03-05-2022

References:
DNA Replication 1.
2.
3.
Watson, molecular biology 7th ed.
Stryer, biochemistry 7th ed.
Voet & Voet, biochemistry 4th ed.
❖ DNA synthesis requires 4.
5.
Lehninger, biochemistry 6th ed
Campbell, biochemistry 7th ed
i. Four deoxynucleosidetriphosphates—dGTP, dCTP, dATP, and dTTP. 6. Snustad, genetics 6th ed.

ii. A particular arrangement of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) called
a primer:template junction.
iii. DNA polymerases and other associated proteins & enzymes.

Substrates required for DNA synthesis

DNA Is Synthesized by Extending the 3’ End of the Primer

➢The phosphodiester bond is formed in an SN2
reaction in which the hydroxyl group at the 3’ end of
the primer strand attacks the α-phosphoryl group of
the incoming nucleoside triphosphate.
✓Hydrolysis of pyrophosphate is the driving force for DNA synthesis

DEC 2017

ANS: 2

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DNA POLYMERASE
✓Unlike most enzymes, DNA pol uses a single active site to catalyze the addition of any of the four dNTPS.

✓DNA polymerases show an impressive ability to distinguish between ribonucleoside and

deoxyribonucleoside triphosphates (rNTPs and dNTPs), though rNTPs are approximately 10-fold higher
inside cell. In DNA polymerase, the nucleotide-binding pocket cannot accommodate a 2’-OH on the in-
coming nucleotide.
✓DNA polymerases resemble a hand that grips the primer:template junction .

➢Based on the hand analogy, the three domains of the

polymerase are called the thumb, fingers, and palm .

➢The palm domain contains the primary elements of the

catalytic site,which binds two divalent metal ions ,Mg2+or
Zn 2+ .The fingers are also important for catalysis.
In contrast to the fingers and the palm, thumb interacts
with the DNA that has been most recently synthesized to
maintain a strong association between the DNA
polymerase and its substrate .
3D structure of DNA polymerase
resembles a right hand

Ans: 3

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DNA POLYMERASE ACTION

➢DNA polymerases are capable of adding as many as

1000 nucleotides/sec to a primer strand due to high
degree of processivity.

Processivity is defined as the average number of

nucleotides added each time the enzyme binds a
primer:template junction.
➢ Processivity is facilitated by sliding of DNA
polymerases along the DNA template. Once bound to a
primer:template junction, DNA polymerase interacts
tightly with much of the double-stranded portion of the
DNA in a sequence-nonspecific manner.
Exonucleases & Proofreading

❖ DNA pol make a mistake in every 1010 bp added in

the growing strand. The mismatched base is removed
by the same DNA pol through proofreading Difference between processive &
exonuclease activity (3’ to 5’ exonuclease). nonprocessive DNA polymerase.

Proofreading...Continue...
➢This “proofreading” of the newly added DNA gives the DNA
polymerase a second chance to add the correct nucleotide.
➢When a mismatched base pair is present in the polymerase active site,
the primer:template junction is destabilized, creating several base pairs
of unpaired DNA. The DNA polymerase active site binds such a
mismatched template poorly, but the exonuclease active site has a 10-
fold higher affinity for single-stranded 3’ends.

✓On average, DNA polymerase inserts one incorrect nucleotide for

every 105 nucleotides added. So, How it achieve 1010 ???

✓Proofreading exonucleases decrease the appearance of incorrect base

pairs to 1 in every 107 nucleotides added.

❖This error rate is still significantly short of the actual rate of mutation
observed in a typical cell (approximately one mistake in every 10 10
nucleotides added). This additional level of accuracy is provided by the
post replication mismatch repair process.

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THE REPLICATION FORK

➢The junction between the newly separated template strands and the unreplicated duplex DNA is known
as the replication fork. In the cell, both strands of the DNA duplex are replicated at the same time
✓The antiparallel nature of DNA creates a complication for the simultaneous replication of the
two exposed templates at the replication fork. Because DNA is synthesized only 5’to 3’direction.
One DNA strand is synthesized continuously, called leading strand and the discontinuous strand
is lagging strand .
➢Although the leading-strand DNA polymerase can replicate its template as soon as it is exposed,
synthesis of the lagging strand must wait for movement of the replication fork to expose a substantial
length of template before it can be replicated.
➢The resulting short fragments of new DNA formed on the lagging strand are called Okazaki fragments &
vary in length from 1000 to 2000 nucleotides in bacteria and from 100 to 400 nucleotides in eukaryotes.

