DNA Replication Overview

Compiled by

V. Magendira ManiAssistant Professor, PG & Research Department of Biochemistry,Islamiah College (Autonomous),Vaniyambadi,Vellore District – 6357512,Tamilnadu, India.magendiramani@rediffmail.com

https://tvuni.academia.edu/mvinayagam

Structure of DNADNA, or deoxyribonucleic acid, is the hereditary

material in humans and almost all other

organisms. Nearly every cell in a person’s body

has the same DNA. Most DNA is located in the

cell nucleus (where it is called nuclear DNA), but

a small amount of DNA can also be found in the

mitochondria (where it is called mitochondrial

DNA or mtDNA). DNA is found in the nucleus of

eukaryotes and the cytoplasm or nucleoid of

prokaryotes and functions as the molecule of

heredity.

In DNA there are four bases: adenine

(abbreviated A), guanine (G), thymine (T) and

cytosine (C). Adenine and guanine are purines;

thymine and cytosine are pyrimidines.

A nucleoside is a pyrimidine or purine base

covalently bonded to a sugar. In DNA, the sugar is

deoxyribose and so this is a deoxynucleoside.

There are four types of deoxynucleoside in DNA;

deoxyadenosine, deoxyguanosine, deoxythymidine

and deoxycytidine.

A nucleotide is base + sugar + phosphate

covalently bonded together. In DNA, where the

sugar is deoxyribose, this unit is a

deoxynucleotide.

In DNA the nucleotides are covalently joined

together by 3’-- 5’ phosphodiester bonds to form a

repetitive sugar–phosphate chain which is the

backbone to which the bases are attached.

The DNA sequence is the sequence of A, C, G and T

along the DNA molecule which carries the genetic

information.

In a DNA double helix, the two strands of DNA are

wound round each other with the bases on the inside

and the sugar–phosphate backbones on the outside.

The two DNA chains are held together by hydrogen

bonds between pairs of bases; adenine (A) always

pairs with thymine (T) and guanine (G) always pairs

with cytosine (C).

Each nucleotide consists of three major parts: (1) a

five-carbon sugar (pentose); (2) a flat, heterocyclic,

nitrogen-containing organic base; and (3) a negatively

charged phosphate group, which gives the polymer its

acidic property. The nitrogenous base in each

nucleotide is covalently attached to the sugar by a

glycosidic bond. The phosphate group is also

covalently linked to the sugar.

Bases in the nucleotides spontaneously form hydrogen

bonds in a highly specific manner. Adenine normally

forms two hydrogen bonds with thymine in a

complementary strand of the DNA double helix like,

Guanine forms three hydrogen bonds with cytosine.

Human DNA consists of about 3 billion bases, and more

than 99 percent of those bases are the same in all

people.

Nucleotides are arranged in two long strands that form

a spiral called a double helix. The structure of the

double helix is somewhat like a ladder, with the base

pairs forming the ladder’s rungs and the sugar and

phosphate molecules forming the vertical sidepieces of

the ladder.

An important property of DNA is that it can

replicate, or make copies of itself. Each strand

of DNA in the double helix can serve as a

pattern for duplicating the sequence of bases.

This is critical when cells divide because each

new cell needs to have an exact copy of the

DNA present in the old cell.

Watson and Crick model of DNA

DNA is a double stranded helix, with the two

strands connected by hydrogen bonds. Adenine

bases are always paired with thymine, and

cytosine is always paired with guanine, which is

consistent with and accounts for Chargaff's

rule. This is called complementary base pairing.

Watson and Crick discovered that DNA had two

sides, or strands, and that these strands were

twisted together like a twisted ladder the

double helix.

Most DNA double helices are right handed, only

one type of DNA, called ZDNA, is left handed.

The G:C and A:T base pairing also maximizes the

number of effective hydrogen bonds that can form

between the bases; there are three hydrogen

bonds between each G:C base pair and two

hydrogen bonds between each A:T base pair. Thus

A:T and G:C base pairs form the most stable

conformation both from steric considerations and

from the point of view of maximizing hydrogen

bond formation.

The sides of the ladder comprise the sugar

phosphate portions of adjacent nucleotides bonded

together. The phosphate of one nucleotide is

covalently bound to the sugar of the next

nucleotide.

The hydrogen bonds between phosphates cause the DNA

strand to twist.

The DNA double helix is antiparallel, which means

that the 5' end of one strand is paired with the 3'

end of its complementary strand (and vice versa).

As shown in Figure, nucleotides are linked to each

other by their phosphate groups, which bind the 3'

end of one sugar to the 5' end of the next sugar.

Along the whole length of the DNA molecule,

there are two depressions—referred to as the

“minor groove” and the “major groove”—that lie

between the strands.

In a DNA molecule, the different nucleotides are

covalently joined to form a long polymer chain by

covalent bonding between the phosphates and

sugars.

For any one nucleotide, the phosphate attached

to the hydroxyl group at the 5’ position of the

sugar is in turn bonded to the hydroxyl group on

the 3’ carbon of the sugar of the next nucleotide.

Since each phosphate–hydroxyl bond is an ester

bond, the linkage between the two

deoxynucleotides is a 3’5’ phosphodiester bond.

Thus, in a DNA chain, all of the 3’ and 5’ hydroxyl

groups are involved in phosphodiester bonds

except for the first and the last nucleotide in the

chain. The first nucleotide has a 5’ phosphate not

bonded to any other nucleotide and the last

nucleotide has a free 3’ hydroxyl. Thus each DNA

chain has polarity; it has a 5’ end and a 3’ end.

