replicons 29 4.8 Alternative Polyadenylation 91
4.9 RNA Editing 92
Chapter 2 4.10 RNA Transport and Regulation 94
4.11 RNA stability 95
DNA Damage and Repair 4.12 Processing of Pre rRNA 97
Mechanism
2.1 Introduction 36 Chapter 5
2.2 Sources of DNA damage 37 Translation
2.3. DNA Repair Pathways 42
5.1. Introduction 99
5.2. Messenger RNA 99
Chapter 3 5.3 Genetic Codon 101
5.4. Transfer RNA 107
Transcription 5.5 Amino-acyl t-RNA Synthetase 109
3.1 Introduction 57 5.6. Ribosomes 112
3.2. Different Type of RNA 59 5.7. Translation Process 116
3.3. RNA Polymerase 60 5.8 mRNA Surveillance Pathways 132
3.4. Transcription in Prokaryotes 63 5.9 Translation Inhibitors 134
3.5. Transcription in Eukaryotes 69 5.10 Post Translational Modifications 136
3.6 Eukaryotic m-RNA processing 74
3.7 Transcription of rRNA genes 76
3.8 Transcription of tRNA genes 77
Chapter 6
Prokaryotic Gene Regulation 7.4 Eukaryotic Transcriptional Repressors 180
6.1. Introduction 141 7.5 Signal transduction and modulation of gene
6.2. Role of Activators and Repressors 142 expression at transcription level 181
6.3. Operon Concept 145 7.6 Transcription Gene Silencing 182
6.4. Alternative σ Factors 154 7.7 RNA Interference and Regulatory RNA in
6.5. Regulation of Gene Expression after Eukaryotes 185
Transcription Initiation 155 7.8 Regulation at translational level 193
6.6. CRISPRs 160
6.7 Regulation in λ-Bacteriophage 162
Chapter 8

Chapter 7 Molecular Techniques

8.1 Analysis of Promoter and Regulatory
Eukaryotic Gene Regulation Elements 197
7.1. Introduction 170 8.2 Analysis of DNA-Protein Interaction
7.2. Eukaryotic Activators 172 200
7.3. Functions of Eukaryotic Activators 175 8.3 Analysis of Protein-Protein Interaction
203
Chapter - 1
DNA Replication
1.1. Introduction  Multiple rounds of replication are possible during
DNA Replication is the process by which DNA makes a copy prokaryotic cell division while in eukaryotes only one
of itself during cell division. DNA is made up of a double round of replication is possible per cell division.
helix of two complementary strands. During replication,  For linear chromosomes, which are most common in
these strands are separated. Each strand of the original eukaryotes, a special problem arises during replication
DNA molecule then serves as a template for the production of chromosomal ends (telomeres).
of its counterpart.
1.2. Basic Concepts of Replication
What is need of replication?
What are possible modes of replication?
DNA carries all of the genetic information for life. One
Theoretically, the template dependent DNA replication
enormously long DNA molecule forms haploid set of the
model can be explained by three modes: semi-conservative,
chromosomes of an organism, 23 of them in a human. The
conservative and dispersive (Figure 1).
fundamental living unit is the single cell. A cell gives rise to
many more cells through serial repetitions of a process
known as cell division. Before each division, new copies
must be made of each of the many molecules that form the
cell, including the duplication of all DNA molecules, so that
it can be equally distributed between two daughter cells.
When DNA replicates during cell cycle?
In prokaryotes DNA replication occurs during C-stage of cell
cycle (ICD Model) while in eukaryotes it occurs during
S-phase of cell cycle (G1SG2M).
What are similarities in replication of prokaryotes and
eukaryotes?
Basic mechanisms of DNA replication are similar across Figure 1: Possible modes of DNA replication
organisms from prokaryotes to multicellular eukaryotes
1. Semi-conservative replication: In this model, the two
 Origin of replication usually contains AT rich sequences.
strands of DNA unwind from each other, and each acts
 Replication is bidirectional from replication origin.
as a template for synthesis of a new, complementary
 Replication is semi-conservative process
strand. This results in two DNA molecules with one
 Replication occurs in 5’3 direction and is template
original strand and one new strand. This is the correct
dependent process
mode of DNA replication in biological organism.
 Replication is semi-discontinuous process.
 DNA polymerase cannot initiate replication, its needs 2. Conservative replication: In this model, DNA
free 3’OH group for extension of strand. replication results in one molecule that consists of both
original DNA strands (identical to the original DNA
What are differences in replication of prokaryotes and molecule) and another molecule that consists of two
eukaryotes? new strands (with exactly the same sequences as the
 In prokaryotes DNA is always accessible to replication original molecule). This mode does not exist in
machinery but in eukaryotes it not directly accessible, it biological organisms.
is bound to nucleosome.
 In prokaryotes, often there is single origin from where 3. Dispersive replication: In the dispersive model, DNA
replication starts while eukaryotes, has multiple origin replication results in two DNA molecules that are
from replication can be initiated. mixtures, or “hybrids,” of parental and daughter DNA.
 Prokaryotic chromosomal DNA replication stops at one In this model, each individual strand is a patchwork of
specific locus while in case of eukaryotes at stops at all original and new DNA. This mode also does not exist in
those locus where two replication fork meets. biological organisms.
2 Molecular Biology