RNA Primer Synthesis & Removal

✓All DNA polymerases require a primer with a free 3’-OH. They cannot initiate a new DNA strand
de novo like RNA pol. Primase is a specialized RNA polymerase dedicated to making short RNA
primers (5–10 nucleotides long) on an ssDNA template containing a particular trimer (GTA in the
case of Escherichia coli primase) during replication.
✓Both the leading and lagging strands require primase to initiate DNA
synthesis. Each leading strand requires only a single RNA primer were
as each okazaki fragment of lagging strand need one new primer.
➢Primase activity is dramatically increased when it associates with
.
another protein that acts at the replication fork called DNA helicase.
❖ RNA primers are replaced with DNA by RNase H to Complete
DNA Replication. RNase H removes all of the RNA primer except
the ribonucleotide directly linked to the DNA end. This is because
RNase H can only cleave bonds between two ribonucleotides. The
final ribonucleotide is removed by a 5’ exonuclease that degrades
RNA or DNA from their 5’ ends.

✓Removal of the RNA primer leaves a gap in the dsDNA that is an

ideal substrate for DNA polymeras , a primer:template junction.
Final nick in DNA is sealed by DNA ligase, makes phospodiester
bond.

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JUNE 2019

Ans: 2

DNA Helicases in bacteria

✓At the replication fork, DNA helicases catalyze the separation of the two strands of duplex DNA
using ATP. Typically, DNA helicases are hexameric proteins that assume the shape of a ring.

✓Like DNA polymerases, DNA helicases act processively. Each time they associate with substrate, they
unwind multiple base pairs of DNA. The ring shaped hexameric DNA helicases found at replication forks
exhibit high processivity because they encircle the DNA.
➢DNA helicases can moves along ssDNA either 5’ to 3’ or 3’ to 5’ direction. The central channel of the
helicase has a diameter of 13 A˚ , large enough to fit ssDNA, but much too small to fit the 20 A˚ diameter
of dsDNA.

Single-Stranded DNA-Binding Proteins

To stabilize the separated strands, ssDNA-binding proteins
(SSBs) rapidly bind to the separated strands in a cooperative
fashion. SSB-SSB interactions is the cause of cooperative
binding of SSB with ssDNA. SSBs primarily contact ssDNA
through electrostatic interactions & not by H-bonding.

DNA helicases separate the two strands of the double helix

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Topoisomerases
➢As the strands of DNA are separated at the replication fork, the
dsDNA in front of the fork becomes increasingly positively
supercoiled
➢If the DNA strands remain unbroken, there can be no reduction
in linking number (the number of times the two DNA strands are
intertwined) ,one DNA link must be removed for every 10 bp of
DNA unwound.

✓The supercoils introduced by the action of the DNA helicase are

removed by topoisomerases that act on the unreplicated dsDNA in
front of the replication fork. These enzymes do this by breaking
either one or both strands of the DNAwithout letting go of the DNA
and passing the same number of DNA strands through the break.

Type IA topoisomerase action

✓Type IA topoisomerase functions via a strand passage mechanism.
✓Type IB topoisomerase functions via a controlled rotation mechanism.
✓Type II Topoisomerases (DNA gyrase) Function via a Strand Passage Mechanism through double
stranded break.

Replication Fork Enzymes

The proteins that act at the replication fork interact tightly but in a sequence-independent manner with the
DNA.

DNA helicase breaks only the hydrogen bonds that hold the two
strands of DNA together without breaking any covalent
bonds. Although topoisomerases break one or two of the targeted
DNA’s covalent bonds, each bond broken is precisely re-formed
before the topoisomerase releases the DNA.

Type II topoisomerase action

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JUNE 2019

Ans: 3

DNA POLYMERASES
❖DNA polymerase III is the primary enzyme (among 5) involved in the replication of the
chromosome. Because the entire 4.6-Mb E. coli genome is replicated by two replication forks, DNA
Pol III must be highly processive. In contrast, DNA polymerase I is specialized for the removal of the
RNA primers by 5’ exonuclease . Unlike DNA Pol III holoenzyme, DNA Pol I is not highly processive,
adding only 20–100 nucleotides per binding event. These properties are ideal for RNA primer removal
and DNA synthesis across the resulting ssDNA gap. The 5’ to 3’ exonuclease of DNA Pol I can remove
the RNA–DNA linkage that is resistant to RNase H.
➢The remaining three DNA polymerases in E. coli are specialized for DNA repair and lack proofreading
activities
Eukaryotic polymerase
➢ Eukaryotic cells also have multiple DNA polymerases, with a typical cell having more than 15. Of
these, three are essential to duplicate the genome: DNA Pol δ, DNA Pol ɛ, and DNA Pol α/primase .