Erwin Chargaff was one of a handful of

scientists who expanded on Levene's work by

uncovering additional details of the structure

of DNA, thus further paving the way for

Watson and Crick.

A, B and Z forms of DNA

The Watson-Crick structure is also referred to as

B form DNA, or B-DNA. The B form is the most

stable structure for a random-sequence DNA

molecule under physiological conditions and is

therefore the standard point of reference in any

study of the properties of DNA. Two structural

variants that have been well characterized in

crystal structures are the A and Z forms. These

three DNA conformations are shown in Figure,

with a summary of their properties.

The A form is favored in many solutions that are

relatively devoid of water. The DNA is still

arranged in a right-handed double helix, but the

helix is wider and the number of base pairs per

helical turn is 11, rather than 10.5 as in B-DNA.

The plane of the base pairs in A-DNA is tilted

about 20with respect to the helix axis. These

structural changes deepen the major groove while

making the minor groove shallower. The reagents

used to promote crystallization of DNA tend to

dehydrate it, and thus most short DNA molecules

tend to crystallize in the A form.

Z-form DNA is a more radical departure from the B

structure; the most obvious distinction is the left handed

helical rotation. There are 12 base pairs per helical turn,

and the structure appears more slender and elongated.

The DNA backbone takes on a zigzag appearance.

Certain nucleotide sequences fold into left handed Z

helices much more readily than others. Prominent

examples are sequences in which pyrimidines alternate

with purines, especially alternating C and G or 5-methyl-

C and G residues. To form the left-handed helix in Z-

DNA, the purine residues flip to the syn conformation,

alternating with pyrimidines in the anti-conformation.

The major groove is barely apparent inZ-DNA, and the

minor groove is narrow and deep. Whether A-DNA

occurs in cells is uncertain, but there is evidence for

some short stretches (tracts) of Z-DNA in both

prokaryotes and eukaryotes. These Z-DNA tracts may

play a role (as yet undefined) in regulating the

expression of some genes or in genetic recombination.

Chargaff Rules The nucleotide composition of DNA varies

among species

The amount of adenine (A) is usually

similar to the amount of thymine (T), and

the amount of guanine (G) usually

approximates the amount of cytosine (C).

The total amount of purines (A + G) and

the total amount of pyrimidines (C + T) are

usually nearly equal.

DNA “semi-conservative”

Replication

 DNA replication is the process by which

the genetic material is copied prior to

distrubution into daughter cells

The original DNA strands are used as

templates for the synthesis of new strands

It occurs very quickly, very accurately and at

the appropriate time in the life cycle of the

cell

 DNA replication relies on the

complementarity of DNA strands

The AT/GC rule or Chargaff’s rule

 

In the semi-conservative model, the two

parental strands separate and each makes a copy

of itself. After one round of replication, the two

daughter molecules each comprise one old and

one new strand. Note that after two rounds, two

of the DNA molecules consist only of new

material, while the other two contain one old and

one new strand. 

The semi-conservative model is the spontaneously

appealing model, because separation of the two

strands provides two templates, each of which

carries all the information of the original

molecule. It also turns out to be the correct one

(Meselson & Stahl, 1958).

The process can be summarized as follows:

The two DNA strands in the parent DNA

molecule come apart

Each “parent strand” then serves as a

template for the synthesis of a new

complementary strand

The two newly-made strands =

daughter strands

The two original ones = parental

strands

This process is called semi-conservative because it

conserves only half of the original (parent) DNA

molecule in the two daughter DNA molecules. One

strand in each daughter molecule is completely

new.

In the late 1950s, three different mechanisms were

proposed for the replication of DNA

Conservative model

• Both parental strands stay together after

DNA replication and one of the daughter

molecules contains all new nucleotides

Semiconservative model

• The double-stranded DNA contains one

parental and one daughter strand following

replication

Dispersive model

• Parental and daughter DNA are interspersed

in both strands following replication

In 1958, Matthew Meselson and Franklin

Stahl devised a method to investigate these

models.

Their experiment can be summarized as follows:

Grow E. coli in the presence of 15N (a heavy

isotope of nitrogen) for many generations

The population of cells now has heavy-labeled

DNA because the DNA bases are rich in

nitrogen

Switch E. coli to medium containing only 14N

(a light isotope of nitrogen)

Collect sample of cells after various times

Analyze the density of the DNA by

centrifugation using a CsCl gradient

 

The actual data from the Mesleson-Stahl

experiment is shown below.

After one generation, DNA is “half-heavy”

After ~ two generations, DNA is of two types: “light” and “half-heavy”

This is consistent with only the semi-conservative model

The Meselson–Stahl experiment was an experiment

by Matthew Meselson and Franklin Stahl in

1958 which supported the hypothesis that DNA

replication was semiconservative. Meselson and

Stahl decided the best way to tag the parent DNA

would be to change one of the atoms in the parent

DNA molecule. Remember that nitrogen is found in

the nitrogenous bases of each nucleotide. So they

decided to use an isotope of nitrogen to distinguish

between parent and newly-copied DNA. The isotope

of nitrogen had an extra neutron in the nucleus,

which made it heavier.

Three hypotheses had been previously proposed for

the method of replication of DNA.

In the semiconservative hypothesis, proposed by

Watson and Crick, the two strands of a DNA

molecule separate during replication. Each strand

then acts as a template for synthesis of a new

strand.

The conservative hypothesis proposed that the

entire DNA molecule acted as a template for the

synthesis of an entirely new one. In the

conservative model, the parental molecule directs

synthesis of an entirely new double-stranded

molecule, such that after one round of

replication, one molecule is conserved as two old

strands. This is repeated in the second round.

