Basics of DNA Replication

DNA REPLICATION
West Bengal State University
cbcs Botany Core viii
Dr. Riddhi Datta
Department of Botany
Dr. A.P.J. Abdul Kalam Government
College

Central Dogma of Molecular Biology
Dr. Riddhi Datta

Replication
• A basic property of genetic material (DNA) is to replicate in a precise way so that
the genetic information can be transmitted from each cell to its progeny.
• DNA Replication is a biological process that occurs in all living organisms and copies
their exact DNA. It involves synthesis of daughter DNA molecules from parent
DNA molecule by the aid of DNA dependent DNA polymerase enzyme.
• It is the basis for biological inheritance.
DNA Lengthening DNA
Dr. Riddhi Datta

Possible models for DNA replication
• Conservative model:
The parental molecule
directs synthesis of an
entirely new double-
stranded molecule. Hence,
after one round of
replication, one molecule
is conserved as two old
strands. This is repeated in
the second round.
• Semi-conservative model: The two parental strands separate and each serves as a template to synthesize its
complementary new strand. After one round of replication, the two daughter molecules each comprises one
old and one new strand. After two rounds, two of the DNA molecules consist only of new material, while the
other two contain one old and one new strand.
• Dispersive model: Material in the two parental strands is distributed more or less randomly between two
daughter molecules. Dr. Riddhi Datta

Mode of DNA replication: Meselson-Stahl experiment
• Meselson and Stahl conducted their famous experiments on DNA replication using E.coli bacteria
as a model system.
• They began by growing E. coli in nutrient broth, containing a "heavy" isotope of nitrogen, N15 .
Bacteria took up the N15 and used it to synthesize new biological molecules, including DNA.
• After many generations growing in the N15 medium, the bacteria were switched to medium
containing a "light" N14 isotope and allowed to grow for several generations. DNA made after
the switch would have to be made up of N14.
• Meselson and Stahl collected small samples of bacteria in each generation and extracted the
DNA. They then measured the density of the DNA (and, indirectly, N14 and N15 content)
using density gradient centrifugation.
• This method separates molecules such as DNA into bands by spinning them at high speeds in the
presence of another molecule, such as cesium chloride, that forms a density gradient from the top
to the bottom of the spinning tube.
Dr. Riddhi Datta

Mode of DNA replication: Meselson-Stahl experiment
• Initially, a single band of N15 DNA was observed.
• After one generation of cell division, the total DNA
of the growing bacterial cells had an intermediate
density, halfway between that of N14 and N15 DNA.
This strongly disproved the conservative model of
DNA.
• After several generations in N14 medium, Meselson
and Stahl observed that the N15 DNA band
disappeared, a band of N14 DNA appeared and then
got progressively darker, and a band of intermediate
density appeared and persisted at about the same
intensity. This strongly discredited the dispersive
model of DNA replication.
• Thus, Meselson and Stahl solidly disproved two of
the possible models of DNA replication
(conservative and dispersive), while strongly
supporting another (semi-conservative).
Dr. Riddhi Datta

DNA-dependent DNA polymerase
• DNA replication is achieved by the enzyme known as DNA-
dependent DNA polymerase which requires single stranded
DNA as template.
• E.coli has 5 types of DNA polymerases:
– DNA polymerase I: The first to be discovered. Required for
excising the primers and filling the gap during replication
– DNA polymerase II: Functions in DNA repair machinery
– DNA polymerase III: Main enzyme for replication
– DNA polymerase IV: Functions in DNA repair machinery
– DNA polymeraseV: Functions in DNA repair machinery
• DNA polymerase was first discovered by Arthur Kornberg
and his colleagues in 1955.
Nobel Prize in
Physiology or
Medicine in 1959
Discovery of mechanism of
biological synthesis of DNA
Arthur Kornberg
Dr. Riddhi Datta