✓ DNA Pol α/primase is specifically involved in initiating new DNA strands.

➢ Because of its relatively low processivity, DNA Pol α/primase is rapidly replaced by the highly
processive DNA Pol δ and Pol ɛ by the process of polymerase switching.

✓ DNA Pol ɛ synthesizing the leading strand where as DNA Pol δ for the lagging strand.

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DNA Polymerases

JUNE 2019

ANS: 3

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DNA polymerase switching during eukaryotic DNA replication

The order of eukaryotic DNA polymerase

function is illustrated. The length of the DNA
synthesized is shorter than in reality for illustrative
purposes. Typically, the combined DNA Pol
α/primase product is between 50 and 100 bp, and
the further extension by Pol ɛor Pol δ is between
100 and 10,000 nucleotides. Although both DNA
Pol δ and ɛ can substitute for DNA Pol α/primase,
recent studies indicate that DNA Pol ɛ substitutes
on the leading-strand template and DNA Pol δ
substitutes on the lagging-strand template.

Sliding Clamps
✓One key to the high processivity of the DNA polymerases that act at replication forks is their
association with proteins called sliding DNA clamps.

➢These proteins are composed of multiple identical subunits that assemble in the shape of a “doughnut.”
The hole in the center of the clamp is large enough to encircle the DNA double helix .

➢In the absence of the sliding clamp, a DNA polymerase dissociates and diffuses away from the
template DNA on average once every 20–100 bp synthesized.

➢Once released from a DNA polymerase, sliding clamps are not

immediately removed from the replicated DNA. Instead, other
proteins interact with the clamp proteins

✓ Enzymes that assemble chromatin in eukaryotic cells are recruited to the sites of DNA replication by
an interaction with the eukaryotic sliding DNA clamp (called “PCNA”).

➢The sliding clamp is a closed ring in solution but must open to encircle the DNA double helix. A special
class of protein complexes, called sliding clamp loaders, catalyze the opening and placement of sliding
clamps on the DNA.
➢Sliding clamp loaders and DNA polymerases cannot interact with a sliding clamp at the same
time because they have overlapping binding sites on the sliding clamp.

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DNA SYNTHESIS AT THE REPLICATION FORK

➢The DNA Pol III holoenzyme includes three copies of the
“core” DNA Pol III enzyme and one copy of the five subunit
sliding clamp loader. Although present in only one copy in
the holoenzyme, the sliding clamp loader includes three
copies of τ protein, each of which binds one DNA Pol III
core enzyme.
❖How do the multiple DNA polymerases remain linked at
the replication fork while synthesizing DNA on both the
leading and lagging template strands? Composition of the DNA Pol III holoenzyme

➢Due to the flexibility of DNA and the τ protein as discussed in “Trombone” model. As the helicase
unwinds the DNA at the replication fork, the leading-strand template is exposed and acted on
immediately by one DNA Pol III core enzyme, which synthesizes a continuous strand of complementary
DNA. In contrast, the lagging-strand template is not immediately acted on by DNA polymerase. Instead,
it is spooled out as ssDNA that is rapidly bound by SSBs.
✓ RNA primer on the lagging-strand (each okazaki fragment) template is recognized by the sliding
DNA clamp loader, a sliding clamp is assembled at this site, and a second DNA Pol III enzyme
initiates lagging-strand synthesis.
✓The third DNA Pol III initiates synthesis of a new Okazaki fragment as soon as a sliding DNA clamp
is assembled on the RNA primer, likely before the completion of the previous Okazaki fragment.

“Trombone” model for coordinating replication by two DNA polymerases at E. coli replication fork.

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Interactions between Replication Fork Proteins

➢Additional protein–protein interactions between replication fork proteins facilitate rapid replication fork
progression. The most important of these is an interaction between the DNA helicase (the hexameric dnaB
protein) and the DNA Pol III holoenzyme .
✓ This association stimulates the activity of the helicase by increasing the rate of helicase movement 10-
fold.
➢ A second important protein–protein interaction occurs between the DNA helicase and primase , this
interaction strongly stimulates primase function(about 1000-fold).