The dispersive hypothesis is exemplified by a

model proposed by Max Delbrück, which

attempts to solve the problem of unwinding the

two strands of the double helix by a mechanism

that breaks the DNA backbone every 10

nucleotides or so, untwists the molecule, and

attaches the old strand to the end of the newly

synthesized one. This would synthesize the DNA

in short pieces alternating from one strand to the

other.

Each of these three models makes a different

prediction about the distribution of the "old"

DNA in molecules formed after replication. In

the conservative hypothesis, after replication,

one molecule is the entirely conserved "old"

molecule, and the other is all newly

synthesized DNA. The semiconservative

hypothesis predicts that each molecule after

replication will contain one old and one new

strand. The dispersive model predicts that

each strand of each new molecule will contain

a mixture of old and new DNA.

Experimental procedure and

results Matthew Meselson and Franklin Stahl were well

acquainted with these three predictions, and

they reasoned that if there were a way to

distinguish old versus new DNA, it should be

possible to test each prediction. Aware of

previous studies that had relied on isotope labels

as a way to differentiate between parental and

progeny molecules, the scientists decided to see

whether the same technique could be used to

differentiate between parental and progeny

DNA. If it could, Meselson and Stahl were

hopeful that they would be able to determine

which prediction and replication model was

correct.

Meselson & Stahl began their experiment by

choosing two isotopes of nitrogen -the common

and lighter 14N, and the rare and heavier 15N (so-

called "heavy" nitrogen) - as their labels and a

technique known as cesium chloride (CsCl)

equilibrium density gradient centrifugation as

their sedimentation method. Meselson and Stahl

opted for nitrogen because it is an essential

chemical component of DNA; therefore, every time

a cell divides and its DNA replicates, it

incorporates new N atoms into the DNA of either

one or both of its two daughter cells, depending on

which model was correct. "If several different

density species of DNA are present," they

predicted, "each will form a band at the position

where the density of the CsCl solution is equal to

the buoyant density of that species. In this way,

DNA labeled with heavy nitrogen (15N) may be

resolved from unlabeled DNA" (Meselson & Stahl,

1958).

The scientists then continued their experiment

by growing a culture of E. coli bacteria in a

medium that had the heavier 15N (in the form

of 15N-labeled ammonium chloride) as its only

source of nitrogen. In fact, they did this for 14

bacterial generations, which was long enough

to create a population of bacterial cells that

contained only the heavier isotope (all the

original 14N-containing cells had died by then).

Next, they changed the medium to one

containing only 14N-labeled ammonium salts as

the sole nitrogen source. So, from that point

onward, every new strand of DNA would be

built with 14N rather than 15N.

Just prior to the addition of 14N and periodically

thereafter, as the bacterial cells grew and

replicated, Meselson and Stahl sampled DNA for

use in equilibrium density gradient

centrifugation to determine how much 15N (from

the original or old DNA) versus 14N (from the

new DNA) was present. For the centrifugation

procedure, they mixed the DNA samples with a

solution of cesium chloride and then centrifuged

the samples for enough time to allow the

heavier 15N and lighter 14N DNA to migrate to

different positions in the centrifuge tube.

By way of centrifugation, the scientists found that

DNA composed entirely of 15N -labeled DNA (i.e.,

DNA collected just prior to changing the culture

from one containing only 15N to one containing

only 14N) formed a single distinct band, because

both of its strands were made entirely in the

"heavy" nitrogen medium. Following a single

round of replication, the DNA again formed a

single distinct band, but the band was located in a

different position along the centrifugation

gradient. Specifically, it was found midway

between where all the 15N and the entire 14N DNA

would have migrated-in other words, halfway

between "heavy" and "light".

Based on these findings, the scientists were

immediately able to exclude the

conservative model of replication as a

possibility. After all, if DNA replicated

conservatively, there should have been two

distinct bands after a single round of

replication; half of the new DNA would have

migrated to the same position as it did

before the culture was transferred to the

14N-containing medium (i.e., to the "heavy"

position), and only the other half would have

migrated to the new position (i.e., to the

"light" position). That left the scientists with

only two options: either DNA replicated

semi-conservatively, as Watson and Crick

had predicted, or it replicated dispersively.

To differentiate between the two, Meselson and Stahl

had to let the cells divide again and then sample the

DNA after a second round of replication. After that

second round of replication, the scientists found that

the DNA separated into two distinct bands: one in a

position where DNA containing only 14N would be

expected to migrate, and the other in a position

where hybrid DNA (containing half 14N and half 15N)

would be expected to migrate. The scientists

continued to observe the same two bands after

several subsequent rounds of replication. These

results were consistent with the semiconservative

model of replication and the reality that, when DNA

replicated, each new double helix was built with one

old strand and one new strand. If the dispersive

model were the correct model, the scientists would

have continued to observe only a single band after

every round of replication.

Rolling circle replication Rolling circle replication describes a process of

unidirectional nucleic acid replication that can

rapidly synthesize multiple copies of circular

molecules of DNA or RNA, such as plasmids,

the genomes of bacteriophages, and

the circular RNA genome of viroids. Some

eukaryotic viruses also replicate their DNA via

a rolling circle mechanism.

