Lecture 5

Introduction to DNA Replication

Replication of a circular
chromosome
DNA polymerase in action

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Introduction to DNA Replication

Lecture Outline: Readings:

1) Overview of DNA Alberts textbook,
replication
li i Ch 55, pp
pp. 263
263-
2) DNA replication in 276, 281-287
bacteria

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 Initial Questions

1) Is DNA replication conservative or

semiconservative?
SEMICONSERVATIVE

2) What
Wh t is
i th
the di
direction
ti off DNA replication?
li ti ?

3) Where does DNA replication start?

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 Is DNA replication conservative or
semiconservative?
Two options:
1) DNA synthesis is 2) DNA synthesis is
conservative
ti semiconservative
i ti ☺
:)

Red strands? parental

parental

mother cell

daughter cells

Blue strands?
 newly synthesized
newly synthesised DNA
(See also Alberts Figure 5-5) 4
 Initial Questions

1) Is DNA replication conservative or

semiconservative?

2) What
Wh t is
i th
the di
direction
ti off DNA replication?
li ti ?

3) Where does DNA replication start?

5
 What is the direction of DNA replication?
Three possible models: All 3 are found in nature

1) Unidirectional growth of single strands from two starting

points. linear
Example: linear virus virus

*keep track of polarity 3’

black, new 3'
5’
of DNA
5'
3' 5'
5’ 3’

2) Unidirectional growth of two strands from one starting point.

p
Example: some plasmids
p
some plasmids (circular)
3’ 5’
5' 3'

3' 5'
5’ 3’
3) Bidirectional growth from one starting point. Example:
3’ 5’ Eukaryotes
Eukaryotes and
5' 3' 3' bacteria
5'
3' 5'
3'
5' and bacteria
5’ 3’ 6
 Initial Questions

1) Is DNA replication conservative or

semiconservative?

2) What
Wh t is
i th
the di
direction
ti off DNA replication?
li ti ?

3) Where does DNA replication start?

7
Where does DNA replication start?
Two possibilities:

1) Always start from the same location on DNA

What are some of the characteristics of the
sequences at replication origins?
 Easy
Easy y to open,
to open; p rich,(only
A-T A-T 2 Hrich
bonds)

 Recognized
Recognised by and by and
bound by binding initiator proteins
initiator proteins

2) Random start

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 How many
y origins
g of replication?
p

1) Single A-T rich

 Bacteria
Bacteria

bacterial chromosome

Alb t Fig.
Alberts, Fi 5-26
5 26
2)) Multiple
 Eukaryotes
Eukaryotes y

yeast red lines are infrequent OoRs

green are frequently used for replication

Alberts, Figure 5-34 9

 An experiment to identify origins of
replication in yeast
small double stranded DNA
budding yeast What does ARS
stand for?
the segment doesn't have an
ARS; will not have the histidine;  Autonomously
Autonomously
replicating
entire plasmid is gone after rep. replicating
sequences (that
sequences
function as origins of
replication)

can survive without histidine - produces its own

Alberts, Figure 5-33

10
 How does DNA replication proceed in
bacteria?
proteins stick here and pull the
strands apart

This style of replication parental strands pulled apart and

nucleotides bond
y applies
only pp to:
 circular
circular genomes
genomes

replication forks:
where two paren-
This is "Bidirectional growth tal strands are
from one starting point" pulled apart and
DNA rep. takes place

semi-conservative; uses a
parental strand in each
new daughter cell
Alberts, Figure 5-26
11
 What happens at the DNA replication
forks?
DNA is anti-parallel
Newly synthesised strands are made by adding to the 3' end
.: The new strand will be the 5' - 3' strand

leading strand template

-----> separation direction

most recently
lagging strand template y
syntheized DNA
On the lagging strand, the replication goes towards 5'
The little gaps are called "Nicks"
which need to be covalently bonded

Alberts, Figure 5-7 12

 Overview of DNA replication
p
Procedure: Ingredients for synthesis:
1) Separate DNA 1) Origin
Origin
strands 2) Primers
ATP
2) Synthesize DNA 3)) dNTPs
d
Primerss
3) Proofread newly 4) ATP
DNAases
as an energy source
synthesized
sy t es ed DNA 5) DNA polymerase
6) Accessory proteins

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 DNA synthesis
y

Base pairing:
A-T
C-G

This reaction is catalysed by

DNA polymerase

Alberts, Figure 5-3

53

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 Steps in bacterial DNA replication

1) Origin of replication
2)) Binding g of initiator p proteins
3) Unwinding by helicaseHELICASE