Kornberg’s discovery
• Kornberg was trying to identify all the ingredients required to synthesize E.coli DNA in vitro.
• First successful DNA synthesis occurred in a mixture containing deoxyribonucleoside 5‘-
triphosphate precursors (dATP, dCTP, dTTP and dGTP, collectively called dNTPs) and E.coli cell
lysate.
• Kornberg analysed the mixture and isolated an enzyme that was capable of synthesizing DNA.
• This enzyme was originally called Kornberg enzyme, but was later named as DNA
polymerase I.
• Subsequently it was identified that DNA synthesis would not occur without the following 4
components:
– All four dNTPs
– A fragment of DNA as template
– DNA polymerase
– Magnesium ions
Dr. Riddhi Datta

General features of DNA Polymerase
• Catalyse polymerisation of deoxyribonucleotide precursors (dNTPs) into a DNA chain.
• At the growing end of the DNA chain, DNA polymerase catalyses the formation of a
phosphodiester bond between the 3‘-OH group of the deoxyribose of the last
nucleotide with the 5‘-PO4 group of the dNTP precursor.
• At each step, DNA polymerase finds the correct precursor dNTP that is
complementary to the nucleotide on the template strand.
• The process doesn‘t occur with 100% accuracy, but error frequency is very low (10-6).
• DNA synthesis always occurs in 5‘ to 3‘ direction.
• DNA polymerase requires RNA primer to initiate DNA synthesis which provides a 3‘-
OH group.
DNA Lengthening DNA
Dr. Riddhi Datta

Process of DNA synthesis
• The entire process of DNA synthesis can be broadly divided into three steps:
– Initiation
– Elongation
– Termination
Dr. Riddhi Datta

Process of DNA synthesis: Initiation
• The initiation of replication is directed by a
DNA sequence called replicator which
includes the origin of replication.
• Replication of E. coli chromosome is initiated
at a 245 bp replication origin called oriC
locus consisting of two series of short
repeats:
– three repeats of a 13bp AT rich
sequence
– four repeats of a 9bp sequence
• There are 8 different enzymes that together
form a pre-priming complex for the
subsequent reactions.
Dr. Riddhi Datta

• A Dna A protein (initiating protein) binds to the four repeats of 9 bp sequence of oriC. With
the help of histone-like HU proteins and ATP, the Dna A causes the AT rich 13 bp repeat region
to denature.
• The hexameric Dna B protein (helicase) now binds to this region with the help of Dna C
protein.
• The helicase then unwinds the DNA molecule bidirectionally creating a replication bubble with
two replication forks. The energy for unwinding is derived from ATP hydrolysis – a reaction
that causes conformational change in helicase enabling it to move along a single strand of DNA.
Dr. Riddhi Datta

• Next, multiple SSB (single strand binding) proteins bind cooperatively to separate the
single stranded DNA and prevents renaturation.
• Continued unwinding by helicase causes supercoiling ahead of the replication forks which is
relieved by gyrase (topoisomerase II).
• Other proteins associated with the initiation complex includes Dna T, Dna J, Dna K, Pri A,
Pri B and Pri C.
Dr. Riddhi Datta

Replicon concept of replication initiation
• Proposed by Francis Jacob, Sydney Brenner and Jacques Cuzin in 1963.
• Replicon: All DNA replicated from a particular origin.
• Two components control initiation of replication:
– Replicator:
• The entire set of cis-acting DNA sequences sufficient to direct initiation of DNA replication. The
origin of replication is a part of replicator.
• It includes a binding site for the initiator protein.
• It includes a stretch of A-T rich DNA sequence that can unwind readily but not spontaneously
– Initiator protein:
• Specifically it recognizes a DNA element in the replicator and unwinds an adjacent stretch of A-
T rich sequence.
• Then it recruits other proteins required to initiate replication.
• This is the only sequence specific DNA binding protein required in replication initiation.
• Replicator of E.coli is oriC and initiator protein is Dna A.
Dr. Riddhi Datta

Process of DNA synthesis: Elongation
• Elongation includes both leading and lagging strand synthesis always in the 5’ to 3’ direction.
• As the two DNA strands are anti-parallel, the template strand is read in the 3‘ to 5‘ direction.
• For simultaneous synthesis of both the strands the synthesis of one strand is continuous while
that of the other strand is discontinuous. Hence, the DNA synthesis, as a whole, is semi-
discontinuous.
Dr. Riddhi Datta