Rolling circle DNA replication is initiated by an

initiator protein encoded by the plasmid or

bacteriophage DNA, which nicks one strand of

the double-stranded, circular DNA molecule at a

site called the double-strand origin, or DSO. The

initiator protein remains bound to the 5'

phosphate end of the nicked strand, and the free

3' hydroxyl end is released to serve as a primer

for DNA synthesis by DNA polymerase III. Using

the un-nicked strand as a template, replication

proceeds around the circular DNA molecule,

displacing the nicked strand as single-stranded

DNA. Displacement of the nicked strand is

carried out by a host-encoded helicase called

PcrA (the abbreviation standing for plasmid copy

reduced) in the presence of the plasmid

replication initiation protein.

Continued DNA synthesis can produce multiple

single-stranded linear copies of the original DNA in

a continuous head-to-tail series called a concatemer.

These linear copies can be converted to double-

stranded circular molecules through the following

process:

First, the initiator protein makes another nick to

terminate synthesis of the first (leading) strand.

RNA polymerase and DNA polymerase III then

replicate the single-stranded origin (SSO) DNA to

make another double-stranded circle. DNA

polymerase I removes the primer, replacing it with

DNA, and DNA ligase joins the ends to make

another molecule of double-stranded circular DNA.

Rolling circle replication has found wide uses in

academic research and biotechnology, and has

been successfully used for amplification of DNA

from very small amounts of starting material.

Some viruses replicate their DNA in host cells

via rolling circle replication. For instance,

human herpesvirus-6 (HHV-6) (hibv) expresses a

set of “early genes” that are believed to be

involved in this process. The long concatemers

that result are subsequently cleaved between

the pac-1 and pac-2 regions of HHV-6's genome

by ribozymes when it is packaged into individual

virions.

John Cairns Experiment

Cairns grew E.Coli bacteria in a medium

containing radioactive thymine, a component of

one of the DNA nucleotides. The radioactivity

was in tritium (31H). The DNA was then carefully

extracted from the bacteria and placed on

photographic emulsion for a period of time. The

emulsion was then developed to produce

autoradiograph that was examined under the

electron microscope. Each grain of silver

represents a radioactive decay. Interpretation of

this autoradiograph reveals several points.

The first, known at the time, is that the E.Coli DNA is a circle. The Second point is that DNA is replicated while maintaining the integrity of the circle i.e., the circle does not appear to be broken in the process of DNA replication; an intermediate theta structure is formed which is due to the formation of replication eye. Third, replication of the DNA seems to be occurring at one or two moving Y-junctions in the circle Replication forks, which further supports the Semiconservative replication.

DNA

REPLICATION

IN

PROKARYOTES

DNA Replication in Bacteria

(E.Coli) DNA synthesis begins at a site termed the origin

of replication (“Ori -C”) Each bacterial chromosome has only one ori- C Synthesis of DNA proceeds bi-directionally

around the bacterial chromosome The “replication forks” eventually meet at the

opposite side of the bacterial chromosome This ends replication

 Bacterial DNA replication has been studied most

extensively in E. coli, the favorite bacterial

“model organism” of molecular geneticists.

The ORI in E. coli is called “oriC” Three types of DNA sequences in oriC are

functionally significant AT-rich region DnaA boxes GATC methylation sites

DNA replication is initiated by the binding of

DnaA proteins to the DnaA box sequences

• This binding stimulates the cooperative

binding of an additional 20 to 40 DnaA

proteins to form a large complex.

• This causes the DNA to twist and the puts

torque on the nearby AT-rich region to

denature and form a replication bubble

AT base pairs are held together by only 2

H bonds

CG base pairs are held together by 3 H

bonds

Therefore, AT-rich regions of DNA

denature more easily than CG-rich regions

of DNA

In the next step, DnaB (also called helicase)

binds to each strand of the separated double

helix. It’s job is to move along the DNA,

progressively expand the replication bubble in

both directions.Travels along the DNA

strand in the 5’ to 3’

direction, using energy

from ATP

As the helicases move on each strand in opposite

directions, two replication forks are created. These

forks move progressively farther and farther in each

direction as the bubble widens.

DNA helicase separates the two DNA strands by

breaking the hydrogen bonds between them

This generates positive supercoiling ahead of

each replication fork so another enzyme,

topoisomerase, travels ahead of the helicase

and alleviates these supercoils

 Single-strand binding proteins (SSBPs) are also

needed to bind to the separated DNA strands and

keep them apart

Otherwise, the strands would simply reanneal

 After the helicase, gyrase, and SSBPs are in place,

short (10 to 12 nucleotides) RNA primers are

synthesized by DNA primase

These short RNA strands start, or prime, DNA

synthesis because DNA polymerase, the enzyme

that copies DNA, cannot start a new strand on its

own

The RNA primers are later removed and replaced

with DNA

Keep the parental strands apart

Breaks the hydrogen bonds between the two strands

Alleviates supercoiling

Synthesizes an RNA primer

DNA Polymerases

DNA polymerases are the enzymes that catalyze the

attachment of nucleotides to make new DNA

 In E. coli there are five proteins with polymerase

activity

DNA pol I

• Composed of a single polypeptide

• Removes the RNA primers and replaces

them with DNA during DNA replication

DNA pol III

Composed of 10 different subunits

The a subunit synthesizes DNA

The other 9 fulfill other functions

The complex of all 10 is referred to as

the “DNA pol III holoenzyme”

Is responsible for most of the DNA

replication process

DNA pol II, IV and V

Specialized DNA polymerases that

replicate short areas of DNA for the

purposes of genome repair

 The numbering of these polymerases was done in

the order they were discovered

 Bacterial DNA polymerases may vary in their

subunit composition. However, they have the same

type of catalytic subunit.

