Priming in E.

coli

E. coli use DnaG (primase) that is coded by

dnaG gene
Also requires DnaB (helicase)
DnaG and DnaB form the primosome
(complex of primase and helicase)
After primase synthesize short primer RNAs
complementary to both strands of duplex
DNA , they dissociate from the single-
stranded template
Replication Mechanism-Summary

https://www.youtube.com/watch?v=4bjerYxOTbU&t=25s
 Replication Mechanism
The replisome consists of: DNA-unwinding proteins, the
priming complex (primosome) and two equivalents of DNA
polymerase III holoenzyme
Elongation involves DnaB helicase unwinding, SSB binding
to keep strands separated, and DNA polymerase grinding
away on both strands
Topoisomerase II (DNA gyrase) relieves supercoiling that
remains.
The two core-polymerase molecules at the fork are linked
together by -subunit dimer
The core polymerase synthesising the leading strand
moves, together with -subunit clamp, along the template in
the direction of the movement of the fork, elongating the
leading strand
 Replication elongation
At the lagging strand, the core polymerase moves with
it -subunit clamp in the direction opposite to the fork
movement.
As elongation proceeds, the size of the DNA loop
between the core-polymerase and the fork increases.
The core polymerase will complete synthesized an
Okazaki fragment, and dissociates from the DNA
template.
However the -subunit dimer continues to tether to the
fork protein complex
Simultaneously a primase binds to a site adjacent to the
DnaB helicase on the single stranded segment of the
lagging strand template.
Initiate synthesis of another RNA primer
 Replication elongation
The new DNA-primer complex attracts another
-clamp to the DNA, followed by re-binding of
the core polymerase, which is still attached to
the fork complex
The polymerase proceeds to elongate the RNA
primer to form another Okazaki fragment.
Near the completion of the Okazaki fragment,
the RNA primer of the previous fragment is
removed by the 5’-3’ exonuclease activity of the
DNA polymerase I.
 Summary So Far

https://www.youtube.com/watch?v=1L8Xb6j7A4w
Consider the fidelity of
DNA replication
DNA Pol makes about 1 mistake
out of 109 nucleotides copied
 How is the high fidelity of
DNA replication
achieved?
Depends on complementary base-pairing

Proofreading mechanisms
Proofreading During Replication
Carried out by DNA Pol
3’-5’ exonuclease activity
Occurs just before a new nucleotide is added to
the growing chain
After nucleotide binding, but before the nucleotide
is covalently added to the growing chain, the DNA
Pol must undergo a conformational change
An incorrect nucleotide is more likely to dissociate
Exonucleolytic
proofreading
 Properties of DNA Polymerase

DNA Pol I – Gap filling during DNA repair

DNA Pol II – Inducible SOS response
DNA Pol III – chain elongation during DNA replication
Eukaryotic DNA Replication
Like E. coli, but more complex
 Eukaryotic DNA Replication

There are several differences between prokaryotic and

eukaryotic DNA replication which include:

1.The structure of the genomes. Eukaryotic genomes are linear,

not circular as prokaryotes
2.Eukaryotic genomes are multipartite, in pieces, as opposed to
the single piece genomes of most prokaryotes.
3.Eukaryotic genomes are much larger than prokaryotic genomes.
For example, the human genome is 3X109 base pairs, 600X larger
than E. coli.

Differences in genome structure lead to differences in the process

of replication.
 Eukaryotic DNA Replication
 Because eukaryotic genomes multipartite, there are many
origins of replication rather than a single origin seen in
prokaryotes.

 Each chromosomes has thousand of replication origins.

 The amount of DNA replicated from one origin (with two

replication forks) is called a replicon. Each replicon is ~50,000
to 300,000 bp in length.

 Useful to have many origins of replication because rate of DNA

replication in eukaryotes is much slower than in prokaryotes.
Prokaryotic replications proceed at a pace of ~ 50,000 bp/min,
whereas eukaryotic replication occurs at rate of ~2000
bp/min.
Replicons
the two lines represent
the two origin of
replication
Labeling doesn’t begin in
the middle as in
prokaryotes
This is because there are
more then one origin of
replication in eucaryote
Each region served by
one origin is called a
replicon
 Eukaryotic DNA Polymerases
In eukaryotes, at least five DNA polymerases have been identified; Polymerase α, δ, ε,
beta and gamma.
.
Polymerase α (Pol α): Forms a complex with primase (Pri protein). This complex
synthesizes a primer that contains a short 10 nucleotide stretch of RNA followed by 10
to 20 DNA bases. Pri subunits act as a primase, synthesizing an RNA primer. Occurs
once at the origin on the leading strand and at the start of each Okazaki fragment on
the lagging strand. After around 20 nucleotides, elongation is taken over by Pol ε on the
leading strand and Pol δ on the lagging strand.

Polymerase δ (Pol δ): Highly processive and has proofreading, 3'->5', exonuclease
activity. The main polymerase involved in lagging strand synthesis.

Polymerase ε (Pol ε): Highly processive and has proofreading, 3'->5', exonuclease
activity. Highly related to pol δ, and is the main polymerase involved in leading strand
synthesis.

Polymerase beta (Pol B): Not the main DNA polymerase in eukaryotes, primarily
involved in DNA repair

DNA polymerase gamma: DNA polymerase in mitochondrial DNA replication

 Eukaryotic DNA Replication
 Replication on the leading and lagging strands is performed by DNA
polymerase ε and DNA polymerase δ.

 Two replicative polymerases synthesize DNA in opposite

orientations. Polymerase ε synthesizes DNA on the "leading" DNA
strand continuously as it is pointing in the same direction as DNA
unwinding by the replisome.