• The strand which is synthesized
continuously in the same direction of
the replication fork movement is
called the leading strand.
• The discontinuous strand whose
synthesis proceeds in the opposite
direction of that of the replication
fork movement is called the lagging
strand
• Each replication bubble has two replication forks moving in opposite directions.
• The anti-parallel nature of DNA strands allows both the strands to serve as templates and both
strands of DNA are synthesized simultaneously.
• Therefore, DNA synthesis occurs simultaneously in both the directions. The process of replication
is, thus, bidirectional.
Dr. Riddhi Datta

• Leading strand synthesis begins with the formation of a short RNA primer at the replication
origin. This is required because DNA polymerase can not start de novo DNA synthesis but
required a 3‘-OH group to begin polymerization. The RNA primer is synthesized by primase
(Dna G).
• The dNTPs are then added one by one to this primer by DNA polymerase III.
• Strand elongation occurs continuously keeping pace with the replication fork movement whose
synthesis and positioning is done by an assembly of proteins called primosome. Primosome
includes a helicase and a primase, along with five other proteins in E.coli.
Dr. Riddhi Datta

• The short stretches of DNA in the lagging
strand are called Okazaki fragments
after the name of its discoverer Reiji
Okazaki.
• A primimg event is required to initiate
each Okazaki fragment during
discontinuous synthesis of the lagging
strand.
• After each round of fragment synthesis,
the RNA primer is removed by the 5‘ to
3‘ exonuclease activity of DNA
polymerase I and the gap is filled with
DNA by the polymerase activity of the
same enzyme. The remaining nicks are
sealed by DNA ligase. Dr. Riddhi Datta

Process of DNA synthesis:Termination
• Ultimately the two replication forks meet at the other
side of the circular DNA of E.coli.
• The terminus is a large region flanked by seven
terminator sites:
– Ter E,Ter D andTer A on one side
– Ter G,Ter F,Ter B andTer C on the other side
• These terminator sites act as one-way valves that allow
replication forks to enter the terminus region but not to
leave it.
• Tus protein arrests the movement of the replication
fork at Ter sites. It prevents unwinding of the helix by
helicase.
• The final step is the topological unlinking of the two
replication products by topoisomerase II. Dr. Riddhi Datta

Proteins required for replication
• DNA polymerase III: Required for dNTP polymerisation and proof-reading
• DNA helicase: Unwinds the helix
• Primase: Synthesizes RNA primer
• Single strand binding proteins: Prevents renaturation of the replication bubble
• Topoisomerase II: Relieves supercoiling ahead of the replication fork as well as causes
topological unlinking of the two replication products at termination.
• RNase H:Removes RNA primer
• DNA polymerase I: Fills the gap after removal of RNA primers with DNA and removes
errors
• DNA ligase: Seals the single stranded nick in DNA
• DNA sliding clamp: slides along the DNA template and keeps the polymersae from
falling off the template.
Dr. Riddhi Datta

DNA helicase
• A class of enzymes that couples ATP
hydrolysis with unwinding of DNA
double helix.
• They are typically hexameric proteins
in the shape of a ring
• As the DNA strands separates, the SSB
proteins bind co-operatively to the
single stranded DNA.
• They prevent renaturation of the
unwound DNA strands.
SSB proteins
Dr. Riddhi Datta

Topoisomerases
• As the DNA unwinds, the DNA strands
get positively supercoiled ahead of the
replication fork.
• This supercoiling is relieved by
topoisomerases (Gyrase in E. coli). The
energy for such action is obtained from
ATP hydrolysis.
• Primase is a specialized RNA
polymerase which use ssDNA as
template to synthesize short RNA
primers.
• It is activated by interacting with DNA
helicase.
Primase
Dr. Riddhi Datta

DNA polymerase
• DNA synthesis is catalyzed by the enzyme DNA polymerase (DNA polymerase III in
E.coli).
• Using a ssDNA as template, it can catalyze formation of phosphodiester bond
between a 3‘-OH of the primer and 5‘-phosphate of the incoming dNTP.
• It can not tart DNA synthesis de novo.
Dr. Riddhi Datta