Structure resembles a human

right hand:

Thumb and fingers wrapped around the

DNA

Template DNA thread through the palm;

All DNA polymerases, whether bacterial or

eukaryotic, share 2 very important limitations:

 1. They cannot initiate DNA synthesis on their

own. They require that an RNA primer be laid

down on the DNA first by DNA primase.

They can only “grow” a new DNA chain in the 5’ to 3’ direction.

It is not fully understood why all DNA

polymerases have these limitations. As will be

demonstrated below, DNA replication would

be much simpler if they did not!

 Because DNA polymerase can only synthesize

a new strand 5’ to 3’, the two new daughter

strands are synthesized in different ways:

Leading strand

One RNA primer is made at the origin

DNA pol III attaches nucleotides in a 5’ to 3’

direction as it slides toward the replication fork

Lagging strand

Synthesis is also in the 5’ to 3’ direction

However it occurs away from the replication fork

Many RNA primers are required

DNA pol III uses the RNA primers to synthesize small

DNA fragments (1000 to 2000 nucleotides each)

These are termed Okazaki fragments after their

discoverers

DNA pol I removes the RNA primers and fills the

resulting gap with DNA

After the gap is filled, a covalent bond is still

missing so

DNA ligase must create this bond

Can be synthesized continuously in the 5’ to 3’ direction

Must be synthesized discontinuously to

maintain 5’ to 3’ synthesis

Note that if DNA polymerase was able to

synthesize a new strand in either direction (5’

to 3’ or 3’ to 5’), lagging strand synthesis and

Okasaki fragments would not be needed.

 The process can also be visualized in 3-D as

follows:

The Synthesis Reaction

 

DNA polymerases catalyze a phosphodiester

bond between the innermost phosphate group of

the incoming deoxynucleoside triphosphate and

the

3’-OH of the sugar of the previous

deoxynucleotide.

  In the process, the last two phosphates

of the incoming nucleotide are released in the

form of pyrophosphate (PPi)

 In E. coli, DNA pol III stays on the DNA

template long enough to polymerize up to 50,000

nucleotides at a rate of ~ 750 nucleotides per

second!

Proofreading

 DNA replication exhibits a high degree of

fidelity.

 Mistakes during the process are extremely rare

In E. coli, DNA pol III makes only one

mistake per 108 bases

There are several reasons why fidelity is high:

 1. Instability of mismatched pairs

Complementary base pairs have much higher

stability than mismatched pairs

This feature only accounts for part of the

fidelity

It has an error rate of 1 per 1,000

nucleotides

 2. Configuration of the DNA polymerase active

site

DNA polymerase is unlikely to catalyze bond

formation between mismatched pairs

This induced-fit phenomenon decreases the

error rate to a range of 1 in 100,000 to 1

million

 

3. Proofreading function of DNA polymeraseDNA polymerases can identify a mismatched nucleotide and remove it from the daughter strandThe enzyme uses its 3’ to 5’ exonuclease activity to remove the incorrect nucleotideIt then changes direction and resumes DNA synthesis in the 5’ to 3’ direction

Termination of Replication in BacteriaDNA replication ends when oppositely advancing forks meet (remember that the chromosome is circular).• DNA replication often results in two intertwined molecules called catenanes• Catenanes and are separated prior to cell division Replicatio

nDecatenization

DNA

REPLICATION

IN

EUKARYOTES

DNA Replication in Eukaryotes Eukaryotic DNA replication is not as well

understood as bacterial replication.

•  The two processes do have extensive

similarities,

• Many of the bacterial enzymes described

above have also been found in eukaryotes

 Nevertheless, DNA replication in eukaryotes

is more complex due to:

• Large linear chromosomes

• Multiple origins of replication per

chromosome

• Tight packaging of the DNA around

proteins

  

Linear eukaryotic chromosomes also have

telomeres at both ends

The term telomere refers to the complex of

repetitive DNA sequences found at the terminal

ends of eukaryotic chromosomes as well as the

proteins that recognize this sequence and bind

the DNA there.

 Telomeric sequences consist of

• Moderately repetitive tandem arrays

• 3’ overhang that is 12-16 nucleotides long that

results from the loss of the RNA primer at the 5’

end of each strand that cannot be replaced

Therefore if this problem is not solved:

• The linear chromosome becomes

progressively shorter with each round of

DNA replication

• Indeed, some cells solve this problem by

adding DNA sequences to the ends of

telomeres following replication

• This requires a specialized mechanism

catalyzed by the enzyme telomerase

• All single-celled eukaryotes have active

telomerase enzyme because if they

didn’t successive generations of the

organism would have shortened

telomeres

Eventually, this would result in the

loss of important genes and the

death of the species

However, most somatic cells in multicellular

organisms do not express telomerase and the

telomeres shorten every time the cells

replicate.

• Most human somatic cells can only

replicate about 30 times before their

telomeres are so shortened that the

cell dies

This sets an upper limit on the life

span of the organism

Telomerase is active in the germ line cells,

maintaining telomere length from one

generation to the next.

 Telomerase is also often abnormally

activated in cancer cells. This is why tumors

don’t eventually replicate themselves to

death. Once a tumor cell has activated

telomerase, it is immortalized.

 

Immortalized somatic cells are extremely

dangerous in multicellular organisms. If a cell

suffers a mutation in a gene controlling the cell

cycle, the cell can begin to replicate much

faster than the surrounding cells.

Normally, such cells will die off when their

telomeres get too short

Immortalized cells will continue to cycle and

the tissue will grow

The result can be a cancerous tumor that is

life-threatening to the organism

 

EUKARYOTIC DNA REPLICATION

DNA replication is the process of producing two

identical replicas from one original DNA molecule.

