Presentation on Replication of Genetic material (DNA) in prokaryotes and eukaryotes
DNA Replicatio n Vipin Shankar
In the early 1930s, biologists beganspeculating as to what sort
of molecule could have the kind of stability that the gene
demanded, yet be capable of permanent, sudden change to the mutant
forms that must provide
What is the genetic material? Is it The proteins, that make up
the enzymes? The complex proteins of the chromosomes? The amino
acids that make up the proteins? Or, the seemingly simple nucleic
acids that make up the chromosomes?
Averys Bombshell Oswald T Avery, Colin M MacLeod and Maclyn
McCarty (Rockefeller Institute, New York), based on original
observations by Griffith. DNA can carry genetic specificity.
The Double Helix
The Cell Cycle
Replication A template directed nucleic acid synthesis reaction.
Replication leads to doubling of the DNA, preserving the genetic
information, for transmission to the next generation. Occurs in the
S phase of the Cell Cycle. Replication requires a template to
provide sequence information.
DNA Replication The possible mechanisms. It has not escaped our
notice that the specific pairing we have postulated immediately
suggests a possible copying mechanism for the genetic material. -
Watson and Crick (in the paper describing DNA structure)
The possible mechanisms The Conservative model Both parental
strands remain together and the two new strands of DNA would form
an entirely new DNA molecule.
The Dispersive model The strands get broken as frequently as ten
base pairs and are used to prime the synthesis of similarly short
regions of DNA, which get subsequently joined to form the complete
The possible mechanisms The Semi-conservative model The two
strands separate during replication and each strand act as the
template for a new strand. Thus the new DNA molecule is made up of
a newly synthesized strand and a strand from the original
Experimental evidence for strand separation during replication
Mathew Meselson and Frank Sthal (1958), at the California Institute
The Meselson - Sthal experiment They grew E. coli in a medium
containing 15NH4Cl as the only source of nitrogen. After growing
for several generations, on the 15N-media, the DNA was found to be
denser. The density of the strands were determined by CsCl-density
gradient centrifugation. Meselson and Sthal, transferred the E.
coli, with the heavy (15N) DNA, to a media containing 14NH4Cl as
More proof for semi-conservative replication Taylor et al,
labeled Vicia fava (broad bean) root tip cells with 3 H-thymidine
and allowed them to grow in unlabelled Dr. J Herbert Taylor medium.
The metaphase chromosomes were analyzed by autoradiography.
Observations: Both chromatids were labeled after one
Chromosome from parent cell labeled with 3Hthymidine. Cells
grown in medium without 3H-thymidine. Chromatids separate during
cell cycle. And each chromatid produces its sister chromatid The
newly produced chromatids are not labeled.
More proof Based on the use of 5bromodeoxyuridine (BrdU), an
analogue of thymidine. DNA with BrdU in place of thymidine, does
not stain with fluorescent dye (33258 Hoechst). When cells labeled
with BrdU are subsequently grown in a medium without the analogue
Only one chromatid takes up the stain while its sister does
Dr. Cairns Experiment Dr. J Cairns (1963) used autoradiography
to demonstrate semi-conservative model of replication. He grew E.
coli on a medium containing 3H-thymine. The DNA was then extracted
and carefully subjected to autoradiography.
Dr. Cairns experiment: inferences The E. coli DNA is a circle.
The DNA is replicated while maintaining the integrity of the
circle. An intermediate theta structure forms (topologically
similar in shape to the Greek letter .) Replication of the DNA
seems to be occurring at one or two moving Yjunctions in the
circle. The DNA is unwound at a given point, and replication
proceeds at a Yjunction, in a semi-conservative
The rolling circle replication This form of replication is
initiated by a break in one of the nucleotide strands that creates
a 3-OH group and a 5-phosphate group. New nucleotides are added to
the 3end of the broken strand, with the inner (unbroken) strand
used as a template. As new nucleotides are added to the 3end, the
5end of the broken strand is displaced from the template, rolling
out like thread being pulled off a spool. The 3end grows around the
circle, giving rise to the name rolling-circle model.
