DNA Replication
Senior Biology
Mrs. Brunone
DNA – Structure
1. A simple yet elegant structure – a double
helix with a sugar phosphate “backbone”
linked to 4 types of nucleotides on the
inside that are paired according to basic
rules. Amazingly this simple molecule
has the capacity to specify Earth’s
incredible biological diversity.
2. The double-stranded structure suggests
a mode of copying (replication) and the
long “strings” of the 4 bases encode
biological life.
3. The human genome is just 3.5 billion
base pairs and greater than 95% is
considered to be non-coding (or “junk”).
History Of DNA Research
Summary
1.
DNA replication is semi-conservative (Meselson-Stahl, 1958).
2.
Replication requires a DNA polymerase, a template, a primer and
the 4 nucleotides and proceeds in a 5’ to 3’ direction (Kornberg,
1957).
3.
Replication is semi-discontinuous (continuous on leading strand
and discontinuous on lagging strand) and requires RNA primers
(Okazaki’s, 1968).
4.
Lagging strand synthesis involves Okazaki fragments.
Replication as a Process
1. Double-stranded DNA unwinds.
2. The junction of the unwound
molecules is a replication fork.
3. A new strand is formed by pairing
complementary bases with the
old strand.
4. Two molecules are made.
Each has one new and one old
DNA strand. “Semi-conservative”
DNA Replication is Semi-discontinuous
Continuous synthesis
Discontinuous synthesis
DNA SYNTHEIS REACTION
5' end of strand
P
P
Base
CH2
CH2
O
P
Base
O
P
CH2
O
Base
CH2
Base
O
products
H20
+
3'
P
P
OH
P
Synthesis reaction
P
5' CH2
3'
OH
P
P
O
CH2
Base
O
Base
OH
3' end of strand
How is DNA primed?
Primase:
• Makes initial nucleotide (RNA
primer) to which DNA
polymerase III attaches
• New strand initiated by adding
nucleotides to RNA primer
• RNA primer later replaced
with DNA
Proteins Involved in DNA Replication
in E. coli
Protein Name
Function
DNA Gyrase
SSB
DnaA
HU
PriA
PriB
PriC
DnaB
DnaC
DnaT
Primase
DNAP III holoenzyme
DNAP I
Ligase
Tus
Unwinding DNA
Single-stranded DNA binding
Initiation factor
Histone-like (DNA binding and bending)
Primosome assembly
Primosome assembly
Primosome assembly
DNA unwinding (helicase)
DnaB chaperone
Assists DnaC in delivery of DnaB
Synthesis of an RNA primer
Elongation (DNA synthesis)
Excises RNA primer, fills in with DNA
Covalently links Okazaki fragments
Termination
Enzymes in DNA replication
Helicase unwinds
parental double helix
DNA polymerase
binds nucleotides
to form new strands
Binding proteins
stabilise separate
strands
DNA polymerase I
(Exonuclease) removes
RNA primer and inserts
the correct bases
Primase adds
short primer
to template strand
Ligase joins Okazaki
fragments and seals
other nicks in sugarphosphate backbone
Replication
3’
3’
5’
5’
3’
5’
3’
5’
Helicase protein binds to DNA sequences called
origins and unwinds DNA strands.
Binding proteins prevent single strands from rewinding.
Primase protein makes a short segment of RNA
complementary to the DNA, a primer.
Replication
Overall direction
of replication
3’
3’
5’
5’
3’
5’
3’
5’
DNA polymerase enzyme adds DNA nucleotides
to the RNA primer.
Replication
Overall direction
of replication
3’
5’
3’
5’
3’
5’
3’
5’
DNA polymerase enzyme adds DNA nucleotides
to the RNA primer.
DNA polymerase proofreads bases added and
replaces incorrect nucleotides.
Replication
Overall direction
of replication
3’
3’
5’
5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
3’
5’
Replication
Overall direction
of replication
3’
3’
5’
5’
Okazaki fragment
3’
5’
3’ 5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication
Overall direction
of replication
3’
3’
5’
5’
Okazaki fragment
3’
5’
3’ 5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication
3’
5’
3’
5’
3’
5’
3’ 5’
3’5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication
3’
5’
3’
5’
3’
5’
3’5’
3’5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication
3’
5’
3’
5’
3’
5’
3’5’
3’5’
3’
5’
Exonuclease activity of DNA polymerase I
removes RNA primers.
Replication
3’
3’
5’
3’
5’
3’5’
3’
5’
Polymerase activity of DNA polymerase I fills the
gaps.
Ligase forms bonds between sugar-phosphate
backbone.
DNA REPLICATION
2
Topoisomerase
nicks DNA to
relieve tension
from unwinding
3 Pol III synthesises leading strand
1 Helicase opens helix
4 Primase synthesises RNA primer
5
6
Pol I excises RNA primer; fills gap
7
Pol III elongates primer;
produces Okazaki fragment
DNA ligase links Okazaki
fragments to form
continuous strand
DNA Synthesis
•Synthesis on leading and lagging strands
•Proofreading and error correction during DNA replication
•Simultaneous replication
occurs via looping of lagging
strand
Simultaneous Replication Occurs via
Looping of the Lagging Strand
•Helicase unwinds helix
•SSBPs prevent closure
•DNA gyrase reduces tension
•Association of core polymerase
with template
•DNA synthesis
•Not shown: pol I, ligase
Replication Termination of the
Bacterial Chromosome
BIDIRECTIONAL REPLICATION
Origin
3’
5’
5’
3’
ori
ter
Procaryotic (Bacterial)
Chromosome Replication
ori
ter
Bidirectional Replication Produces
a Theta Intermediate
Replication
Forks
Summary

DNA replication proteins:
DNA Pol III
DNA Pol I
DNA Ligase
Primase
Helicase
SSB
Gyrase
Exonuclease (DNAP II)