This biological process occurs in all living organisms

and is the basis for biological inheritance. DNA is

made up of two strands and each strand of the original

DNA molecule serves as template for the production

of the complementary strand, a process referred to as

semiconservative replication. The fundamental

mechanism of eukaryotic replication is same as

prokaryotic DNA Replication but some variation also

there.

The replications in eukaryotes are more complex. Because

DNA molecule of eukaryote

Eukaryotic genomes are quite complex

Considerably larger than bacterial DNA

Organized into complex nucleoprotein structure

(chromatin)

Essential features of DNA replication are the same in

prokaryotes and eukaryotes, Similarities of prokaryotes and

eukaryotic replication

Replication process is fundamentally similar in both

prokaryotes and eukaryotes. Process that are similar Include

Formation of replication fork

Simi conservative replication

Movement of replication fork bidirectional

Primer synthesis

Okazaki fragment synthesis in lagging strand

Primer removal

Gap bridging between newly synthesized DNA

fragments.

Difference between prokaryotic and eukaryotic replication

Overall process of eukaryotic replication is bit more complex.

Important differences are due to

• Larger size of eukaryotic DNA (105-106 Kb) compared to

prokaryotic DNA 15x103 kb in E.Coli

• Distinct package of eukaryotic DNA in the term of chromatin

• Slower rate of fork movement in eukaryotes

For DNA to become available to DNA polymerase, nucleotide

must dissemble. This step slows the Rate of fork movement.

Replication rate:

Prokaryotes: An E.Coli replication fork progresses at

approximately 1000 bp / sec.

Eukaryotes: Replication rate ten times slower than prokaryotes

50 nucleotides / sec.

Enzymes and proteins required for eukaryotic DNA

replication

Eukaryotic DNA polymerase:

In eukaryotes there are five different polymerases and they

differ in

Intracellular compartmentation

Kinetic property

Response to inhibitor

DNA pol location functionDNA Pol α nucleus lagging strand

synthesisDNA Pol β nucleus DNA repair

DNA Pol ϒ mitochondria mitochondrial replication

DNA Pol δ nucleus leading strand synthesis

DNA Pol Ɛ nucleus gap filling between okazaki fragments

DNA polymerases location function

DNA Pol alpha nucleusDNA replication initiation (both leading

and lagging strand)

DNA Pol Delta nucleus lagging strand synthesis

DNA Pol Epsilon nucleusleading strand synthesis

DNA polymerase Alpha

- Located in nucleus

- Catalysis the initiation of replication on both

leading and lagging strand synthesis

- Tetramer – 4 subunits POLA 1 (catalytic) POLA 1

(regulatory) POLA 3 ,4 (Primase)

- larger subunit - 5´-3´ polymerization activity

-Two smaller subunit – primase activity

- one subunit – assist in other three

subunits

- RNA primer 5-15 nucleotides are subsequently

extended by DNA Pol α.

DNA polymerase Delta

- Located in nucleus

- Catalyzes the synthesis of lagging strand

- Having four subunits – POLD 1,2,3,4

- larger subunits catalyzes 5´-3´ polymerization

activity

- Smaller subunits catalyzes 3´-5´ exonuclease

activity (proof reading activity)

- High processivity when interacting with PCNA

(Proliferating cell nuclear antigen).

PCNA

- Molecular weight 25,000; PCNA is important for both

DNA synthesis and DNA repair

- Multimeric protein

- Found in large amount in nuclei of proliferating

cells.

- Act as “clamp” to keep DNA pol δ from

dissociating off the leading DNA strand. “Clamp”

consist of 3 PCNA molecules each containing two

topologically identical domains that are tightly associated to

form closed ring.

- PCNA helps hold DNA polymerase epsilon (Pol ε)

to DNA.

- DNA pol δ improves fidelity of replication by a

factor of 102 due to its proof reading action. It contributes in

limiting the rates of overall error to 10-9 to 10-12.

- DNA Pol δ is also associated with helicase activity.

DNA polymerase Epsilon - Є

located in nucleus

Having four subunits – POLE 1, (Catalytic)

2,3,4 (subunits)associated with - 5´- 3´ polymerization activity 5’- 3’ exonuclease activity (to remove RNA primer) 3’- 5’ exonuclease activity (to proof read)

DNA pol Є catalyzes the repair mechanism

and catalyzes the removal of primer and

filing the primer gap in Okazaki fragments.

Replicating factor A/ Replicating protein

A (RPA/RFA)

RPA/ RFA are similar to single strand binding

protein. They bind to SS DNA and prevent

the re-annealing of parental DNA.

Replication factor C (RFC)

RFC also called as clamp loader or match

maker.

RFC assist in DNA pol δ to form clamp

between DNA and PCNA.

RFC also plays important role in setting up a

link between DNA pol δ and DNA pol α, so

that the leading strand synthesis and lagging

strand synthesis in eukaryotes can take place

simultaneously.

Histone Dissociation and Association Since DNA is present in packaged form as chromatin, DNA replication is sandwiched between two additional steps in eukaryotes.1. Carefully ordered and in complete dissociation of the chromatin.2. Re-association of DNA with the histone octomers to form nucleosome.Dissociation of histone: methylation at the fifth position of cytosine residues by a DNA methyl transferase appears to functioning by loosening up the chromatin structure. This allows DNA access to proteins and enzymes needed for DNA replication.Synthesis of histone: the synthesis of new histone occurs simultaneously with DNA replication.

