DNA Polymerases

Functions of different polymerases and difference between prokaryotic

and eukaryotic replication

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The Specialization of DNA Polymerases
The efficient & accurate replication of the genome requires that cells
have multiple specialized DNA polymerases.

Prokaryotic DNA Polymerases:

E.coli has at least five DNA
polymerases that are
distinguished by their enzymatic
properties , subunit composition
and abundance.

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Translesion synthesis (TLS)
• Translesion synthesis (TLS) is one of
the ways to overcome stalled
replication in which specific
polymerases (TLS polymerase)
perform bypass synthesis across
DNA damage.

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DNA POL III
DNA POL III is the primary enzyme involved in the replication of E.coli
chromosome. It is generally found to be a part of large complex that
confers very high processivity –a complex known as DNA POL III
Holoenzyme. DNA POL III carry an associated proof reading
exonuclease (3’-5’ exonuclease).

DNA POL III HOLOENZYME include 2

copies of core DNA Pol III & 1 copy of
five protein γ complex.

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DNA POL I
• DNA POL I: is specialized for the removal of the RNA primers that are used
to initiate DNA synthesis. DNA Pol I has a 5’ exonuclease that allows DNA
Pol I to remove RNA or DNA immediately upstream of the site of DNA
synthesis.
• The 5’ exonuclease of DNA Pol I can remove the RNA-DNA linkage that is
resistant to RNAse H.
• DNA POL I is not highly processive, adding only 20-100 nucleotides per
binding, ideal for replacing the short region previously occupied by RNA
Primers (< 10 nt).
• Pol I & Pol III carry an associated proof reading exonuclease for high
replication accuracy.
• The remaining three DNA polymerases in E.coli are specialized for DNA
repair & lack proof reading activities.

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Eukaryotic DNA Polymerases:

• 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 polymerase δ
• DNA polymerase ε
• DNA polymerase α/Primase
Each of these eukaryotic DNA polymerases is composed of multiple
subunits.

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DNA Polα/Primase
• It is specifically involved in initiating DNA strands.
• DNA Pol α/ Primase is a four-subunit protein complex. It consists of a
two-subunit DNA Pol α & a two-subunit Primase.
• After the synthesis of RNA primer by primase, primer- template
junction is handed off to the associated DNA Pol α to initiate DNA
synthesis.
• It has low processivity so DNA Pol α/ Primase is rapidly replaced by
highly processive DNA polymerase delta & epsilon.
• So three different polymerases function at replication fork. While rest
of the polymerases are involve in DNA repair.

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Eukaryotic & Prokaryotic DNA Polymerases

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 DNA REPLICATION IN
EUKARYOTES
Initiation, elongation and termination phases of DNA replication in
eukaryotic cells

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EUKARYOTIC REPLICATION
• In eukaryotes, clusters of about 20-50 replicons initiate
simultaneously at defined times throughout S-phase.
• Those which replicate early in S-phase comprise predominantly
euchromatin which includes transcriptionally active DNA.
• Those activated late in S-phase are mainly within heterochromatin.
• Centromeric & telomeric DNA replicates last.

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Phases of Cell Cycle

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 Cycle of Cell Reproduction

Fig. 18-1
 Reproduction Rates Vary

early Drosophila embryo nuclei (not cells yet) 6 minutes!

Phases of Cell Cycle

Fig. 18-2
There are quality control checkpoints along the way.

Fig. 18-3
Cyclin/Cdk Complexes
Regulate Progression

Fig. 18-4
 Cyclin Abundance Regulates Cdk Activity

Fig. 18-8

Cdks present at all times; cyclin levels “cycle”.

INITIATION
• The initiation of replication in eukaryotic cells requires two steps to
occur at distinct times in the cell cycle:
A) Replicator Selection
B) Origin Activation

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REPLICATOR SELECTION
• Replicator selection is mediated by the formation of pre-replicative
complexes (Pre-RCs).
• The Pre- RC is composed of four separate proteins that assemble in an
ordered fashion at each replicator.

