DNA polymerases

Replication
Unwinding of DNA helix
Hybridization of Template and Primer
Cell Molecular
Biology, N Dhami,
Pokhara
Extension of primer
(addition
of
dNTPs)
University Nepal

DNA polymerase is a key enzyme catalyzing DNA synthesis

Possess single catalytic
site
Distinguish appropriate
dNTPs
Active site of DNA
polymerase is
responsible for selection
of correct nucleotides
rNTPs are >10 fold higher
in concentration in a cell
rNTPs rarely added to
DNA chain >1,000 fold
less chances
Nucleotide binding
pocket of DNA
polymerase allow only
one OH group in NTP
Watson et al. page 185

Cell Molecular Biology, N Dhami, Pokhara

University Nepal

DNA polymerase allows nucleotide with only one OH group

Watson et al. page 185

Cell Molecular Biology, N Dhami, Pokhara

University Nepal

Diversity of DNA polymerases

Cell Molecular Biology, N Dhami, Pokhara

University Nepal

Diversity of DNA polymerases

Cell Molecular Biology, N Dhami, Pokhara

University Nepal

DNA polymerase resembles a hand that grips the

primer:template junction

Watson et al. page 186

Palm domain contains 2

divalent metal ions typically
Mg++ or Zn++ that alter chemical
environment around dNTP and
3 OH of the primer
Cell Molecular Biology, N Dhami, Pokhara
University Nepal

Two metal ions (Mg++ or Zn++)

facilitate NTP addition

Watson et al. page 187

One Mg++ ion reduces affinity of

the 3 OH for its hydrogen and
generates O- nucleophile .
O- nucleophile (3 OH) of the
primer attacks on Phosphate
2nd ion neutralize negative
Cell Molecular Biology, N Dhami, Pokhara
University Nepal charge of and Phosphorous

Palm domain monitors dNTP

addition

Palm domain monitors

accuracy of recent base
pairing
Mismatched nucleotides
slows the catalysis and
reduces affinity for further
extension
Reduced affinity for new
nucleotide causes release of
primer: template complex
from active site
Cell Molecular Biology, N Dhami, Pokhara
University Nepal

Finger domain grips the template and incoming dNTPs

Finger domain bind to incoming dNTPs

As soon as correct base pairing occurs between incoming dNTP and template, the finger
domain enclose the dNTPs
Finger domain associates with the template and expose only the first template base after
primer at the catalytic site. Thus,Cell
eliminates
the ambiguity
of the template base to be copied
Molecular Biology,
N Dhami, Pokhara
University Nepal

Thumb domain involves in post DNA synthesis regulation

Thumb domain interacts with

the most recently synthesized
DNA
It maintains correct position of
primer and active site
Helps to maintain strong
association between DNA
polymerase and its substrate
Cell Molecular Biology, N Dhami, Pokhara
University Nepal

DNA polymerases are processive enzymes

Cell Molecular Biology, N Dhami, Pokhara

University Nepal

DNA polymerases are processive enzymes

Capable of adding many nucleotides
Processivity of a DNA polymerase is defined as the number
of nucleotides added each time the enzyme binds a
primer:template junction
Each DNA polymerase has specific processivity few
nucleotides to many nt
The initial binding of DNA pol to primer:template junction
is rate limiting step in DNA synthesis
Efficient DNA pol can add ~1000 nt per second
Many rounds of reactions can add up to 1000 times nt
The processivity of a DNA pol determined by association of
DNA pol to primer:template junction and its ability to slide
along the template

Cell Molecular Biology, N Dhami, Pokhara

University Nepal

DNA polymerases possess 3 5 exonuclease activity

DNA synthesis maintains

high level accuracy
1 mistake per 1010 nt is
possible because of
tautomeric forms of
bases or other reasons
Mismatched bases are
duly removed
(proofreading) by the
exonuclease action of
DNA polymerase

Cell Molecular Biology, N Dhami, Pokhara

University Nepal