How we can say that mode of DNA replication is semi- centrifugation involves spinning a solution of cesium
conservative? chloride (CsCl) – a heavy metal salt – and the DNA samples
In semi-conservative mode of DNA replication two parental in an ultracentrifuge at high speed for several hours.
strands unwind and are used for synthesis of new strands Eventually, there occurs an equilibrium between centrifugal
following the rule of complimentary base pairing. The semi- force and diffusion, such that there is formation of a
conservative mechanism of DNA replication can be best bottom to top gradient of CsCl. DNA forms a band at a
explained by a classic experiment conducted by M. concentration of CsCl having density in the gradient equal
Meselson and W. F. Stahl, outlined in Figure 2. Their to that of the density of DNA. The bands are detected by
experiment significantly explains differences between the checking for the absorbance under ultraviolet light at a
semiconservative, conservative, and dispersive modes of wavelength of 260 nm where DNA has maximum
replication. absorption.
First Meselson and Stahl, labelled DNA in a bacterium by After growing for many generations in 15N containing
flowing cells in medium containing either 14N nitrogen or medium, Meselson and Stahl transferred the bacteria to an
the heavier isotope, 15N (It has an additional neutron than only 14N contacting medium. They found that DNA
the naturally occurring 14N but not radioactive). replicated in the 14N medium for one generation was of
Furthermore, they isolated pure DNA from these organisms, intermediate density between light (14N) and heavy (15N). In
and subjected it to CsCl density gradient centrifugation the next generation, only DNA of intermediate and light
leading separation of light (14N) and heavy (15N) forms of density was present. The results shown in figure 2a are
DNA to different locations in the centrifuge tube (Figure 2). consistent only with semiconservative replication.
To determine density of the strands a technique known as
density-gradient centrifugation was used. Density-gradient

Figure 2: Experiment demonstrating that DNA replication is bacteria is (a) semiconservative. (b) and (c) Results if DNA replication
has been conservative or dispersive