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STEPS IN THE FORMATION OF PRE-RC
• Recognition of replicator by the Origin Recognition Complex (ORC).
• Recruitment of two helicase loading proteins namely Cdc6 (Cell
Division Cycle 6) & Cdt1 (Chromatin Licensing And DNA Replication
Factor 1) by ORC.
• Recruitment of Mcm2-7 (mini-chromosome maintenance) complex
by ORC & the loading proteins.
• Pre-RCs that are formed during G1 phase are only activated to
initiate replication after cells pass from G1 to the S phase of the cell
cycle.

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ORIGIN ACTIVATION
• Pre-RCs are activated to initiate replication by two protein kinases
namely Cdk & DdK.
• Each of these kinases is inactive in G1 & is activated only when cell
enter S phase. Once activated, these kinases target the Pre-RC &
other replication proteins.
• Phosphorylation of these proteins (i.e., CdK and Ddk) results in the
assembly of additional replication proteins at the origin & initiation of
replication.
• The newly formed proteins includes three eukaryotic DNA
polymerases & a number of other proteins required for their
recruitment.
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• The polymerases assemble at the origin in a particular order.
• DNA Polδ & Polε associate first, followed by DNA Polα/Primase.
• This order ensures that all three DNA polymerases are present at the
origin prior to the synthesis of the first RNA Primer by DNA
Polα/Primase.
• Only a subset of the proteins that assemble at origin go on to function
as part of the eukaryotic replisome.
• Three DNA polymerases, the Mcm complex & many of the factors
required for DNA polymerase recruitment become part of the
replication fork machinery.
• Similar to the E.coli DNA helicase loader (DnaC), the other factors
such as Cdc6 & Cdt, are released or destroyed after their role is
complete.
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Three different DNA polymerases are
involved in elongation
• The leading strands and each lagging strand fragment are initiated with
RNA by a primase activity that is an integral part of DNA polymerase α.
• DNA polymerase α continues elongation with DNA but is quickly replaced
by DNA polymerase ε on the leading strand and by DNA polymerase δ on
lagging strand.
• Both these enzymes have associated proofreading activity.
• The ability to synthesize long DNA is conferred on DNA polymerase by
proliferating cell nuclear antigen (PCNA), the functional equivalent of the
β subunit of E.coli DNA polymerase III holoenzyme.
• In addition to doubling the DNA, the histone content of the cell is also
doubled during S-phase.
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TERMINATION
• The ends of linear chromosomes cannot be fully replicated by semi-
discontinuous replication.
• In case of leading strand duplication, a single internal RNA primer can
direct the initiation of a DNA strand that can be extended to the
extreme 5’ terminus of its template.
• The requirement for multiple primers to complete lagging strand
synthesis means that a complete copy of its template cannot be made
as there is no DNA available for further primer attachment or to
replace RNA removed from the 5’ end.

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• To overcome this, the ends of eukaryotic chromosomes, called
telomeres, consist of hundreds of copies of a simple, non-
informational repeat sequence with the 3’ end overhanging the 5’
end.
• This repeat sequence in humans is TTAGGG. The enzyme telomerase
contains a short RNA molecule, part of whose sequence is
complementary to this repeat.
• This RNA acts as a template for the addition of these repeats to the 3’
overhang by repeated cycles of elongation & translocation.

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• The complementary strand is then synthesized by normal lagging
strand replication machinery, leaving a 3’ overhang.

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• Telomerase activity is repressed in the somatic cells of multicellular
organisms, resulting in a gradual shortening of the chromosomes with
each cell generation.
• As this shortening reaches informational DNA, the cells die. This
phenomenon may be important in cell aging.
• Furthermore, the unlimited proliferative capacity of many cells is
associated with reactivation of telomerase activity.

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