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Lecture 13 – Protein/DNA interaction: DNA Polymerase
DNA Polymerase Families
Based on sequence homology (the degree of similarity between sequences), DNA
Polymerases can be subdivided into 7 different families:
Family
A

B
C
D
X

Y

RT

Types of DNA Polymerases
Replicative
Repair - involved in excision repair and
processing of Okazaki fragments
Mostly replicative – many have 3’-5’
proofreading exonucleases activity
Multimeric replicative polymerase
Replicative polymerases (polymerases
in this family are not very well
characterised)
Polymerases for:
Base excision repair (BER)
Non-homologous end joining
Addition of ‘n nucleotides’ to V, D and J
exons during antibody gene
recombination
Translesion synthesis polymerases –
they can bypass damage done to DNA
RNA and DNA dependent DNA
polymerases

Examples
T7 DNA Polymerase
Eukaryotic mitochondrial
DNA Polymerase I
E
...
aquaticus Pol I
DNA polymerases α, δ, ε,
DNA polymerase ζ
...
coli Pol III

Eukaryotic Pol β
Eukaryotic Pol λ and µ
Terminal deoxynucleotidyl
transferase (TdT)
Pol ι (iota)
Pol κ (kappa)
Pol IV
Pol V
Telomerase

DNA Polymerase Structures
Despite differences in
sequence homology, most
DNA polymerases have a
similar three-dimensional fold
that resembles the right hand
with three sub-domains: the
fingers, palm and thumb
domains, that together make
the polymerisation domain
...


•
•
•

Fingers domain - binds NTPs to template base
Thumb domain – processivity, translocation and DNA positioning
Palm domain - houses the active site residues responsible for 5’ to 3’ polymerase
activity

This lecture will discuss the conformational
changes that occur during DNA replication in
Klentaq I DNA polymerase, which belongs to
Family A and is the most understood
...


Fidelity
Accurate replication of genomic DNA is imperative for the successful proliferation of an
organism
...

DNA polymerase must be able to choose the correct nucleotide to insert into the growing
strand from a pool of nucleotides that are somewhat similar in structure
...

• Geometric constraints imposed by the polymerase – the geometries of AT and GC
base pairs are remarkably similar, but differ from mismatched base pairs
...

DNA Polymerase Conformational Changes

Step 1: the primer/template DNA (p/t) binds to the unliganded enzyme (E) forming the
E;p/t complex
...
Binding of the DNA substrate causes a conformational change to
occur in the thumb domain, in essence to grip the substrate
...


The binding of dNTP causes a large conformational change, predominantly affecting the
fingers domain
...

This is because in the E:p/t:dNTP complex, the dNTP is too far from the active site
residues and therefore, this conformational change delivers the nucleotide to the active
site
...

There is indirect evidence that this transition may not be the rate-limiting step
...
This suggests that the rate-limiting
step actually occurs in the closed conformation
...
It has then been shown that that the
formation of the closed complex is stable and relatively fast, dismissing it as the RDS
...


The above diagram is a close up of the active site
...
Whilst one
metal ion binds both the dNTP and the 3’-hydroxyl group of the primer, the other interacts
only with the dNTP
...
The metal ion bound to the primer
activates the 3’-hydroxyl group of the primer, facilitating its attack on the α-phosphate
group of the dNTP substrate in the active site
...

Step 5: a second conformational change occurs by which the pyrophosphate product s
released
...

What follows is the dissociation of the complex (distributive synthesis) or translocation of
the substrate to form a new 3’ terminus for a new round of incorporation (processive
synthesis)

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