IFAS Publications www.ifasonline.com

DNA Replication 3

Figure 3: Experimental demonstration that DNA replication occurs semi conservatively in eukaryotes
If replication would be conservative, then there would be (a point where replication starts). From origin local
presence of two bands after first generation an original 15N unwinding (melting) of DNA starts. There is a formation of
(heavy) double helix and a new 14N (light) double helix. And replication “eye” or “bubble” that contains two replication
also the original DNA would have continued to show up as a forks that proceed in opposite directions around the
15
N (heavy) band throughout the experiment. If the method circular DNA. During replication, the chromosome looks like
of replication had been dispersive, there would be multiple- the Greek letter theta (θ) by electron microscopy.
banding patterns, depending on the degree of Replication intermediates are thus termed “theta
dispersiveness. structures.” Cairns’ findings have subsequently been
verified by both autoradiographic and genetic analysis
How we can say that replication is also semi-conservative
(Figure 4).
in eukaryotes?
DNA replication is semi-conservative in eukaryotes was What is direction of DNA synthesis?
demonstrated by J. Herbert. Experiments were performed The new strand of DNA is synthesized from 5’ to 3’ during
to explain the mechanism as show in figure 3. Cultured semiconservative replication. New nucleotides are added
mammalian cells were allowed to multiply in the presence one at a time to the 3’ hydroxyl end of the DNA chain,
of bromodeoxyuridine (BrdU); BrdU is thus gets forming new phosphodiester bond. The 3’-5’ parental
incorporated in newly produced strands at the place of strand act as template.
thymidine. After replication, a chromosome, which is having
What is substrate for DNA synthesis?
two chromatids are produced. After one round of
Deoxynucleoside 5’ triphosphates (dNTPs) are the building
replication in BrdU, both chromatids of each chromosome
blocks of DNA from which the terminal two phosphates are
contained BrdU (Figure 3).
lost during phosphodieseter bond formation, making the
After two rounds of replication in BrdU, one chromatid of reaction essentially irreversible.
each chromosome was composed of two BrdU-containing
strands, whereas the other chromatid was a hybrid
consisting of a BrdU-containing strand and a thymidine-
containing strand (Figure 3). The thymidine containing
strand is the original parental strand which was present
prior to addition of BrdU.

What is proof that DNA replication is bidirectional?

J. Cairns in 1963 verified that DNA replication is semi-
conservative using the technique of autoradiography. In
this technique there involves the exposure of radioactive
emissions on a photographic film. Quantification of
radioactive material can be done by counting the silver
colored dots on photographic film. Cairns extracted DNA
from E.coli culture grown on tritium labeled thymine.
Autoradiographs were made for the radiolabelled DNA. By
Figure 4: Theta replication in E. coli
analysis of DNA at different time points during replication
(it takes approximately 42 minutes to replicate the entire
genome), Cairns showed that replication of the circular
genome was bidirectional. There is an origin of replication

IFAS Publications www.ifasonline.com

4 Molecular Biology

Why DNA polymerase need template: primer Junction for satisfies both the requirements and thus promotes
DNA synthesis? nucleotide incorporation (Figure 5).
The process of replication is template dependent that is the
DNA replication is semi-discontinuous?
sequential addition of nucleotides in new strand is
Besides with being semi-conservative, DNA replication is
complementary to that of the parental strand.
semi-discontinuous. The major form of replication that
DNA polymerases are the key enzymes involved in DNA occurs in nuclear DNA (eukaryotes), some viruses (e.g. the
replication. Initially Arthur Kornberg and his colleagues papovavirus SV40), and bacteria is called semi-
purified an enzyme from bacterial extracts that discontinuous DNA replication. Term semi-discontinuous
incorporated radioactively labeled DNA precursors into an implies that on one strand DNA replication runs without any
acid-insoluble polymer identified as DNA. The enzyme was pause while on other strand synthesis takes place in the
later on named as DNA polymerase (and later, after the form of fragments. This is due to the fact that, DNA
discovery of additional DNA-polymerizing enzymes, it was polymerase can only add nucleotides in the 5’ 3’
named DNA polymerase I). direction.
DNA polymerase I requires the presence of template DNA Synthesis of complimentary strands require that DNA
and all four deoxyribonucleoside triphosphates (dTTP, synthesis proceeds in opposite direction, while the double
dATP, dCTP, and dGTP) for the reaction to proceed. helix is progressively unwinding and replicating in only one
Kornberg used radiolabeled dNTPs and an unlabeled direction. one of the DNA strands is continuously
template DNA. The newly synthesized, radioactively labeled synthesized in the same direction as the advancing
DNA had the same base composition as the original replication fork and is called leading strand whereas the
unlabeled DNA, which strongly suggested that the original other strands is synthesized discontinuously in segments
DNA strands had served as templates for the polymerization and is referred to as lagging strands. (Figure 6).
reaction.
With increasing knowledge of DNA replication it became
clear that DNA replication was more complex than
previously thought. An intact, double-stranded DNA
molecule, for example, does not stimulate incorporation. A
completely single stranded, circular DNA or linear molecule
must prove itself a best template since replication requires
a separated single stranded DNA, but it was found that no
replication was initiated from these templates. Even double
stranded DNA cannot be utilized by DNA polymerase for
DNA synthesis. A DNA strand must fulfill some structural
requirements for being template strand.
Later on it became clear that single stranded circular DNA
cannot serve as template because the DNA polymerase
enzyme can only add nucleotides to a pre-existing free 3’ –
OH end of nucleotides hydrogen bonded to template
strand. Thus, the enzyme DNA polymerase whether
Figure 5: Templates and non-templates for DNA
prokaryotic or eukaryotic; needs a primer. A primer is a
polymerase activity (a) Examples of DNA structures that
strand of nucleotides/deoxyribonucleotides that provides a
do allow DNA synthesis (b) Examples of DNA structures
free 3’ –OH end. Both the prokaryotic as well as eukaryotic
that stimulates DNA synthesis. The molecules that
DNA polymerase have two basic requirements that is a
allow DNA synthesis by DNA polymerase contain a
template strand to copy and a primer strand with free
single stranded template strand to copy and a primer
3’OH group to which can be extended by adding
strand with a 3’OH on which new nucleotides are
nucleotides.
added.
DNA molecules that do not fulfill these two basic
Continuous DNA Synthesis occurs on leading strand
requirements fail to promote DNA synthesis. Linear single
Two strands of DNA run are antiparallel (i.e., running in
stranded DNA has a free 3’-OH end but lacks a template
opposite directions). Thus, problem might have been
while circular single strand of DNA can act as template but
occurred since DNA polymerase can only add nucleotides in
lacks a free 3’-OH end. Partially double stranded DNA