4) Binding of single-strand binding proteins

5) RNA primers made by primase PRIMASE

6) DNA polymerase
7) Sliding clamp holds polymerase onto DNA
SLIDING CLAMP

8) Nick sealing by DNA ligase

DNA LIGASE

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 Initiator proteins for replication in E.coli

1) Binds to origin
BINDS TO THE ORIGIN

2) Helps helicase
HELPS HELICASE BIND bind

3) Requires
REQUIRES ATP ATP

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Alberts, Figure 5-27
 Steps in bacterial DNA replication

1) Origin of replication
2)) Bindingg of initiator p
proteins
3) Unwinding by helicase
4) Binding of single-strand binding proteins
5) RNA primers made by primase
6) DNA polymerase
7) Sliding clamp holds polymerase onto DNA
8) Nick sealing by DNA ligase

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Unwinding
g DNA
Helicases unwind and separate the two strands; "unzipping"
-> HELICASE REQUIRES ATP

1) Two types of
helicases exist. The
predominant one
p
moves in which
direction?
5’5' to-3'3’ along the lagging
along lagging strand template

strand template
1) How many subunits
does the helicase
have?
 66
18
Alberts, Figure 5-14
Helicase structure

Alberts, Figure 5-15 19

 Steps in bacterial DNA replication

1) Origin of replication
2)) Bindingg of initiator p
proteins
3) Unwinding by helicase
4) Binding of single-strand binding proteins
5) RNA primers made by primase
6) DNA polymerase
7) Sliding clamp holds polymerase onto DNA
8) Nick sealing by DNA ligase

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Following the action of helicase, single strand
binding proteins keep DNA strands separated

1) Separates the
strands d b by:
 Binding
Binding ssDNA ssDNA

2) Prevents strands
from:
 H-bonding
H-bonding

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Alberts, Figure 5-16
 Structure of SSB’s
Single strand binding proteins straighten DNA and
prevent formation of:
 Hairpins p kinks & kinks
Hairpins and

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Alberts, Figure 5-17
 Steps in bacterial DNA replication

1) Origin of replication
2)) Bindingg of initiator p
proteins
3) Unwinding by helicase
4) Binding of single-strand binding protein
5) RNA primers made by primase
6) DNA polymerase
7) Sliding clamp holds polymerase onto DNA
8) Nick sealing by DNA ligase

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 RNA primers made by primase
1) In order to begin, DNA
polymerase
l requires:
i
 Bound primer
Bound primase

2) What is the purpose

of the primase in
replication?
 Synthesize an RNA
Synthesise an RNA primer

primer
makes an RNA
3) Primase proceeds in primer!

which direction?
 5'5’- 3' 3’

Alberts, Figure 5-11 24

 Steps in bacterial DNA replication

1) Origin of replication
2)) Bindingg of initiator p
proteins
3) Unwinding by helicase
4) Binding of single-strand binding protein
5) RNA primers made by primase
6) DNA polymerase
7) Sliding clamp holds polymerase onto DNA
8) Nick sealing by DNA ligase

25
DNA polymerase
p y

Alberts, Figure 5-4 26

 Steps in bacterial DNA replication

1) Origin of replication
2)) Bindingg of initiator p
proteins
3) Unwinding by helicase
4) Binding of single-strand binding protein
5) RNA primers made by primase
6) DNA polymerase
7) Sliding clamp holds polymerase onto DNA
8) Nick sealing by DNA ligase

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Sliding clamp holds polymerase onto
DNA

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Alberts, Figure 5-18
 Steps in bacterial DNA replication

1) Origin of replication
2)) Bindingg of initiator p
proteins
3) Unwinding by helicase
4) Binding of single-strand binding protein
5) RNA primers made by primase
6) DNA polymerase
7) Sliding clamp holds polymerase onto DNA
8) Nick sealing by DNA ligase

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 How are the Okazaki
fragments on the lagging
strand linked together?

A special DNA repair system is

responsible for removal of the
RNA primer and replacing it
with a correctly matched DNA
sequence

Figure 5-12 Molecular Biology of the Cell (© Garland Science 2008)

Summary of bacterial DNA replication

slinding clamp
DNA polymerase on
leading strand

DNA primase } both called

Helicase primosome

single-stranded DNA binding

proteins (not on lead)

clamp loader

DNA ploymerase on lagging strand (just finishing

an Okazaki fragment)

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An active bacterial DNA replication fork

Alberts, Figure 5-19a 32

 Two useful animations

http://www.youtube.com/watch?v=4jtmOZaIvS0&NR=1&feature=endscreen
p y j

http://www.youtube.com/watch?v=teV62zrm2P0
p y
The End

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