INITIATION OF DNA REPLICATION

❖The specific sites at which DNA unwinding and initiation of replication occur are called origins of
replication. Depending on the organism, there may be as few as one or as many as thousands of origins
per chromosome. The replicon model proposed two components that controlled the initiation of
replication: the replicator and the initiator.

➢ The replicator is defined as the cis-acting DNA sequences that are sufficient to direct the initiation of
DNA replication.
➢ Initiator protein specifically recognizes a DNA element in the replicator and activates the initiation of
replication.
✓ The single replicator required for E. coli chromosomal replication is called oriC. Two repeated
motifs are critical for oriC function. The 9-mer motif is the binding site for the E. coli initiator, DnaA,
and is repeated five times at oriC. The 13-mer motif, repeated three times, is the initial site of ssDNA
formation during initiation.

The DNA elements that make up three well characterized replicators

are shown, taken from E.coli.

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BINDING AND UNWINDING

➢Initiator proteins perform at least two different functions during the initiation of replication. First,
these proteins bind to the replicator DNA, often via a specific binding site. Second, initiator proteins
interact with additional factors required for replication initiation, thus recruiting them to the replicator .

✓DnaA includes two DNA-binding domains. One domain binds the repeated 9-mer elements in oriC in
their double-stranded form . When bound to ATP (but not ADP), DnaA also interacts with DNA in the
region of the repeated 13-mer repeats of oriC and result in the separation of the DNA strands over more
than 20 bp within the 13-mer repeat region.

✓DnaA recruits additional replication proteins to the ssDNA formed at the replicator including the DNA
helicase. E. Coli DNA Replication is regulated by DnaA.ATP levels and SeqA .

➢In eukaryotic cells, the initiator is a six-protein complex called the origin recognition complex (ORC) .

➢ORC recognizes a conserved sequence found in yeast replicators, called the “A element,” as well as
a second, less-conserved B1 element. Like DnaA, ORC binds and hydrolyzes ATP.

✓Helicase loading Is the First Step in the Initiation of Replication in Eukaryotes.

Protein–Protein and Protein–DNA Interactions During Initiation

❖After the initiator (DnaA) has bound to oriC and unwound the 13-mer DNA, the combination of
ssDNA and DnaA recruits a complex of two proteins: the DNA helicase (DnaB) and helicase
loader (DnaC). Importantly, binding to the helicase loader inactivates the DNA helicase,
preventing it from functioning at inappropriate sites.

➢Interestingly, like the subunits of the sliding clamp loader, DnaC is an ATP-utilizing AAA + protein.

➢Helicase recruits DNA primase to the origin DNA, resulting in the synthesis of an RNA primer on
each strand of the origin and release the helicase loader.

✓The DNA Pol III holoenzyme is brought to the origins through interactions with the primer:template
junction and the helicase. Once the holoenzyme is present, sliding clamps are assembled on the RNA
primers, and the leading-strand polymerases are engaged .

➢As new ssDNA is exposed by the action of the helicase, it is bound by SSBs, and DNA primase
synthesizes the first lagging-strand primers and second DNA recruited and ultimately the third pol for
the synthesis of first and second Okazaki fragments.
➢At this point, two replication forks have been assembled, and initiation of replication is complete.

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Model for E. coli initiation of DNA replication.

Single DNA duplication per Generation in E coli

❖E. coli prevents runaway chromosome duplication by inhibiting recently initiated origins from
reinitiating.
➢In E. coli cells, an enzyme called Dam methyltransferase adds a methyl group to the A within every
GATC sequence (note that the sequence is a palindrome). Typically, the genome is fully methylated at
GATC sequences.
➢This situation is changed after each GATC sequence is
replicated. Because the A residues in the newly synthesized
DNA strands are unmethylated, recently replicated sites will
be methylated on only one strand (referred to as
hemimethylated).

✓The hemimethylated state of newly replicated oriC DNA

is detected by a protein called SeqA. SeqA binds tightly to
the GATC sequence, but only when it is hemimethylated.
There is an abundance of GATC sequences within and near
oriC. Once replication has initiated, SeqA binds to these
sites before they can become fully methylated by the Dam
methyl transferase.

