It talks about how a DNA can be replicated

1. DNA replication
Learning objectives
Understand the basic rules governing
DNA replication
Introduce proteins that are typically
involved in generalised replication
Reference: Any of the recommended texts
Optional
Nature (2003) vol 421,pp431-435
http://www.bath.ac.uk/bio-sci/cbt/
http://www.dnai.org/lesson/go/2166/1973

2. `It has not escaped our notice that the
specific pairing we have postulated
immediately suggests a possible
copying mechanism for the genetic
material’
Watson & Crick
Nature (1953)
Original drawing by Francis Crick

3. Four requirements for DNA to
be genetic material
Must carry information
 Cracking the genetic code
Must replicate
 DNA replication
Must allow for information to change
 Mutation
Must govern the expression of the phenotype
 Gene function

4. Much of DNA’s sequence-specific information is
accessible only when the double helix is unwound
Proteins read the DNA sequence of nucleotides as
the DNA helix unwinds. Proteins can either bind to a
DNA sequence, or initiate the copying of it.
Some genetic information is accessible even in
intact, double-stranded DNA molecules
Some proteins recognize the base sequence of DNA
without unwinding it.
One example is a restriction enzyme.
DNA stores information in the sequence of
its bases

5. DNA Replication
Process of duplication of the entire genome prior to cell
division
Biological significance
 extreme accuracy of DNA replication is necessary in
order to preserve the integrity of the genome in
successive generations
 In eukaryotes , replication only occurs during the S
phase of the cell cycle. The slower replication rate in
eukaryotes results in a higher fidelity or accuracy of
replication in eukaryotes

6. Basic rules of replication
A. Semi-conservative
B. Starts at the ‘origin’
C. Can be uni or bidirectional
D. Semi-discontinuous
E. Involves DNA templating
F. RNA primers required

7. A) DNA replication – three possible models
 Semiconservative replication – Watson
and Crick model
 Conservative replication: The parental
double helix remains intact; both strands
of the daughter double helix are newly
synthesized
 Dispersive replication: At completion,
both strands of both double helices
contain both original and newly
synthesized material.

8. Meselson-Stahl experiments confirm
semiconservative replication
Semi-conservative replication An
ingenious experiment by Meselson and
Stahl showed that one strand of each
DNA strand is passed on unchanged to
each of the daughter cells. This
'conserved' strand acts as a template for
the synthesis of a new, complementary
strand by the enzyme DNA polymerase

10. Meselson-Stahl experiments confirm
semiconservative replication

11. Double helix
unwinds
Each strand acts
as template
Complementary
base pairing
Two daughter
helices produced
after replication

12. Starts at origin
Initiator proteins identify specific base sequences on
DNA
Prokaryotes – single ori site E.g E.coli - oriC
Eukaryotes – multiple sites of origin (replicator)
E.g. yeast - ARS (autonomously replicating sequences)
Prokaryotes
Eukaryotes

13. Uni or bidirectional

14. Bidirectional replication
 Replication forks move in opposite directions
 In linear chromosomes, telomeres ensure the
maintenance and accurate replication of
chromosome ends
 In circular chromosomes, such as E. coli, there
is only one origin of replication.
 In circular chromosomes, unwinding and
replication causes supercoiling, which may
impede replication
 Topoisomerase – enzyme that relaxes
supercoils by nicking strands

15. The bidirectional replication of a circular
chromosome

17. Semi-discontinuous replication
Anti parallel strands replicated simultaneously
 Leading strand synthesis continuously in 5’– 3’
 Lagging strand synthesis in fragments in 5’-3’

18. Semi-discontinuous replication
New strand synthesis always in the 5’-3’ direction

19. DNA templating
Nucleotide recognition
Enzyme catalysed
polymerisation
Complementary base pair
copied
Substrate used is dNTP

20. RNA primers required

21. Topoisomerases
Helicases
Single strand
binding proteins
Primase
DNA polymerase
Tethering protein
DNA ligase
- Prevents torsion by DNA breaks
- separates 2 strands
- prevent reannealing
of single strands
- RNA primer synthesis
- synthesis of new strand
- stabilises polymerase
- seals nick via phosphodiester
linkage
Core proteins at the replication fork

22. The mechanism of DNA replication
Arthur Kornberg, a Nobel prize winner and
other biochemists deduced steps of
replication
 Initiation
 Proteins bind to DNA and open up double helix
 Prepare DNA for complementary base pairing
 Elongation
 Proteins connect the correct sequences of
nucleotides into a continuous new strand of DNA
 Termination
 Proteins release the replication complex

25. Core proteins at the replication fork
Nature (2003) vol 421,pp431-435 Figure in ‘Big’ Alberts too

26. Topoisomerase I
Cause temporary
single strand breaks in
DNA
Allows free rotation of
helix
Monomeric enzymes
(~100kD)
Reversible catenation
(knotting)
Acts by breaking
phosphodiester
linkage via tyrosine
active site

27. Topoisomerase II (DNA gyrase)
Cause temporary double strand breaks in DNA

28. Topoisomerase II (DNA gyrase)
~ 375 kD
2 pairs of subunits: A
& B
ATP hydrolysis
mediated double
strand break
Catenates double
stranded circles
Inhibited by antibiotics