Sliding DNA clamps
• Surrounds the DNA and binds the polymerae.
• Slides along the DNA template and keeps the
polymerase from falling off the template.
• Increases processivity of the polymerase.
Rnase H
• The RNA primers are
removed by Rnase H.
• H stands for ‗hybrid‘ since they
degrade RNA from the RNA-
DNA hybrids.
Dr. Riddhi Datta

DNA polymerase
• Removal of the RNA primers leaves single
stranded gaps in the DNA.
• These gaps are filled by DNA polymerase
(DNA polymerase I in E.coli)
DNA ligase
• DNA ligase seals the nick.
• It catalyzes phosphodiester
bond formation using energy
derived from ATP hydrolysis.
Dr. Riddhi Datta

DNA Polymerase I
• Encoded by polA gene.
• Consists of a single polypeptide of 109 kDa.
• Possess following catalytic activities:
– 5‘ to 3‘ polymerase activity
– 3‘ to 5‘ exonuclease activity
– 5‘ to 3‘ exonuclease activity
• Involved in proof-reading of replicated DNA
by 3‘ to 5‘ exonuclease activity.
• Involved in repairing damaged DNA by
unique 5‘ to 3‘ exonuclease activity as well as
removing RNA primers and filling the gaps.
• If an incorrect nucleotide is
incorporated by DNA polymerase
(frequency of error is 10-6), it is
recognized immediately.
• The 3‘ to 5‘ exonuclease activity of
the polymerase then excise the
incorrect nucleotide from the new
strand.
• The polymerase then resumes its
forward motion and inserts the
correct nucleotide.
Proof-reading activity
Dr. Riddhi Datta

DNA Polymerase III
• A multimeric enzyme with molecular mass
900 kDa
• Minimal core (has catalytic activity in
vitro) contains 3 subunits: α, ε and θ.
• Catalytic core synthesizes short DNA
fragments and falls off the template
• The τ subunit results in dimerization of
the catalytic core and increased activity.
• The β subunit forms a dimeric clamp,
keeps the catalytic core from falling off the
template and slides along it.
• A group of 5 other subunits (γ, δ, δ’, χ and ψ) forms the γ complex which loads the enzyme
onto the template at the replication fork.
• Holoenzyme possesses 5’ to 3’ polymerase activity and 3’ to 5’ exonuclease (proof reading)
activity. Dr. Riddhi Datta

Processivity
• The ability of an enzyme to catalyze many reactions before releasing its substrate is called
processivity.
• The sliding clamp loader loads the sliding clamp to DNA polymerase and increases its processivity.
• DNA polymerase can add upto 1000 nucleotides / second to the growing nucleotide chain.
http://media.pearsoncmg.com/bc/bc_martini_
ap_slim/assets/animations/ch08_replication.ht
ml
Enzymes involved in DNA synthesis: quick recap!
Dr. Riddhi Datta

Chemistry of DNA synthesis
Substrates for DNA
synthesis:
• Deoxyribonucleotide
triphosphates (dNTPs)
• Primer:template
junction:
Primer provides a free 3‘-OH
while template provides a
single stranded DNA to be
copied.
Dr. Riddhi Datta

• DNA polymerization is a NUCLEOPHILIC SUBSTITUTION (SN2)
REACTION.
Dr. Riddhi Datta

• DNA polymerase has 3 main
domains:
– Palm domain
– Thumb domain
– Finger domain
Dr. Riddhi Datta

Palm domain:
• Possess the active site of DNA synthesis and is composed of a β-sheet.
• The active site in the palm domain can distinguish between rNTPs and dNTPs.
• The rNTPs are present in around 10 fold higher concentration in cell.
• But the nucleotide binding pocket is too small for the 2‘-OH on the incoming rNTP.
• Thus the polymerase can exclude the rNTPs by steric constraining.
x
Dr. Riddhi Datta

Palm domain:
• Correct base pairing is also required
for catalysis.
• If an incorrect dNTP comes, its
α-phosphoryl group cannot
properly align with the 3’-OH
of the growing strand.
• Once the correct dNTP is bound in
the pocket, the reaction can
continue.
• The palm domain also binds Zn2+
and Mg2+ which are crucial for
catalysis.
x
Dr. Riddhi Datta