IFAS Publications www.ifasonline.com

DNA Replication 5

the 5’  3’ direction. To avoid this, two strands are 1.3. Proteins/ Enzymes Involved In Replication:
replicated in 5’ to 3’ direction by different mechanisms.
Some fundamental properties of replication are conserved
After primer binding on 3’→5’ template strand replication
from E. coli to humans while specific enzymes and other
takes place in continuous manner without any pause.
proteins are the major distinguishing characters (Table 1).
This strand is called as “leading strand”. Synthesis of
Table 1: Important proteins involved in replication of E.coli
leading strand is in the direction of proceeding replication
and eukaryotes
fork. Thus, the polymerase gets easy access of single
stranded template DNA. DNA polymerase on leading strand E. coli Eukaryotic Function
is highly processive i.e., it does not get released until it Protein Protein
meets up a replication fork running in opposite direction or DnaA ORC proteins Recognization of origin
entire strand is replicated (Figure 6). of replication
Gyrase Topoisomerase Relieves positive
II supercoil ahead of
replication fork
DnaB Mcm DNA helicase that
unwinds parental
duplex
DnaC Cdc6, Cdt1 Load DNA helicase to
origin
SSB RPA Maintain DNA in single
stranded state
γ- RFC Clamp loader, load
complex clamps on to the DNA,
Subunit of DNA
polymerase
Pol III pol δ/ε Major replicating
Figure 6: Semi-discontinuous DNA synthesis core enzymes, synthesize
leading and lagging
Why on Lagging Strand DNA Replication Is Discontinuous?
strand
The lagging strand is the strand of new DNA whose
direction of synthesis is opposite to the direction of the β clamp PCNA Increase processivity of
growing replication fork (Figure 6). On the “lagging strand”, DNA polymerase; works
DNA replication does not occurs as a continuous stretch but with pol III or pol δ/ε
it takes place in the form of short fragments. Primase Primase Synthesize RNA primer
- pol α Extend RNA primer with
Since the direction of polymerase (also the direction of short DNA
synthesis) is opposite to that of the direction of migration of oligonucleotides
replication fork, the DNA is copied in short segments (1000– DNA DNA ligase Seals Okazaki fragments
2000 nt in prokaryotes and 100–200 nt in eukaryotes). ligase into continuous strand
These short segments are called as “Okazaki fragments” as pol I FEN-1 Removes RNA primers;
they were first described by Reiji and Tuneko Okazaki in pol I of E.coli also fills
1969. gap with DNA
Replication of lagging strand in bacteria requires the
repetition of four basic steps: primer synthesis by primase,
What are initiator proteins?
elongation by DNA polymerase III, primer removal with gap
filling by DNA polymerase I, and joining of the Okazaki The proteins which recognize replication origin and assist in
fragments by DNA ligase. Despite these extra steps, loading of helicase to origin are referred as initiator
synthesis of both new strands occurs concurrently. proteins. The E. coli replication origin (oriC) is recognized by
Nucleotides are added to the leading and lagging strands at DnaA protein while yeast replication origins (ARS) is
the same time and rate, by two DNA polymerases, one for recognized by origin recognition complex (ORC 1-6).
each strand.