SEQUENTIAL STEPS IN EUKARYOTIC DNA REPLICATIONDNA replication is a very complicated process that involves several enzymes and other proteins. It occurs in following stages

• Pre-initiation• Initiation• Elongation• Termination• Telomerase function

PRE-INITIATION

Actually 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

multiprotein complex at each replicator in the

genome. Origin activation only occurs after cells enter

S phase and triggers the Replicator – associated

protein complex to 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 (cell division cycle protein - 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 Gl

to the S phase of the cell cycle.

Figure - The steps in the formation of pre-replicative complex (pre-RC)The assembly of the pre-RC is an ordered process that is initiated by the association of the ORC with the replicator. Once bound to the replicator ORC recruits at least two additional proteins Cdc6 and Cdt1 (cell division cycle proteins). These three proteins function together to recruit the putative eukaryotic DNA helicase- the MCM 2-7 (multi-chromatin maintenance protein) complex to complete the formation of the pre-RC

INITIATION

ARS (Autonomously Replicating

Sequences)

In eukaryotes the DNA replication is initiated at

specific site known as ARS

(Autonomously Replication Sequences) or

replicators.

ARS (Origin of chromosome in eukaryotes)

contains

- A central core sequence which contains

highly conserved 11 bp sequence (AT rich

sequence)

- Flanking sequences.

ARS – is 100- 150 long (generally it span about

150 bp)

There are multiple origins in eukaryotes. Eg:

yeast contains 400 ARS. The multiple origins are

spaced 30 -300 kb apart. The sequence between

two origins of replication is called replicons. An

average human chromosome contains as many as

100 replicons and replication may proceed

simultaneously at as many as 200 forks.

- The central core sequence contains 11 bp

elements known as “ARS consensus sequence”

rich in AT pair (It is similar to AT rich 13 mers

present in E.Coli Ori C). It is also

called as ORE (Origin replication element)

- The flanking sequences consist of

overlapping sequence that include varients of core

sequences

ORE (Origin Replicating Element) and

ORC (Origin Recognition Complex)

At the origin there is an association of

sequence specified – ds DNA binding

sequence.

ORE (11 bp sequence in core sequence) binds

to a set of proteins (DNA pol α, DNA pol δ,

RFC, PCNA, RFA, SSB and helicase)

collectively called as ORC Origin Recognition

Complex

ORC is a multimeric protein. Initiation of

replication in all eukaryotes requires this

multimeric subunit protein (ORC) which binds

to several sequences within the replicator.

DUE (DNA Unwinding Element)

ORE located adjacent to approximately 80 bp

AT rich sequence that is easy to unwind.

This is called DUE (DNA Unwinding

Element) Binding of ORC to ORE causes

unwinding at DUE.

Events in replication fork:

When ORC (DNA pol α, DNA pol δ, RFC,

RFA, PCNA, SSB helicase into the origin of

replication especially at ORE, the DNA

synthesis is initiated. The replication fork

moves bi-directionally and replication

proceeds simultaneously as many as 200

forks.

Formation of replication fork:

The replication fork in eukaryotes consists of

four components that form in the following

sequence.

DNA helicase and DNA pol δ (due to its

associated helicase activity) unwinds short

segment of parental DNA at 80 bp AT rich

sequence called DUE (DNA unwinding elements)

which is located adjacent to ORE.

DNA pol α initiated the synthesis of RNA primer.

(DNA pol α is also having primase activity) The

primer is approximately 10 bp.

DNA pol ε in lagging strand and DNA pol δ in

leading strand initiates the daughter strand

synthesis.

SSB and RFA bind to SS DNA and prevent re-

annealing of SS DNA.

In addition to the above, two additional factors play important role in replication of eukaryotesPCNA (proliferating cell nuclear antigen) act as a ‘’clamp’’ to keep DNA pol δ from dissociating off the leading strand and thus increasing the processing of DNA pol ε.RFC also called as ‘clamp loader’ or ‘match maker’.RFC assist in - DNA pol δ to form clamp between DNA and PCNA and - setting up a link between DNA pol δ and DNA pol ε so that the leading Strand and lagging strand synthesis in eukaryotes can take place simultaneously. 

Rate of Replication fork Movement

The rate of replication fork movement in

eukaryote (approximately 50 nucleotide /sec) is

only one tenth that observed in E.Coli at this

rate, replication of an average human

chromosome proceeding from a single origin

would take more than 500 hours. Instead of

that, replication of human chromosome

proceeds bi-directionally from multiple origins

spaced 30-300 kb apart and completed within an

hour.

DNA sequence between two origins of

replication is called replicons. An average

chromosome contains nearly 100 replicons and

thus replication proceeds simultaneously at as

many as 200 forks.

ELONGATION

During elongation, an enzyme called DNA

polymerase adds DNA nucleotides to the 3' end

of the newly synthesized polynucleotide strand.

The template strand specifies which of the four

DNA nucleotides (A, T, C, or G) is added at each

position along the new chain. Only the

nucleotide complementary to the template

nucleotide at that position is added to the new

strand. For example, when DNA polymerase

meets an adenosine nucleotide on the template

strand, it adds a thymidine to the 3' end of the

newly synthesized strand, and then moves to

the next nucleotide on the template strand. This

process will continue until the DNA polymerase

reaches the end of the template strand.