Finger domain:
• Composed of α-helix.
• Once the correct dNTP is bound in the
pocket, the finger domain moves to enclose
the base paired dNTPs.
• This conformational change brings the
dNTP and the primer (or growing DNA
strand) into correct orientation with
the divalent metal ions.
• The O-helix of the finger domain moves
40° to enclose the base by stacking
interaction with its tyrosine residue.
Dr. Riddhi Datta

Finger domain:
• Metal ion A helps to deprotonate the 3’-
OH of the primer producing an oxyanion.
• This oxyanion attacks the α–
phosphoryl group of the incoming dNTP.
• Metal ion B coordinates the negative
charge of the β- and γ-phosphate
groups and stabilizes the pyrophosphate
leaving group.
• Lysine and arginine residues on the
finger domain also help to stabilize the
pyrophosphate and the tyrosine residue
hold the dNTP in place for catalysis
(stacking interaction).
Dr. Riddhi Datta

Finger domain:
• The finger domain also associates with the template region resulting in a 90° turn in the template which
helps to avoid confusion in the active site.
• This ensures that only one template nucleotide remains in the active site.
Thumb domain:
• It is not intimately involved in catalysis.
• It interacts with the DNA that has been synthesized
most recently and hold the primer:template
junction in the active site.
• This reduces the dissociation of the polymerase from
the template.
Dr. Riddhi Datta

Proof reading
• The palm domain also has proof reading activity.
• It H-bonds with the base pairs in the minor groove. It is not sequence specific but occurs only when the nucleotides
are correctly base paired.
• If mismatch occurs, replication slows down and the palm domain is not able to make contact with the minor groove.
• This frees the primer:template junction to move and make contact with the exonuclease site.
• The exonuclease site removes the incorrect base from 3‘ to 5‘ direction in a process called proof reading.
• After excision is complete, the primer:template junction slides back to the replication active site.
Dr. Riddhi Datta

http://media.pearsoncmg.com/bc/bc_martini_ap_slim/assets/animations/ch
08_polymerization.html
Chemistry of DNA synthesis: quick recap!
Dr. Riddhi Datta

Dr. Riddhi Datta
Theta (θ) replication
• The bidirectional replication of a circular chromosome
in prokaryotes occur through θ mode of replication.
• A θ structure is an intermediate structure formed during
the replication of a circular DNA molecule.
• Two replication forks can proceed independently around the
DNA ring and when viewed from above the structure resembles
the Greek letter "theta" (θ).
• Semiconservative mode of DNA replication in bacterial
chromosome begins at the origin of replication called oriC.
• DNA polymerases then move bi-directionally from
origin to the termination site of DNA replication
abbreviated as ter.
• This results in the two replication forks moving in
opposite directions around circular chromosome.

Dr. Riddhi Datta
• The bidirectional
replication forks move
at identical speed after
initiation, therefore,
both replication
forks meet at the
termination site.
• As a result, the two
synthesized circular
DNA molecules remain
interlocked with each
other.
• The process ends by topological unlinking of the two replicated
DNA molecules by the action of topoisomerases.

Dr. Riddhi Datta
• Originally discovered by John Cairns, this model led to the understanding that
bidirectional DNA replication.
• The replicating cells were exposed to a pulse of tritiated thymidine, quenched rapidly and then
autoradiographed.
• Results showed that the radioactive thymidine was incorporated into both forks of the theta
structure, not just one, indicating synthesis at both forks in opposite directions around the loop.