 Polymerase δ synthesizes DNA on the "lagging" strand, in a

discontinuous manner.

 RNAse H recognizes the DNA:RNA hybrids that are created by the

use of RNA primers and is responsible for removing these from the
replicated strand.

 DNA polymerase α, recognizes these sites and elongates the breaks

left by primer removal.
Note: most of the other aspects of prokaryotic replication, such
as the need for RNA primers, the synthesis of Okazaki
fragments, the removal and replacement of RNA primers and
ligation of adjacent daughter fragments by DNA ligase are
similar in eukaryotes.
Summary of Eukaryotes DNA polymerase
 Telomeres
Linear chromosome of eukaryotic cell at both
ends in protective caps called telomeres
During replication, lagging strand requires DNA
site for primer to bind…created problem
If there is no mechanism to overcome the
problem, chromosome in successive
generations of cells would become shorter and
shorter, losing crucial genes as their DNA
diminished
 What are
Telomeres

http://
learn.genetics.utah.edu/
content/chromosomes/
telomeres/
Telomere Replication
https://www.youtube.com/watch?v=it8g9RU8KMM
 Telomerase
Telomeres consist of special repetitive DNA sequences.
Human telomeres composed of the base sequence
TTAGGG repeated 250-1500 times.
Telomeres DNA helps maintain the replicate
chromosome ends by binding to enzyme called
telomerase
Elongate the lagging strand template from its 3’-hydroxyl
end
Telomerase is an unusual enzyme consisting of protein
in association with RNA
Because of this mix, it is called nucleoprotein
 Telomeres and telomerase
The RNA portion of the telomerase contains
CCCUAA repeats that are complementary to the
TTAGGG repeats in the telomeres
Serve as a template for adding new TTAGGG
repeats to the end of the telomere
Telomerase is a modified reverse transcriptase
Once the 3’ end of the lagging strand template
is sufficiently elongated, synthesis of the lagging
strand can take place
 Mechanism
• Telomerase binds to chromosome
because complementarity between
CCCUAA repeats of telomerase
RNA and TTAGGG repeats of
telomeres.

• Telomerase RNA CCCUAA

repeats serve as templates for
adding TTAGGG repeats to the end
of telomeres

• After a telomere has acquired new

repeat, the enzyme moves
(translocate) to newly synthesised
end, allowing additional rounds of
telomere elongation

• Once the lagging strand template

is sufficiently elongated, synthesis
of the lagging strand can take place
 The human genes expressing the
telomerase proteins and the telomerase
associated RNA are active in germ cells and
stem cells, but turned off in most cells of
adult tissues.
DNA METHYLATION and REPAIR

Methyl directed mismatch repair

DNA METHYLATION

• Attachment of
methyl group (- Representation of a DNA molecule that is
CH3) after DNA methylated. The two white spheres
replication represent methyl groups. They are bound to
two cytosine nucleotidemolecules that make
up the DNA sequence.
• Post-replication
event Note: Two of DNA's four bases, cytosine and
adenine, can be methylated.
 DNA modification after replication: DNA
methylation
Bacteria use site-specific DNA
methylation to mark their own
DNA and cleavage at the
same site to destroy invading
viral DNA
Would inhibit bacteria
restriction endonuclease from
cutting bacterial DNA
Viral DNA is destroyed but a
small amount can be
methylated, making it resistant
This form can then invade the
same host strain and
overcome the bacterial
defense system
Methyl directed mismatch repair
In bacteria methylation also helps in DNA repair –
methyl directed mismatch repair
Proofreading/editing activity of DNA Pol I and
DNA Pol III does not always correct mismatched
base pairs.
An efficient mechanism to detect and eliminate
mismatched base pairs relies on the
hemimethylated (parent strand methylated while
newly synthesized strand does not) state of newly
replicated DNA.
Involves the activity of the MutH, MutL and MutS
complex (also known as MutHLS) and In E. coli
encoded by mutH, mutL and mutS genes.
Methyl directed mismatch repair
MutHLS methyl directed mismatch repair recognizes
lesions that cause only minor distortions in the DNA
molecules, including those caused by mismatched base
pairs, frame-shifts mutation, intercalated dyes.
Four steps process:
 Only one strand in the DNA molecule is methylated

immediately after replication – the parent strand

 Mismatched base pair is recognized by MutS

 MutS interacts with two MutH proteins and one MutL

protein
 MutH cuts non methylated daughter strands
Methyl directed mismatch repair in E. coli
https://www.youtube.com/watch?
v=p3MXIKWAi2w&t=153s
 MutHLS directed mismatch repair
Newly replicated DNA
consists of a methylated
parent strand and non-
methylated daughter strand –
hemimethylated DNA
molecule.
Daughter strand will be
methylated by Dam
methyltransferase, only after a
lag of several minutes
During this period, MutS scan
for mismatched base pairs,
find the mismatched and binds
to it.
Two MutH protein and one
MutL protein bind to the bound
MutS protein and adjacent
methylated site
 MutHLS directed mismatch repair
MutH (endonuclease) cuts
the non-methylated daughter
strand at GATC
5’-3’exonuclease (RecJ or
exonuclease VII) removes
nucleotides from the non-
methylated, cut daughter
strand. The nucleotide from
GATC through the mismatch
are removed.
DNA Pol III polymerizes the
nucleotide, filling the gap by
the removal of nucleotides
Ligase catalyzes the
formation of phosphodiester
bond to give an intact strand
 Class Discussion

How the knowledge on DNA replication applied

to industry?
Steps in PCR
What are the Differences between PCR Components and DNA
replication in vivo?
Thank You