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20 min vs 40 min problem in Bacteria

➢The DNA methylation, action of SeqA and Dna A rapidly and dramatically reduce the ability of E. coli
to initiate replication from new copies of oriC. Although these mechanisms prevent rapid reinitiation,
this inhibition does not necessarily last until cell division is complete .
✓Indeed, for E. coli cells to divide at the maximum rate, the daughter copies of oriC must initiate replication
before the completion of the previous round of replication. This is because E. coli cells can divide every 20
min, but it takes more than 40 min to replicate the E. coli genome. Thus, under rapid growth conditions, E.
coli cells reinitiate replication once and sometimes twice before the completion of previous rounds of
replication.

➢Even under such rapid growth conditions, initiation does

not occur more than once per round of cell division

❖So, in E.coli the chromosomes that are segregated into

the daughter cells are being actively replicated. This is in
contrast to eukaryotic cells, which do not start chromosome
segregation until all of the chromosomes are completely
replicated.

DEC 2017

ANS: 3 Ans: 2

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FINISHING REPLICATION- Termination

➢ Eventually, the two replication forks of the circular E. coli
chromosome meet at a terminus region containing multiple copies
of a 20 bp sequence called Ter. The Ter sequences are arranged on
the chromosome to create a trap that a replication fork can enter
but cannot leave. The Ter sequences function as binding sites for
the protein Tus (terminus utilization substance). The Tus-Ter
complex can arrest a replication fork from only one direction first
encountered (among two). when either replication fork encounters
a functional Tus-Ter complex, it halts; the other fork halts when it
meets the first (arrested) fork.
➢ The final few hundred base pairs of DNA between Tus-Ter
complexes are then replicated (by an unknown mechanism), completing
two topologically interlinked (catenated) circular chromosomes . DNA
circles linked in this way are known as catenanes. Separation of the
catenated circles in E. coli requires topoisomerase IV (a type II
topoisomerase). The separated chromosomes then segregate into
daughter cells at cell division. The terminal phase of replication of other
circular chromosomes, including many of the DNA viruses that infect
eukaryotic cells, is similar. Topoisomerase II catalyzes
the decatenation

Eukaryotic Chromosomes Are Replicated Exactly Once per Cell Cycle

➢The need to replicate the DNA once and only once is a particular challenge for eukaryotic
chromosomes because they each have many origins of replication. Origins are typically separated by 30
kb, thus even a small eukaryotic chromosome may have more than 10 origins and a large human
chromosome may have thousands .

✓Helicase loading is the first step in the initiation of replication in eukaryotes. Helicase loading
occurs at all replicators during G1 (before S phase). Replicator or origin activation, including
helicase activation and replisome assembly, only occurs after cells enter S phase.

❖The first step in helicase loading is the recognition of the replicator by the eukaryotic initiator, ORC,
bound to ATP. As cells enter the G1 phase of the cell cycle, ORC bound to the origin recruits two
helicase loading proteins (Cdc6 and Cdt1) and two copies of the minichromosome maintenance
(MCM) proteins, Mcm2-7 helicase to the origin.
➢ATP hydrolysis by Cdc6 results in the loading of a head-to-head dimer of the Mcm2-7 complex such that
they encircle the double-stranded origin DNA.

✓Consistent with the Mcm2–7 complex encircling dsDNA instead of ssDNA, eukaryotic helicase loading
does not lead to the immediate unwinding of origin DNA

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❖ The loaded helicases in G1 are only activated to unwind DNA and initiate replication after cells pass
from the G1 to the S phase of the cell cycle. Loaded helicases are activated by two protein kinases:
CDK (cyclin dependent kinase) and DDK (Dbf4-dependent kinase). These kinases are activated when
cells enter S phase .
❖Phosphorylation of these proteins results in the Cdc45 and GINS proteins binding to the Mcm2-7
helicase . Importantly, Cdc45 and GINS strongly stimulate the Mcm2-7 ATPase and helicase activities
and together form the Cdc45–Mcm2-7–GINS (CMG) complex, which is the active form of the Mcm2-7
DNA helicase.

Eukaryotic helicase loading

Activation of loaded helicases

leads to the assembly of the
eukaryotic replisome.

Helicase Loading is Critical

❖How do eukaryotic cells control the activity of hundreds or even thousands of origins of replication such
that not even one is activated more than once during a cell cycle ???

