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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”).
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.
History Of DNA Research
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 theold strand.
4. Two molecules are made.
Each has one new and one old
DNA strand. “Semi-conservative”
Continuous synthesis
Discontinuous synthesis
DNA Replication is Semi-discontinuous
P
P
P
P
P
P
P
PP P
CH2
CH2
CH2
OH
OH
O
O
OBase
Base
Base
CH2
CH2
CH2
OH
O
O
OBase
Base
Base
5' end of strand
3' end of strand3'
5'
3'
H20+
Synthesis reaction
DNA SYNTHEIS REACTION
products
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
Enzymes in DNA replication
Helicase unwinds parental double helix
Binding proteinsstabilise separatestrands
DNA polymerase binds nucleotides to form new strands
Ligase joins Okazaki fragments and seals other nicks in sugar-phosphate backbone
Primase adds short primer to template strand
DNA polymerase I (Exonuclease) removes RNA primer and inserts the correct bases
Binding proteins prevent single strands from rewinding.
Helicase protein binds to DNA sequences called origins and unwinds DNA strands.
5’ 3’
5’
3’
Primase protein makes a short segment of RNA complementary to the DNA, a primer.
3’ 5’
5’ 3’
Replication
Overall directionof replication
5’ 3’
5’
3’
5’
3’
3’ 5’
DNA polymerase enzyme adds DNA nucleotides to the RNA primer.
Replication
DNA polymerase enzyme adds DNA nucleotides to the RNA primer.
5’
5’
Overall directionof replication
5’
3’
5’
3’
3’
3’
DNA polymerase proofreads bases added and replaces incorrect nucleotides.
Replication
5’
5’ 3’
5’
3’
3’
5’
3’Overall directionof replication
Leading strand synthesis continues in a 5’ to 3’ direction.
Replication
3’ 5’ 5’
5’ 3’
5’
3’
3’
5’
3’Overall directionof replication
Okazaki fragment
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
Replication
5’ 5’
5’ 3’
5’
3’
3’
5’
3’Overall directionof replication
3’
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
Okazaki fragment
Replication
5’
5’ 3’
5’
3’
3’
5’
3’
3’
5’ 5’ 3’
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’
5’ 3’
5’
3’
3’
5’ 5’ 3’
Leading strand synthesis continues in a 5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
Replication
5’
5’
3’ 3’
5’
3’
5’ 3’
5’
3’
3’
5’
Exonuclease activity of DNA polymerase I removes RNA primers.
Replication
Polymerase activity of DNA polymerase I fills the gaps.
Ligase forms bonds between sugar-phosphate backbone.
3’
5’
3’
5’ 3’
5’
3’
3’
5’
Replication
Topoisomerase nicks DNA to relieve tension from unwinding
23
1
4
56
7
Pol III synthesises leading strand
Helicase opens helix
Primase synthesises RNA primer
Pol III elongates primer; produces Okazaki fragment
Pol I excises RNA primer; fills gap
DNA ligase links Okazaki fragments to form continuous strand
DNA REPLICATION
DNA Synthesis
•Synthesis on leading and lagging strands
•Simultaneous replication occurs via looping of lagging strand
•Proofreading and error correction during DNA replication
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
ori
ter
Origin
5’3’
3’5’
BIDIRECTIONAL REPLICATION
Procaryotic (Bacterial)Chromosome Replication
ori
ter
Bidirectional Replication Produces a Theta Intermediate
Replication Forks
Summary
DNA replication proteins:
DNA Pol IIIDNA Pol IDNA LigasePrimase Helicase SSBGyraseExonuclease (DNAP II)