The rolling circle mode The replication fork may continue around
the circle a number of times, producing several linked copies of
the same sequence. With each revolution around the circle, the
growing 3 end displaces the nucleotide strand synthesized in the
preceding revolution. Eventually, the linear DNA molecule is
cleaved from the circle, resulting in a double stranded circular
DNA molecule and a single-stranded linear DNA molecule. The linear
molecule circularizes either before or after serving as a template
The replicon Stretches of DNA with a single origin of
replication (Francois Jacob, Sydney Brenner & Jacques Cuzin
(1963)). The entire DNA replicated from a single origin. 2
components: The initiator The replicator
The replicon The initiator A protein that specifically recognize
the replicator. Recruits the replication machinery to the origin of
The replicator The entire set of DNA sequences sufficient to
direct the initiation of replication. Composed of two parts A
recognition site for the initiator
The origin of replication A stretch of AT rich DNA, that unwinds
readily but not spontaneously. Called oriC in E. coli. Contains 2
repeated motifs. A 9-mer motif repeated 5 times & A 13-mer
motif repeated 3 times.
The mechanism of replication Tightly controlled process, occurs
at specific times during the cell cycle.
Requires: A set of proteins and enzymes, Energy.
Two basic steps: Initiation Elongation.
Two basic components: Template Primer.
Process of semi-conservative replication Identification of the
origins of replication. Unwinding (denaturation) of ds-DNA to
provide an ss-DNA template. Formation of the replication fork.
Initiation of DNA synthesis and elongation. Formation of
replication bubbles with ligation of the newly synthesized DNA
Enzymology of DNA replication DNA Helicase: Unwinds DNA ahead of
replication fork DNA Polymerase: DNA synthesis and repair of gaps
DNA Ligase: Joins fragments of DNA DNA Primase: Syntheses primers
DNA Topoisomerases: Releases torsional strain caused by helicase
activity SSBs: Stabilizes single stranded
DNA Polymerases First identified in lysates of E. coli by Arthur
Kornberg (1963). The first polymerases to be isolated was named DNA
Pol I Isolation of polymerases represent a landmark discovery in
molecular biology, since the ability of these enzymes to accurately
copy a DNA template provided biochemical basis of the mode of
replication provided by Watson & Crick.
DNA Polymerases: more studies Cultures of E. coli were treated
with chemical mutagens. Mutants deficit in Pol-I activity were
isolated and sub-cultured. These strains grew normally in normal
media. But, these strains were extremely sensitive to agents that
cause DNA damage.
DNA Polymerases: more studies Inferences: Pol-I may not be
required for DNA replication. Pol-I may be involved in the repair
of DNA damage, rather than in DNA replication.
DNA Polymerases: further studies DNA polymerases II & III
were isolated from E. coli. The potential role of these enzymes
were studied by the isolation of appropriate mutants. Its been
confirmed that Pol-III & Pol-I are the major enzymes
responsible for replication. Pol-II is responsible for error-prone
Eukaryotic cells contain 5 different
DNA Polymerases: Types
DNA Polymerases: Functions
DNA Polymerase: Mechanism Requirements: All 4 dNTPs; viz, dATP,
dCTP, dGTP & dTTP. A primer template junction.
The new chain is synthesized by adding appropriate dNTPs at the
3 end of the primer at the primer template junction. A
phosphodiester bond is formed between the 3OH of the primer and the
-phosphate group of the
DNA Polymerases: Mechanism Hydrolysis of the pyrophosphate
drives the reaction. XTP + (XMP)n (XMP)n+1 + P ~ P P ~ P 2Pi XTP +
(XMP)n (XMP)n+1 + 2Pi
DNA Polymerases: Mechanism Have a single active site to catalyze
the addition of all four dNTPS. This is done by exploring the
nearly identical geometry of the A:T & G:C base pairs. The DNA
polymerase monitors the ability of the incoming nucleotide to form
a correct base pair. Only when a correct base pair is formed a
phosphodiester bond is formed between the 3OH of the
DNA Polymerase: Mechanism Incorrect base-pairing leads to lower
rates of nucleotide addition, due to catalytically unfavorable
alignment of the substrates : Kinetic selection. The rate of
incorporation of an incorrect nucleotide is 10,000 fold slower. DNA
polymerase can distinguish between rNTPs and dNTPs, by steric
DNA Polymerase: Mechanism DNA Polymerase resembles a Hand that
Grips the Primer : Template junction 3 domains The palm: contains
the primary elements of the catalytic site. Has 2 divalent metal
ions Mg2+ or Zn2+. Catalysis of the phosphodiester bond. Monitors
the accuracy of base
DNA Polymerase: Mechanism Domains The Fingers: Bind to the
incoming nucleotide. Once the correct base pair is formed, the
finger domain moves and enclose the dNTP, thus enhancing catalysis.