➢Upon entry into S phase and continuing throughout G2 and M phase, helicases loaded during G1 can be
activated, but new helicase loading is strictly inhibited
➢ In each cell cycle there is only one opportunity for helicases to load onto origins (during G1) and only
one opportunity for those loaded helicases to be activated (during S, G2, and M—although in practice, all
loaded helicases are activated or disrupted by replication forks during S phase)

✓In the budding yeast S. cerevisiae, the regulation is tightly coupled to the function of CDKs.

✓CDK levels are low during G1, allowing helicase loading but preventing helicase activation. Entry into
the S phase of the cell cycle is coupled with a rapid increase in CDK activity, driving activation of
loaded helicases but simultaneously preventing new helicase loading.

➢Importantly, CDK levels remain elevated during the remainder of the cell cycle (S, G2, and M phases)
that inhibit the function of ORC, Cdc6, and Cdt1 and thus helicase loading is ceased further.

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Cell cycle regulation of

CDK activity controls replication

JUNE 2019

Ans: 2

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The End Replication Problem

➢ Lagging-strand synthesis is unable to copy the extreme ends
of linear chromosomes. The last RNA primerforOkazaki
fragment once removed, there will remain a short region
(the size of the RNA primer) of un replicated ssDNA at the end
of the chromosome. Slowly, but surely, genes at the end of the
chromosomes would be lost.
❖ This difficulty is not observed during the duplication of the
leading-strand template, as the single RNA primer can be
extended extreme 5’ terminus of its template.
Solutions: 1. Many viruses use a “priming protein” instead of an
RNA as the primer for the last Okazaki fragment at each end of
the chromosome. Here, OH group of a amino acid ( tyrosine
often) is used for reaction initiation.
2. Most eukaryotic cells recruits a specialized DNA polymerase
called telomerase for chromosome end (telomeres) repairing.

When lagging-strand replication machinery reaches the end of the chromosome, primase no longer has
sufficient space to synthesize a new RNA primer. This results in incomplete replication and a short
ssDNA region at the 3’- end of the lagging-strand DNA product.

Telomerase Solves the End Replication Problem

➢The ends of eukaryotic chromosomes are called telomeres, and they are
generally composed of head-to-tail repeats of the sequence 5’-TTAGGG-3’.
✓ Telomerase is a template independent, ribonucleoprotein that includes
multiple protein subunits and an RNA component (TER). Telomerase
specifically elongates the 3’-OH of telomeric ssDNA sequences using its own
RNA as a template.
✓ In mammals, the short sequence, 5’-AAUCCCAAUC-3’ of the RNA can
anneal to the ssDNA at the 3’ end of the telomere and form primer:template
junction that can be acted on by telomerase.
➢ The “telomerase reverse transcriptase”(TERT) of one subunit add dNTPs
on RNA primer. Telomerase then displaces the RNA from the DNA product
using its RNA-DNA helicase activity and rebinds at the end of the telomere
and repeats the process.
❖ When telomerase acts on the 3’ end of the telomere, it extends only
one of the two strands of the chromosome. How is the 5’- end extended?
This is done by the lagging-strand DNA replication machinery.
By providing an extended 3’ end, telomerase provides additional template
for the lagging-strand replication machinery & thus increase the length of
the 5’-end of the chromosome.

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❑ Even after the action of the lagging-strand machinery, there remains a

short ssDNA region at the end of the chromosome. Indeed, the presence of
a 3’overhang may be important for the end protection function of the telomere.
Because of the repetitive and non-protein-coding nature of the telomeric DNA,
variations in the length of the telomere are easily tolerated by the cell.

Telomere-binding proteins act to regulate the activity of telomerase

and protect the ends of chromosomes from degradation and recombination.
Telomere-binding proteins are illustrated for S.cerevisiae and human cells.
(a) S. Cerevisiae cells contain binding protein Rap1 , Rif1 and Rif 2. All three
proteins have been implicated in the inhibition of telomerase activity where as
Cdc13 binds to the single-stranded telomere repeat DNA and is involved in
telomerase recruitment.
(b) Human cells. TRF1 andTRF2 bind directly to the double-
stranded telomere repeat DNA. The human homolog of
Rap1 as well as TIN2, TPP1, and POT1 all associate with
either TRF1 or TRF2. Together these proteins form a
complex that is called Shelterin for its ability to “shelter” the
telomeres from the action of DNA repair enzymes. POT1
also binds directly to the single-stranded telomere repeat
DNA and inhibits telomerase activity.