IFAS Publications www.ifasonline.com

6 Molecular Biology

Figure 7: Polarity of helicase 3’5’ template for leading strand, 5’3’ template for lagging strand
What are functions of helicase loader? How Single-Stranded DNA-Binding Proteins (SSB/RP-A)
stabilize ssDNA before Replication?
Helicase loaders recognize proteins bound at replication
Once the helicase has passed replication origin, the two
origin (DnaA/ORC) and recruits helicase (DnaB/MCM) to
separated strands may reanneal together as there is high
origin. The role of helicase loader is prokaryotes is
degree of complementarity. Thus, to avoid pairing amongst
performed by DnaC protein while in eukaryotes same
themselves, these single strands must be kept occupied by
function is done by cdt1 and cdc6 proteins.
some other protein until the DNA polymerase adds
How DNA duplexes unwind at replication forks? complementary sequences.
Unwinding or separation is done by specialized enzymes
To stabilize the separated strands, ssDNA-binding proteins
called as helicases in prokaryotes as well as in eukaryotes.
(SSBs in bacteria and RPA in yeast) rapidly bind to the
DNA helicases are the enzymes that use ATP as energy to
separated strands. Binding of one SSB promotes the binding
separate (melt) the DNA duplex in the direction of
of another SSB to the immediately adjacent ssDNA. This is
replication fork. They progressively catalyze the transition
called ‘cooperative binding’ and occurs because SSB
from double stranded DNA to single stranded DNA. The role
molecules bound to immediately adjacent regions of ssDNA
of helicase in E. coli is done by DnaB while in eukaryotes it is
also bind to each other. Cooperative binding ensures that
performed by MCM 2-7 proteins.
ssDNA is rapidly coated by SSB as it emerges from the DNA
What is polarity of helicases? helicase. Once coated with SSBs, ssDNA is held in an
The E.coli helicases (DnaB) migrates in 5’→3’ direction elongated state that facilitates its use as a template for DNA
bound to the lagging strand and unwinds DNA. Whereas or RNA primer synthesis. SSBs interact with ssDNA in a
helicases that are functional in eukaryotic system like SV40 sequence-independent manner. SSBs primarily contact
T antigen or the MCM2-7 move in 3’→5’ direction, thus are ssDNA through electrostatic interactions with the
bound on leading strand template during DNA replication phosphate backbone and stacking interactions with the
(Figure 7). DNA bases.

