1. Decoding the Genetic Code
Replication
RNA Synthesis
Decoding the Genetic Code

2. DNA – an emblem of the 20th century.
A simple yet elegant structure – a double helix with a sugar phosphate “backbone” linked to 4 types of nucleotide 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. These lectures cover the research that led to the elucidation of the replication (copying/ reproduction) of DNA, how DNA can generate protein products and the code that lies within DNA to generate proteins.
Summary of Lecture 1
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 of the Escherichia coli genome (a single circular DNA) starts at a specific site (ori) and is bi-directional (Cairns, 1963).
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.
DNA polymerase III is the replicative enzyme of E. coli (Cairns and deLucia, 1969).

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.

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

6. Lecture 2 Outline
The replicative enzyme, DNA polymerase III
Evidence that RNA primers are required for replication.
Additional features of the replication process.
The central dogma in biology: DNA RNA Protein.
Transcription.

7. 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

8. The subunits of E. coli DNA polymerase III
Function
Holoenzyme a e q t b g d d’ c y
5’ to 3’ polymerizing activity
3’ to 5’ exonuclease activity
a and e assembly (scaffold)
Assembly of holoenzyme on DNA
Sliding clamp = processivity factor
Clamp-loading complex
Core
Enzyme
dimer

9. DNA Must Be “Primed” Before DNA Polymerase Can Replicate It
DNA polymerase cannot initiate polymerisation de novo.
Okazaki and colleagues provided evidence for short stretches of RNA linked to nascent chains of DNA during replication.
Sugino et al., (1972) isolated Okazaki fragments after pulsing with 3H-U (incorporates into RNA and not DNA) and found it associated with newly replicated DNA.
Initiation of DNA replication, but not continuation, was shown to be sensitive to rifampicin (an antibiotic that inhibits RNA polymerases).
In follow up experiments Sugino et al., (1973) isolated Okazaki fragments after a short pulse (3H-dT) by banding on a CsCl gradient.
Treatment of the Okazaki fragments with alkali (hydrolyses RNA but not DNA) or ribonuclease resulted in a small shift in density (size).

10. Conclusions
There is a covalent linkage between ribonucleotides and deoxyribonucleotides in the newly synthesised DNA.
RNA fragments (10 to 20 nt) are located at the 5’ end of the nascent fragments and are required for priming de novo DNA synthesis.
How is the DNA primed?

11. Primase:
Makes initial nucleotide (RNA primer) to which DNA polymerase attaches
New strand initiated by adding nucleotides to RNA primer
RNA primer later replaced with DNA

12. Proteins Involved in DNA Replication in E. coli

13. 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

14. 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.

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

16. 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.

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

18. 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.

19. 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.

20. 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.

21. 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.

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

23. 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.

24. 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

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

26. 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

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

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

29. Replication Termination of the
Bacterial Chromosome
Termination: meeting of two replication forks
and the completion of daughter
chromosomes
Region 180o from ori contains replication fork
traps:
ori
Ter sites
Chromosome

30. Replication Termination of the
Bacterial Chromosome
TerA
TerB
One set of Ter sites arrest DNA forks
progressing in the clockwise direction, a
second set arrests forks in the
counterclockwise direction:
Chromosome

31. Replication Termination of the
Bacterial Chromosome
Ter sites are binding sites for the Tus protein
Tus: 35.8 kD
DNA binding at Ter
Monomer
Tus
DNA
Ter
Replication fork
arrested in polar
manner
Tus may inhibit replication fork progression
by directly contacting DnaB helicase,
inhibiting DNA unwinding

32. Summary DNA replication proteins: DNA PolIII DNA PolI DNA Ligase
Primase (DnaG)
Helicase (DnaB)
SSB
Replication termination
Replication fork traps opposite oriC
Ter sites
Tus protein