1. DNA Replication
Senior Biology
Mrs. Brunone

2. DNA – Structure
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.
The double-stranded structure suggests a mode of copying (replication) and the long “strings” of the 4 bases encode biological life.
The human genome is just 3.5 billion base pairs and greater than 95% is considered to be non-coding (or “junk”).
Consider the human genome as a 3.5 gigabyte hard drive filled with information of which greater than 95% is rubbish, corrupted etc.

3. History Of DNA Research
Summary
DNA replication is semi-conservative (Meselson-Stahl, 1958).
Replication requires a DNA polymerase, a template, a primer and the 4 nucleotides and proceeds in a 5’ to 3’ direction (Kornberg, 1957).
Replication is semi-discontinuous (continuous on leading strand and discontinuous on lagging strand) and requires RNA primers (Okazaki’s, 1968).
Lagging strand synthesis involves Okazaki fragments.

4. 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”

5. DNA Replication is Semi-discontinuous
Continuous synthesis
Discontinuous synthesis

6. DNA SYNTHEIS REACTION products 5' end of strand P P CH2 Base CH2 Base+ P 3' P P
Phosphodiester bonds OH P P
Synthesis reaction
CH2
Base P O 5'
CH2
Base O OH 3'
3' end of strand OH

7. 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

8. Proteins Involved in DNA Replication in E. coli

9. Enzymes in DNA replication
Primase adds
short primer
to template strand
Helicase unwinds
parental double helix
Binding proteins
stabilise separate
strands
DNA polymerase
binds nucleotides
to form new strands
DNA polymerase I (Exonuclease) removes RNA primer and inserts the correct bases
Ligase joins Okazaki
fragments and seals
other nicks in sugar-phosphate backbone

10. Replication Helicase protein binds to DNA sequences called
Primase protein makes a short segment of RNA
complementary to the DNA, a primer. 5’ 3’
Binding proteins prevent single strands from rewinding. 3’ 5’
Helicase protein binds to DNA sequences called
origins and unwinds DNA strands.

11. Replication DNA polymerase enzyme adds DNA nucleotides
Overall direction
of replication 5’ 3’
DNA polymerase enzyme adds DNA nucleotides
to the RNA primer.

12. Replication DNA polymerase enzyme adds DNA nucleotides5’
Overall direction
of replication 3’
DNA polymerase enzyme adds DNA nucleotides
to the RNA primer.
DNA polymerase proofreads bases added and
replaces incorrect nucleotides.

13. Replication Leading strand synthesis continues in a5’ 3’
Overall direction
of replication
Leading strand synthesis continues in a
5’ to 3’ direction.

14. Replication Leading strand synthesis continues in a3’ 5’
Overall direction
of replication
Okazaki fragment
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.

15. Replication Leading strand synthesis continues in a
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.

16. Replication Leading strand synthesis continues in a3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.

17. Replication Leading strand synthesis continues in a5’ 3’ 3’ 3’ 5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.

18. Replication 3’ 5’ 5’ 5’ 3’
Exonuclease activity of DNA polymerase I removes RNA primers.

19. Replication Polymerase activity of DNA polymerase I fills the gaps.3’ 5’
Polymerase activity of DNA polymerase I fills the gaps.
Ligase forms bonds between sugar-phosphate backbone.

20. DNA REPLICATION 3 Pol III synthesises leading strand 2 1
Helicase opens helix
Topoisomerase nicks DNA to relieve tension from unwinding 4
Primase synthesises RNA primer 5
Pol I excises RNA primer; fills gap 6 7
Pol III elongates primer; produces Okazaki fragment
DNA ligase links Okazaki fragments to form continuous strand

21. DNA Synthesis •Synthesis on leading and lagging strands
•Proofreading and error correction during DNA replication
•Simultaneous replication occurs via looping of lagging strand

22. 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

23. Replication Termination of the Bacterial Chromosome
ori
ter
Origin 5’ 3’
BIDIRECTIONAL REPLICATION

24. Procaryotic (Bacterial) Chromosome Replication
ori
ter
Replication
Forks
Bidirectional Replication Produces
a Theta Intermediate

25. Summary DNA replication proteins: DNA Pol III DNA Pol I DNA Ligase
Primase
Helicase
SSB
Gyrase
Exonuclease (DNAP II)