The Thumb: Not intimately involved in catalysis. Maintains the
correct position of the primer in the active site. Maintains a
strong association between the polymerase and the
Processivity of DNA Polymerase Processivity is a characteristic
of enzymes that operate on polymeric substrates. DNA polymerases
are capable of adding as many as 1,000 nts per second. The degree
of processivity is defined as the average number of nucleotides
added each time the enzyme binds a primer : template junction. Each
DNA polymerase has a
Processivity. The initial binding of the polymerase to the
primer : template junction is the rate limiting step. The
processivity of DNA Pol is increased by a Sliding Clamp. The
Sliding clamp is an association of proteins that assemble in the
shape of a doughnut. The clamp encircles the newly synthesized ds
DNA and keeps DNA Pol associated with the primer :
Proof Reading by Polymerase Mediated by nuclease activity that
remove the incorrectly base paired nucleotides. The 3 to 5
exonuclease activity of the palm domain, checks for the incorrect
base pairing. The proof reading gives a second chance to correct
mistakes and to add the correct nucleotide.
DNA Replication: The process The initiator protein recognizes
and binds to the replicator sequence. The initiator protein
recruits DNA Helicase to the Origin of replication. DNA Helicase
unwinds the ds DNA at the origin of replication formation of the
Replication Fork. Other proteins of replication machinery assemble
at the replication fork.
DNA Replication: The process Single Strand Binding Proteins
(SSBs) stabilize the replication fork by binding to the newly
opened ss DNA, preventing the recoil. Topoisomerases prevent the
supercoiling formed during helicase activity. 2 classes of
topoisomerases are present. Class II topoisomerases also called
Gyrases are the important ones in DNA
The need for topoisomerases
DNA Replication: The process Primase synthesizes RNA primer for
the action of DNA polymerase. PROBLEM. DNA Polymerase can
synthesize DNA only in the 5 to 3 direction. So synthesis on one
strand is continuous. What happens on the other strand?
The strand on which continuous DNA synthesis proceeds is called
the Leading Strand.
DNA Replication: The process The second strand is called the
Lagging Strand. On the lagging strand DNA synthesis happens in
short fragments (1000 2000 nts in bacteria and 100 400 in Ek.cells)
called Okazaki Fragments. Each Okazaki fragment requires a new
primer. The RNA primers are removed by RNAase H. The single
stranded nicks produced
DNA Replication: Trombone Model At the replication fork, the
leading strand and the lagging strand are synthesized
simultaneously. This limits the amount of ss DNA present in the
cell during replication. To co-ordinate the replication of both the
strands, multiple DNA Polymerases act together. A large
multi-protein complex called the DNA Pol III holoenzyme in which
the core enzyme is associated with
Finishing replication Completion of replication is different in
circular and linear chromosomes. In a circular chromosome (like in
E. coli), the 2 replication forks meet at a region called the
termination region or ter region. Termination utilization substance
(tus protein), forms a complex with the ter region and stops the
progress of the replication fork. This results in two daughter
molecules linked to one another (Catenane). These are seperated by
the action of class II
Replication of circular chromosomes
Finishing Replication The requirement of a RNA primer for the
synthesis of all Okazaki fragments on the lagging strand, creates a
dilemma for the replication of the end of the linear chromosome the
end replication problem. Once RNA molecule is removed from the last
Okazaki fragment, a short region of un-replicated DNA will remain
on the lagging strand. This means that each round of replication
would result in the
Telomerase Telomerase is a DNA polymerase that does not require
a exogenous template. Telomerase is an enzyme which includes both
protein and RNA components (ribonucleoprotein). The RNA component
acts as a template for adding the telomeric sequence to the 3
terminus at the end of the chromosome and thus solves the end
The End Replication Problem
Assembling newly replicated DNA into nucleosomes When eukaryotic
DNA is replicated, it complexes with histones. This requires
synthesis of histone proteins and assembly of new nucleosomes.
Transcription of histone genes is initiated near the end of G1
phase, and translation of histone proteins occurs throughout S
Assembly of nucleosomes