Ans: 1

Ans: 3

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Telomere length regulation by telomere-binding proteins

❖When telomeres are relatively short, few telomere binding proteins
will be present, and inhibition of telomerase is weak. Under these
conditions, telomerase can extend the 3’ end of the telomere. When
these regions are made double-stranded by the action of the lagging-
strand DNA synthesis machinery, additional telomere-binding
proteins can associate with the telomere. Binding of these proteins
increases the level of inhibition, preventing further elongation by
telomerase.

Telomeres isolated from human cells were

observed by electron microscopy and found to
form a loop rather than a linear structure, called
a t-loop, was formed by the 3’-ssDNA end of
the telomere invading the dsDNA region of the
telomere . Telomere binding protein, TRF2
direct t-loop formation.
The loop structure may protect the telomere from
DNA repair enzymes, it is also likely that telomerase
cannot recognize this form of the telomere, because it
lacks an obvious single-strand 3’-end.

Aging, Cancer, and the Telomere Hypothesis---Fountain of youth

➢ Leonard Hayflick’s studies led to the idea that cells contain an intrinsic countdown clock that
limits the number of divisions in which a cell can participate.

➢ Telomere DNA isolated from young people is longer than that isolated from older people. This
observation led to the hypothesis that the length of telomeric DNA limited the number of times a cell
could divide.
➢ Many normal cells have limited or no telomerase activity. In contrast, cells that have increased
proliferative capacity, such as stem cells and cells derived from tumors, have higher levels of
telomerase activity.

✓ Indeed, studies of cancer cells in culture indicate that they can divide indefinitely. A important
experiment in support of the model showed that expression of telomerase in normal cells
effectively immortalized the cells.

❖ The elevation of telomerase activity in cancer cells also suggests that globally activating
telomerase would not be a wise method to seek immortality!

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Eukaryotic Vs Prokaryotic Replication (Review topic)

VVI for net exam.
❖The Eukaryotic Replication Fork .The general features of DNA
replication in eukaryotes are similar to those in prokaryotes. An
RNA primer is formed by a specifc enzyme in eukaryotic DNA
replication, as is the case with prokaryotes, but in this case the
primase activity is associated with Pol α. After the RNA primer is
made and a few nucleotides are added by Pol α, the polymerase
dissociates and is replaced by Pol δ and its attached PCNA
protein (proliferating cell nuclear antigen, eukaryotic version of
sliding clamp). Another protein, called RFC (replication factor C),
is involved in attaching PCNA to Pol δ.

➢The RNA primer is eventually degraded,

but, in the case of eukaryotes, the
polymerases do not have the 5‘ to 3'
exonuclease activity to do it. Instead, separate
enzymes, FEN-1 and RNase H1, degrade the
RNA. Continued movement of Pol δ fills in
the gaps made by primer removal.
The basics of the eukaryotic replication fork

DEC 2017

Ans: 2

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Semiconservative Replication
✓Each new DNA molecule contains one strand from the
original DNA and one newly synthesized strand. This
situation is called semiconservative replication.
Semiconservative replication of DNA was established in the
late1950s by Matthew Meselson and Franklin Stahl.

➢E. coli bacteria were grown with 15NH4Cl as the sole

nitrogen source, 15N being a heavy isotope of nitrogen. The
15N-labeled cells were then transferred to a medium that

contained only 14N. The cells continued to grow in the new

medium. With every new generation of growth, a sample of
DNA was extracted and analyzed by CsCl density-gradient
centrifugation.

Θ- SHAPED REPLICATION
Replication of the E. coli chromosome occurs bidirectionally from the
unique origin of replication. Each Y-shaped structure is a replication fork,
and the two replication forks move in opposite directions sequentially
around the circular chromosome . The circular E. coli chromosomes
formed θ shaped intermediates during replication.

ROLLING-CIRCLE REPLICATION
Rolling-circle replication is used (1) by many viruses to duplicate their
genomes, (2) in bacteria to transfer DNA from donor cells to recipient
cells during one type of genetic exchange, and (3) in amphibians to
amplify extrachromosomal DNAs carrying clusters of ribosomal RNA
genes during oogenesis. The enzymes involved in rolling-circle
replication and the reactions catalyzed by these enzymes are basically
the same as those responsible for DNA replication involving θ-type Rolling circle replication in virus φX174
intermediates.

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