All newly synthesized polynucleotide strands

must be initiated by a specialized RNA

polymerase called primase. Primase initiates

polynucleotide synthesis and by creating a short

RNA polynucleotide strand complementary to

template DNA strand. This short stretch of RNA

nucleotides is called the primer. Once RNA

primer has been synthesized at the template

DNA, primase exits, and DNA polymerase extends

the new strand with nucleotides complementary

to the template DNA. Eventually, the RNA

nucleotides in the primer are removed and

replaced with DNA nucleotides. Once DNA

replication is finished, the daughter molecules

are made entirely of continuous DNA nucleotides,

with no RNA portions.

The Leading and Lagging Strands

DNA polymerase can only synthesize new

strands in the 5' to 3' direction. Therefore, the

two newly synthesized strands grow in opposite

directions because the template strands at each

replication fork are antiparallel. The "leading

strand" is synthesized continuously toward the

replication fork as helicase unwinds the template

double stranded DNA.

The "lagging strand" is synthesized in the

direction away from the replication fork and

away from the DNA helicase unwinds. This

lagging strand is synthesized in pieces because

the DNA polymerase can only synthesize in the 5'

to 3' direction, and so it constantly encounters

the previously synthesized new strand. The

pieces are called Okazaki fragments, and each

fragment begins with its own RNA primer.

Leading strand synthesis:- Leading strand synthesis is initiated upon RNA primer, synthesized by the primase subunit of DNA pol α. The RNA primer contains 10-15 nucleotides. - Then DNA pol α adds a stretch of DNA to the primer.- At this point replication factor C (RFC) carries out a process called polymerase switching.- RFC removes DNA pol α and assembles PCNA in the region of primer strand terminus.- Then DNA pol epsilon binds to PCNA and carries out highly processive leading strand synthesis due to its 5’-3’ polymerization activity.- After the addition of several nucleotides in the daughter strand, primer is removed. DNA pol Є due to its 5’-3’ exonuclease activity removes the primer and the gap is filled by the same DNA pol Є due to its 5’-3’ polymerization activity.- Then the nick is sealed by DNA ligase. DNA pol δ improves the fidelity of replication due to its proof reading activity.

Lagging strand synthesis:Lagging strand synthesis of Okazaki fragment initiated same way as leading strand synthesis. An Okazaki fragment contains 150-200 nucleotides.RNA primer is synthesised by DNA pol α due to its primase activity.The primer is then extended by DNA pol delta due to its 5’-3’ polymerization activity (lagging strand synthesis), using deoxy ribonucleotides (dNTPs).Priming is a frequent event in lagging strand synthesis with RNA primers placed every 50 or 80 nucleotides.All but one of the ribonucleotides in RNA primer is removed by RNase H1.Then exonuclease activity of FEN 1/ RTH 1 complex removes the one remaining nucleotide. The gap is filled by DNA pol Є by its 5’-3’ polymerase activity.DNA ligase joins the Okazaki fragment of the growing DNA strand.

Combined activity of DNA pol delta and DNA

pol epsilon:-

Looping of lagging strand allows a combined

polymerase delta and polymerase epsilon

asymmetric dimer to assemble and elongate both

leading and lagging strands in the same overall

direction of fork movement.

 

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.

TELOMERES

Eukaryotic chromosomes are linear. The ends

of chromosomes have specialized structures

known as ‘Telomeres’.

Telomeres are – short (5-8 bp)

- tandem repeated and

- GC rich nucleotide

sequence.

- Telomeres form protective cap 7-12 kbp

long in the ends of chromosome. Telomeres

are necessary for chromosome maintenance

and stability. They are responsible for

maintaining chromosome integrity by

protecting against DNA degradation and

rearrangement.

Problem in the completion of replication of

lagging strand:

- Linear genomes including those of

several viruses as well as the chromosomes of

eukaryotic cells force a special problem

completion of replication of the lagging strand.

- Excision of an RNA primer from the 5’

end of a linear molecule would leave a gap

(primer gap). This gap cannot be filled by DNA

polymerase action, because of the absence of a

primer terminus to extend. If the DNA could not

be replicated, the chromosome would shorten a

bit with each round of replication.

- This problem has been solved by

Telomerase.

Telomerase:- Telomerase is ribonucleoprotein. It contains a RNA component which has repeat of 9 to 30 nucleotides long. This RNA component serves as the template for the synthesis of telomeric repeats at the parental DNA ends.- Telomerase is a RNA dependent DNA polymerase with a RNA component.

Telomerase uses the - 3’ end of parental DNA strand as primer,- RNA component of telomerase as template,- adds successive telomeric repeats to the parental DNA strand at its 3’ end due to its 5’-3’ RNA dependent DNA polymerase activity.

Regeneration of telomeres:Telomeric DNA consists of simple tandemly repeated sequences like those shown as below:Telomeric repeats sequence at 5’end -

Organism RepeatHuman AGGGTTHigher plant AGGGTTTAlgae AGGGTTTTprotozoan GGGGTTTTYeast GGGT

These sequences are repeatedly added to the 3’ termini of chromosomal DNAs by ‘Telomerase’. Telomerase uses its RNA component as template and parental DNA as primer. Then by its RNA dependent DNA polymerase activity it repeatedly adds telomeric sequences to the 3’ termini of parental DNA. - Then the telomerase is released.- Finally the RNA primer, (of telomerase) is bound near the lagging strand and it is extended by DNA polymerase. Thus the lagging strand synthesis is completed.

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

basepairs 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

V. Magendira ManiAssistant Professor, PG & Research Department of Biochemistry,Islamiah College (Autonomous),Vaniyambadi,Vellore District – 6357512,Tamilnadu, India.magendiramani@rediffmail.com ;

https://tvuni.academia.edu/mvinayagam