IFAS Publications www.ifasonline.com

DNA Replication 7

What are total number of DNA polymerases? phosphodiester bond) in the DNA sugar–phosphate
All the known DNA polymerases can add nucleotides in backbone. This property has been used in the laboratory to
5’→3’ direction. They add nucleotides at 3’OH of a growing make radio-labeled DNA by a technique called “nick
strand of DNA. There are five DNA polymerases in bacteria translation”.
as compared to the fourteen DNA polymerases in
DNA polymerase III: It is very surprising that though being
eukaryotes and perform the similar overall function as the
the most abundant polymerase in E.coli, DNA polymerase I
eukaryotic DNA polymerases. (Table 2).
is not the major replicative polymerase. In fact, a much less
Bacterial DNA dependent DNA polymerases abundant enzyme, DNA polymerase III, catalyzes genome
DNA polymerase I: The very first polymerase purified and replication. DNA polymerase III is a holoenzyme complex
characterized from any organism was DNA polymerase I. made up of 10 different polypeptide subunits. The α-
Although it is not the major DNA polymerase involved in subunit has the replicase (5’ 3’ polymerase) activity and
prokaryotic DNA replication; it is the most abundant the ε-subunit has the proofreading activity (3’5’
polymerase in E.coli. It is involved in nucleotide excision exonuclease). DNA polymerase III also plays a role in
repair pathway and primer removal followed by gap filling nucleotide excision repair pathways.
between two Okazaki fragments.
DNA polymerases II: DNA Pol II is single subunit protein
On enzymatically cleaved by the protease subtilisin, DNA encoded by the polB (dinA) gene. The enzyme has 5′→3′
polymerase I give two fragments. DNA synthesis capability as well as 3′→5′ exonuclease
proofreading activity. It is involved in replication on lagging
 The larger fragment called as the Klenow fragment that
strand and base excision repair mechanisms.
has 5’ 3’ polymerase activity and 3’5’ exonuclease
DNA polymerases IV and V: DNA polymerases IV and V are
activity (proof reading).
members of Y family DNA polymerases that mediate
 The other smaller fragment has 5’3’ exonuclease translesion DNA synthesis. These polymerases are known
activity which is used for the primer removal. for their ability to bypass the DNA damage that has blocked
the DNA replication by DNA polymerase III. These
The Klenow fragment is also widely used for labeling DNA
polymerases are error prone enzymes and thus may play a
by a technique called “random priming”. Klenow fragment
role in adaptive mutagenesis.
has unique ability to start replication at a nick (broken
Table 2: List of DNA polymerase in prokaryotes and eukaryotes

Prokaryotic (E. coli) DNA polymerases Eukaryotic DNA polymerases

Sub Units Function Sub Units Function
RNA primer removal, Primer synthesis during DNA
Pol I 1 Pol α 4
DNA repair replication
Pol II (Din A) 1 DNA repair Pol β 1 Base excision repair
Mitochondrial DNA replication and
Pol III core 3 Chromosome replication Pol γ 3
repair
Lagging strand synthesis; nucleotide
Pol III holoenzyme 9 Chromosome replication Pol δ 2-3
and base excision repair
DNA repair, translesion Leading strand synthesis; nucleotide
Pol IV (DinB) 1 Pol ε 4
synthesis and base excision repair
Pol V (UmuC, 3 Pol Ѳ
Translesion synthesis 1 DNA repair of cross links
UmuD2’C)
Pol σ 1 TLS
Pol λ 1 Meiosis associated DNA repair
Pol μ 1 Somatic hypermutation
Pol κ 1 TLS
Relatively accurate TLS past cis-syn
Pol η 1
cyclobutane dimers
Pol ι 1 TLS, somatic hypermutaion
Rev l 1 TLS