• For some virus chromosomes, such as that of
bacteriophage , a circular, double-stranded
DNA replicates to produce linear DNA; the
process is called rolling circle replication.
• Steps:
• The first step is generation of a specific nick in one
of the two strands at the origin of replication.
• The end of the nicked strand is then displaced from
the circular molecule to create a replication fork.
• The free end of the nicked strand acts a primer for
DNA polymerase to synthesize new DNA, using the
single-stranded segment of the circular DNA as a
template.
Rolling circle replication
Dr. Riddhi Datta

• The displaced single strand of DNA rolls out as a
free ―tongue‖ of increasing length as replication
proceeds.
• New DNA is synthesized by DNA polymerase on
the displaced DNA in the -to- direction, meaning
from the circle out toward the end of the displaced
DNA.
• With further displacement, new DNA is
synthesized again, beginning at the circle and
moving outward along the displaced DNA strand.
• Thus, synthesis on this strand is discontinuous
because the displaced strand is the lagging-strand
template.
Dr. Riddhi Datta

• As the single-stranded DNA tongue rolls out, new
DNA synthesis proceeds continuously on the
circular DNA template.
• Because the parental DNA circle can continue to
―roll,‖ a linear double stranded DNA molecule can
be produced that is longer than the circumference
of the circle.
• Example: Replication of λ phage DNA.
Dr. Riddhi Datta

• Phage genome has a linear, mostly double-
stranded DNA with 12-nucleotide-long,
single-stranded ends.
• The two ends have complementary
sequences—they are referred to as ―sticky‖
ends and can base pair with one another.
• When phage infects E. coli, the linear
chromosome is injected into the cell and the
complementary ends pair.
• To produce copies of the chromosome to
package in progeny phages, the now-
circular phage chromosome replicates by
the rolling circle mechanism.
Rolling circle replication of λ phage DNA
Dr. Riddhi Datta

• The result is a multi-genome-length ―tongue‖
of head-to-tail copies of the chromosome.
• A DNA molecule like this, made up of
repeated chromosome copies, is called a
concatamer.
• The phage chromosome has a gene called ter
(terminus-generating activity), which codes
for a DNA endonuclease.
• The endonuclease binds to the cos sequence
and makes a staggered cut such that linear
chromosomes with the correct
complementary, 12-base-long, single-
stranded ends are produced.
• The chromosomes are then packaged into the
progeny phages.
Rolling circle replication of λ phage DNA
Dr. Riddhi Datta

• Each eukaryotic chromosome consists
of one linear DNA double helix.
• Eukaryotic chromosomes replicate efficiently
and quickly because DNA replication is
initiated at many origins of replication
throughout the genome.
• Eventually, each replication fork runs into an
adjacent replication fork, initiated at an
adjacent origin of replication.
• The stretch of DNA from the origin of
replication to the two termini of replication
(where adjacent replication forks fuse) on
each side of the origin is called a replicon
or replication unit.
• [The E.coli genome consists of one replicon.]
Replication of linear double stranded DNA in eukaryotes
Dr. Riddhi Datta

Initiation of replication:
• In the Saccharomyces cerevisiae, replicators are
approximately 100-bp sequences called
autonomously replicating sequences
(ARSs).
• Replicators of more complex, multicellular
organisms are less well characterized.
• The initiator protein in eukaryotes is the
multisubunit origin recognition complex
(ORC).
• The ORC binds to two different regions at one end
of the replicator and recruits other replication
proteins (auxiliary proteins).
• The origin of replication is between the binding
sites of ORC and other replication proteins.
Dr. Riddhi Datta

Restricting replication once per cell cycle:
• DNA replication takes place during S phase of cell cycle.
• For correct duplication of the chromosomes, each origin of replication
must be used only once in the cell cycle.
• This is accomplished by two steps.
• The first step is replicator selection, where ORC binds to each
replicator and recruits other proteins to form pre-replicative
complexes (pre-RCs).
• The pre-RCs are activated only when the cell progresses from G1 to S, the
unwinding of DNA starts and then they initiate replication.
• Limiting replication initiation to the S stage is controlled by licensing
factors (MCM helicase) that are synthesized in G1 phase.
• After one cycle of replication begins in the S phase, the licensing factors are
degraded and thus, a second cycle of replication can not initiate.
Dr. Riddhi Datta

