Dna replication part i

DNA REPLICATION PART-I
By
Dr. Ichha PuraK
University Professor
Department of Botany
Ranchi Women’s College,Ranchi
www.dripurak.com
6/15/2013DNA REPLICATION Part-I1

DNA REPLICATION
INTRODUCTION
IMPORTANCE OF REPLICATION
THE REPLICATION FACTORY
•MODE OF REPLICATION : SEMI CONSERVATIVE
•EXPERIMENTAL EVIDENCE
•PHASE OF REPLICATION
•PLACE OF REPLICATION
•DIRECTION OF REPLICATION
•INITIATION OF REPLICATION : PROKARYOTES
•EUKARYOTIC INITIATION OF REPLICATION
•PROTEINS AND ENZYMES INVOLVED IN REPLICATION
•REPLICATION FORK

DNA replication is a fundamental process by which the parent DNA
duplex copies itself and become duplicated and produce two daughter
DNA duplexes .
Both strands of DNA ladder separate and act as template, onto which
new daughter strands are assembled, for which complementary
deoxyribonucleotides are taken from pool of deoxyribonucleotides.
The two DNA duplexes that emerge from this process receive only one
side of original DNA and other side newly synthesized.

IMPORTANCE OF REPLICATION
It is one of the most vital processes. It provides means by which
genetic instructions can be transmitted from one (parent ) cell to its
two daughter cells or from one individual to its offsprings because
during Replication Parent DNA duplex is able to make its two
identical copies or Replica and It is now well known that DNA is the
Genetic Material,able to transmit information over generations.

DNA Template
New DNA
PARENTAL DNA
Semi-conservative
Model:
Watson and Crick
predicted : the two
strands of the
parental molecule
separate, and each
functions as a
template for synthesis
of a new
complementary
strand.

THE REPLICATION FACTORY
Replication proteins are clustered together in particular locations in the
cell and may therefore be regarded as Replication Factory that
manufactures DNA copies.
The DNA to be copied is fed through the factory, much as a reel of film is
fed through a movie projector. The incoming DNA double helix is split
into two single strands and each original strand becomes half of a new
double helix. Because each resulting DNA double helix retains one
strand of the original DNA. DNA replication is said to be semi-
conservative

MODE OF REPLICATION :
Three possibilities have been suggested as mode for replication :
•Conservative Of the two duplexes produced after replication one is
entirely parental and other entirely new
•Semi-conservative Both the two duplexes formed after replication
contain one parental strand and one newly synthesized strand. Parent
DNA double helix is not conserved as entity, But one parental strand is
conserved in both daughter double helices in each generation and it
continues for many generations.
•Dispersive Both strands of parental molecule break at random, after
replication the parental and daughter pieces are joined randomly.

Of the various ideas proposed regarding mode of replication viz.
conservative, semi-conservative and dispersive, the semi-
conservative mode is highly accepted , which indicates that one
half of the parent duplex is transmitted to each daughter duplex. It
has been theoretically predicted by Watson and Crick ( 1953) on
the basis of double helical nature and complementary sequence of
bases on two strands of DNA.
Semi conservative mode of replication has been experimentally
proved by Maselson and Stahl (1958) using 15N in the nutrient
medium for culturing E.coli and monitoring the density of DNA after
each replication

EXPERIMENTAL EVIDENCE
Steps in the Experiment
1.E. coli cells were grown in culture medium having NH4Cl (with15N)
as Nitrogen source for 14 generations, so that all DNA bases
become labeled. This heavier ( High density) DNA settles
differently after centrifugation than the lighter (Low Density ) DNA
having 14 N.
2. E.coli cells were then removed ,washed and transferred to
culture medium having NH4Cl ( with 14 N) as Nitrogen source.
3. Some cells of E. coli were removed after every 30 minutes and
DNA was extracted and examined for density by centrifugation
with Cesium Chloride solution followed by sedimentation. ( E coli
takes 30 minutes for one division)

The results were observed :
4. After first generation only one band was observed by ultraviolet
absorption(260 nm) and it showed density intermediate between
heavier ( DNA 15 N) and lighter (DNA 14 N ) DNA s.
5. After two generations two bands were seen, one comparable to
Intermediate (Hybrid) DNA density and other of Normal ( DNA 14 N )
density.
6. In all subsequent generations two bands appeared, but intensity of
Hybrid density band gradually decreased and band of lighter DNA
gradually increased as expected by Semiconservative mode of
replication.