IFAS Publications www.ifasonline.com

8 Molecular Biology

DNA polymerase IV, also called DinB, is encoded by the dinB short RNA chain which must be synthesized on the DNA
gene. DNA polymerase V, also known as the UmuD2’C template before DNA polymerase can start elongation of a
complex, is encoded by the umuDC operon. new DNA chain. DNA polymerases recognize and add dNTPs
to the free 3’-hydroxyl group at the end of the primer. Later
Eukaryotic DNA dependent DNA polymerase:
on same RNA primer is removed and replaced by DNA with
In eukaryotes there are four replicative DNA polymerases
efficient proof reading of DNA polymerase I.
that are involved in DNA replication; out of the hour, three
are DNA polymerase α, δ and DNA polymerase ε involved in How Primase initiates DNA synthesis?
nuclear DNA replication. DNA polymerase α initiates DNA As soon as DNA at origin of replication is opened, the
replication, DNA polymerase δ works on lagging strand and enzymes helicase and primase are recruited for replication
DNA polymerase ε works on leading strand. initiation. For addition of nucleotide by DNA polymerase a
free 3’OH end is needed. This free 3’-OH end is provided by
DNA polymerase γ is involved in mitochondrial and
a short sequence of nucleotides which is referred as primer.
chloroplast DNA replication. Rest of the DNA polymerases
The first step in DNA replication is thus synthesis of primers.
are involved in repair processes as shown in table 2. All
Primase is a specialized RNA polymerase dedicated to
eukaryotic DNA polymerase has 5’3’ polymerase activity
making short RNA primers (5–10 nucleotides long) on an
but 3’5’ proof reading (exonuclease) activity is present
ssDNA template. Primase binds to single-stranded DNA that
only in DNA polymerase α, γ, δ and ε.
is coated with single-stranded binding proteins. Thus, a
What is rate of DNA replication? DNA dependent RNA polymerase can act as primase. On
The rate of bacterial DNA replication is around 500-1000 leading strand, primer is added only once. While Priming
nt/second and mammalian DNA polymerases can add occurs more frequently on lagging strand since DNA
around 50-100 nt/second. The reason for slow rate of replication is semi-discontinuous.The bacterial primers are
polymerization in eukaryotes is that DNA is not readily short RNA stretches of around 5–10 nucleotides that are
accessible for DNA replication (It is bound with histone synthesized by a primase that then hands off replication
proteins). directly to DNA polymerase III. However in eukaryotes, the
How DNA polymerase distinguish ribonucleotides from primase complex contains four subunits: two subunits that
dexoyribonucleotides? function as a primase, bound in a complex with the DNA
Although ribonucleoside triphosphates (rNTPs) are present polymerase α catalytic subunit and an accessory subunit.
at approximately 10-fold higher concentration than (Figure 8)
deoxyribo-nucleoside triphosphates (dNTPs) in the cell but The polymerase α–primase complex synthesizes a stretch of
they are incorporated into DNA at a rate that is more than 10–30 nucleotides of RNA, which the polymerase α subunit
1000-fold lower than dNTPs. This is because DNA of the complex elongates with a short stretch of DNA. After
polymerase efficiently discriminates between rNTPs and this primer synthesis, the replicative polymerases δ or ε
dNTPs, because its nucleotide binding pocket cannot take over and carry out the rest of the elongation phase of
accommodate a 2'-OH on the incoming nucleotide. DNA replication. This handover from polymerase α–primase
Why no DNA polymerase has evolved which can initiate to polymerase δ on lagging strand or polymerase ε on
the replication? leading strand is known as ‘polymerase switching’.
DNA polymerases cannot initiate DNA synthesis is on their Polymerase switch occurs each time a new DNA strand is
own. DNA polymerases can extend the pre-existing strand started on both the leading and the lagging strands (Figure
but they cannot initiate synthesis. An exception to this is 9).
DNA polymerase α, the eukaryotic polymerase which has What is clamp loader?
associated sub-unit which acts as primase involved in Clamp loaders are the special class of protein complexes
primer synthesis. During primase activity proof reading that open up the clamp and also place it on the DNA. The γ,
cannot be performed by DNA polymerase α. δ, and δ’ subunit of DNA polymerase constitute the clamp
If DNA polymerase would have started replication process, loader in prokaryotes while in eukaryotes the protein
then during addition of first two deoxyribonucleotides, Replication Factor C (RFC) is clamp loader. The RFC consists
proof reading cannot be performed as there is no DNA to of five different protein subunits, called Rfc1–5. Clamp
back track. This lack of proofreading would have decreased loader recruits sliding clamp (β-clamp/PCNA) at the primer
fidelity of DNA replication process. So, it better evolutionary DNA junction at the double stranded stretch near the free 3′
decision that DNA replication is initiated by Primase which end which is going to be elongated. The sliding clamp in
add RNA primer without proof reading. A primer is usually a turn recruits the replicative polymerase at the time of

IFAS Publications www.ifasonline.com