Eukaryotic replication enzymes:
• Eukaryotic cells may have 15 or more DNA polymerases.
• Typically, replication of nuclear DNA requires three of these:
– Pol α/primase: initiates new strands in replication by primase, making about 10 nucleotides of an
RNA primer; then Pol α adds 10–20 nucleotides of DNA.
– Pol δ:Pol δ synthesizes the lagging strand.
– Pol ε: Pol ε appears to synthesize the leading strand,
• Other eukaryotic DNA polymerases are involved in specific DNA repair processes, and yet others replicate
mitochondrial and chloroplast DNA.
• Primer removal does not involve the progressive removal of nucleotides, as is the case in prokaryotes. Rather,
Pol δ continues extension of the newer Okazaki fragment; this activity displaces the RNA/DNA ahead of the
enzyme, producing a flap. Nucleases remove the flap. The two Okazaki fragments are then joined by the
eukaryotic DNA ligase.
Dr. Riddhi Datta

• Because DNA polymerases can synthesize new DNA only
by extending a primer, there are special problems in
replicating the ends—the telomeres—of eukaryotic
chromosomes .
• Replication of a parental chromosome produces two new
DNA molecules, each of which has an RNA primer at
the end of the newly synthesized strand in the
telomere region.
• Removal of these RNA primers leaves a single-stranded
stretch of parental DNA—an overhang—extending
beyond the end of each new strand.
• DNA polymerase cannot fill in the overhang.
• If nothing were done about these overhangs, the
chromosomes would get shorter and shorter with each
replication cycle.
Replication of 5’ ends of linear chromosomes
Dr. Riddhi Datta

• Most eukaryotic chromosomes have species-
specific, tandemly repeated, simple sequences at
their telomeres.
• Elizabeth Blackburn and Carol W. Greider
have shown that an enzyme called telomerase
maintains chromosome lengths by adding
telomere repeats to one strand (the one
with the end), which serves as template on
previous DNA replication at each end of a linear
chromosome.
• The complementary strand to the one
synthesized by telomerase must be added by the
regular replication machinery.
• The repeated sequence in humans and all other
vertebrates is 5’–TTAGGG–3’.
Dr. Riddhi Datta

• Telomerase is an enzyme made up of both
protein and RNA.
• The RNA component includes an 11-base
template RNA sequence that is used for the
synthesis of new telomere repeat DNA.
• The telomerase binds specifically to the overhanging
telomere repeat on the strand of the chromosome
with the 3‘ end .
• The end of the RNA template sequence in the
telomerase—here, 3‘-CAAUC-5‘— base-pairs with
the 5‘-GTTAG-3‘ sequence at the end of the
overhanging DNA strand.
• Next, the telomerase catalyzes the addition of
new nucleotides to the end of the DNA using
the telomerase RNA as a template.
Dr. Riddhi Datta

• The telomerase then slides to the new end of the
chromosome so that the end of the RNA template
sequence—3‘-CAAUC-5‘—now pairs with some of
the newly synthesized DNA.
• Then, as before, telomerase synthesizes telomere
DNA, extending the overhang.
• If the telomerase leaves the DNA now, the
chromosome will have been lengthened by two
telomere repeats.
• But, the process can recur to add more telomere
repeats.
Dr. Riddhi Datta

• Then, when the chromosome is replicated using the
elongated strand as a template, and the primer of the
new DNA strand is removed, there will still be an
overhang—but any net shortening of the
chromosome will have been more than compensated
for due to the action of telomerase.
• In most cells, the telomere DNA then loops back on
itself to form a t-loop, with the single stranded end
invading the double-stranded telomeric repeat
sequences to form a D-loop.
• The synthesis of DNA from an RNA template is
called reverse transcription, so telomerase is
an example of a reverse transcriptase enzyme.
Dr. Riddhi Datta

• Telomere length is regulated to an average length for the every organism and cell type.
• Mutants with a deletion in the TLC1 gene (encodes the telomerase RNA) or mutation in the EST1
or EST3 (ever shorter telomeres) genes causes telomeres to shorten continuously until the cells
die.
• This phenotype provides evidence that telomerase activity is necessary for long-term cell
viability.
• Mutations of the TEL1 and TEL2 genes cause cells to maintain their telomeres at a new, shorter-
than-wild-type length, making it clear that telomere length is regulated genetically.
Dr. Riddhi Datta

Basics of DNA Replication

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