15 N 14 N
15 N+14 N
Hybrid

15 N
Hybrid
14 N

PHASE OF REPLICATION
Takes place during ‘S’ (Synthetic) phase of the preparatory stage of
cell cycle which commences after G 1 and is followed by G 2. The
preparatory stage is also known as Resting stage as cell is supposed to
be not active in division. It is also known as Interphase as it is the
period in between two cell divisions. During this period cell is
metabolically active.

Eukaryotic Replication having many origins
PLACE OF REPLICATION
It starts at origins of replication (Ori), appears as bubble under
electron microscope, ultimately extend in the form of ‘Y’
shaped replication fork.

Bubble : 2 Replication Forks

CIRCULAR BACTERIAL
CHROMOSOME UNDERGOING
BIDIRECTIONAL
SEMICONSERVATIVE
REPLICATION

PROKARYOTIC DNA REPLICATION
Parent duplex Daughter duplexes

DIRECTION OF REPLICATION
Replication takes place in bi-directional way, proceeds on both
sides of origin of replication ( Ori), both in pro as well as
eukaryotes. DNA synthesis takes place in 5’--------> 3’ direction and
the template is read in 3’ --------> 5’ direction so two newly
synthesized stretches of nucleotide chains must grow in opposite
direction . On one direction new strand grows towards fork and on
other strand away from the fork.

Thetalike configuration
assumed by
replicating,circular,no-end
double helical DNA
molecule of Escherichia
coli. Arrows indicate the two
replication forks. These
have progressed
bidirectionally from a single
replication bubble
In Bacteria, as DNA is circular and there is single (Ori), bi-directional
replication gives a shape.

Pattern of replication
of long DNA
molecule with more
than one origin of
replication.
Replication proceeds
out bidirectionally
from each origin

Origin of replication in E.coli
Replication initiates at a Unique site on
the E.coli chromosome ,designated as
ori.
First event is the binding of an initiator
protein to ori DNA , which leads to
partial unwinding of the DNA double
helix giving two templates
DNA continues to unwind by the
action of Helicase and single
stranded DNA binding proteins and
RNA primers synthesized by Primase
The two replication forks formed at
the origin then move in opposite
directions along the circular DNA
molecule

INITIATION OF REPLICATION : FACTORS INVOLVED
Prokaryotes : Eubacterial cell : E.coli
Initiation begins at A -T rich sequences at Ori C in E.coli. The main
components of the replication complex formation are ORC
(Origin Recognition Complex), factors Dna A, Dna B, Dna C, HU
and enzyme helicase. Dna A binding site consists of 9 bp repeats of
5’TGTGAATAA 3’, it binds to the Ori C , sets the platform and promotes
double helical opening, it also loads Dna B .

Binding of Dna A protein appears to be the key event ,it appears as
ellipsoidal mass (Dna A-Ori C ) under electron microscope. Dna B
has 5’ 3’ helicase activity and is also activator of primase Dna C
complex. HU is another double stranded binding protein.
As ORC binds DNA with the help of different protein factors, the
double helical structure opens in the form of small bubble by the
enzyme helicase. Once double helical structure is opened, it is
stabilized by single stranded DNA binding proteins.

EUKARYOTIC INITIATION OF REPLICATION : FACTORS
INVOLVED
Eukaryotic DNA is complexed with proteins, is assembled as
chromosomes. Eukaryotic DNA is many times larger than
prokaryotic DNA and therefore have multiple origin of
replication (ori) sites. The average human chromosome contains
150x106 nucleotide pairs, which are copied at about 50 bp per
sec speed. But due to presence of multiple origin of replication,
whole genome is replicated in 1 hour. In order to initiate
replication at these multiple sites a pre replicative complex is
formed having the following components :

ORC ( Origin Recognition Complex ) ,these remain bound to
DNA through out the Initiation process and is a six subunit
complex. It has affinity for single stranded DNA. Other factors
help ORC in identifying the ori sites. Its binding is coupled with
ATP hydrolysis.
CdC6 protein associates with ORC and help MCM proteins to
associate with chromatin.
Cdt 1 protein is identified as key factor in Pre-Rc assembly, so
mutations in Cdt 1 (In vitro studies ) results in a block to DNA
replication.

MCM (Mini chromosome Maintenance ) proteins - DNA is coated
with these proteins with the help of CdC6 and Cdt1. Once
replication begins in S phase,Cdt1 and CdC6 leave the ORCs and
MCM proteins remain in front of Replication fork on both sides
and all these help in stabilizing replication fork and its extension
having single stranded DNA.
DNA synthesis begins with the activity of DNA helicase which
causes melting of hydrogen bonds between base pairs ( A- T and
G-C) . As a result two single stranded structures with their
exposed nucleotides are produced.

These single strands have a tendency to wound again (make
base pairs) and to avoid this single stranded DNA binding
proteins play important role. They also protect the single
stranded structures from nucleases. After this DNA polymerase
comes into action. It selects the complementary nucleotide
from a mixture of dNTPs ( dATP,dCTP, dGTP,dTTP) and add to
the template strand and also establish phosphodi-ester bonds
between successive nucleotides.

PROTEINS AND ENZYMES INVOLVED IN
REPLICATION
Many enzymes and proteins are involved in the process of DNA
Replication to unwind double helix,replication fork stabilization
and synthesizing new DNA strand reading the template.viz.
Helicase, SSB Protein, Primase, The sliding Clamp, DNA
Polymerase, Rnase H and DNA Ligase. Each enzymes has a
specific role.
DNA replication requires a variety of proteins. Each protein
performs a specific function in the production of the new DNA
strands.
Helicase, made of six proteins arranged in a ring shape, unwinds
the DNA double helix into two individual strands.

Single-strand binding proteins, or SSBs, are tetramers that coat the
single-stranded DNA. This prevents the DNA strands from
reannealing to form double-stranded DNA. Primase is an RNA
polymerase that synthesizes the short RNA primers needed to start
the strand replication process. DNA polymerase is a hand-shaped
enzyme that strings nucleotides together to form a DNA strand. The
sliding clamp is an accessory protein that helps hold the DNA
polymerase onto the DNA strand during replication. RNAse H
removes the RNA primers that previously began the DNA strand
synthesis. DNA ligase links short stretches of DNA together to
create one long continuous DNA strand.

Enzymes and Proteins Involved in DNA
Replication

DNA Gyrase ( Type II Topoisomerase). Helps unwinding by DNA
helicase. Reduces the tension caused by super coil formation during
unwinding. For it single nick is created by breaking the phosphodiester
bond on one of the strand, which helps to release tension .
Topoisomerases have both nuclease (strand cutting) and ligase (strand
resealing) activities.
DNA helicase Helps in dissolving ‘H’ bonds between base pairs and
result in separating the two strands near origin of replication, which
gradually extends. Helicase as it requires energy to open the duplex is
associated with hydrolysis of ATP.
Singe stranded DNA binding Proteins Maintain the stability of
replication fork by binding the separated strands on both sides and
keeping them apart to avoid rewinding.These proteins also protect the
exposed nucleotides on the separated strands against nucleases.

DNA Polymerase Proceeds along the single stranded
templates , recruit complementary dNTPs, form Hydrogen
bonds with their appropriate complementary base present on
the template and catalyze phosphodiester bond with previous
nucleotide of the same strand.In prokaryotes DP III is
responsible for synthesis of new DNA strands .DP I is involved
in replacing deoxyribonucleotides after removal of RNA primers
on the lagging strand.
The sliding clamp is an accessory protein that helps hold the
DNA polymerase onto the DNA strand during replication.
RNA Primase Is actually part of aggregates of proteins called
primosomes. This enzyme attaches a small RNA primer to the
single stranded DNA onto which DP III can add
deoxyribonucleotides.

RNase This enzyme dismantles the RNA primers
present at the 5’ end of each Okazaki fragment on
lagging strand and also single RNA primer present on
the leading strand at the 5’ end.
DNA ligase Can catalyse the formation of
phosphodiester bond between 3’OH and 5’
phosphate groups of two DNA fragments on lagging
strand after removal of RNA primers. They seal the
gaps between two such DNA pieces.

REPLICATION FORK
The point where the DNA is separated into single strands, and
where new DNA will be synthesized, is known as the replication
fork.
For replication to take place the two strands of DNA double
helix at weak spots ( Where more A------- T base pairing is present )
separate by dissolving Hydrogen bond. The separated strands
appear as bubble under Electron Microscope. The two halves of
this bubble look as Y shaped Replication Fork with Neck and Mouth.
In prokaryotes there is single origin (Ori) , where as in Eukaryotes
several Ori are present.

REPLICATION FORK

DNA replication
“It has not escaped our
notice that the specific
pairing we have postulated
immediately suggests a
possible copying mechanism
for the genetic material.”
James Watson
Francis Crick
1953

Dna replication part i

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