Lecture 5

Introduction to DNA Replication

Replication of a circular
chromosome
h
DNA polymerase in action

Introduction to DNA Replication

Lecture Outline:

Readings:

1) Overview of DNA
replication
2) DNA replication in
bacteria

Alberts
Alb
t ttextbook,
tb k
Ch 5, pp. 263276 281
276,
281-287
287

Initial Questions
1) Is DNA replication conservative or
semiconservative?
2) What is the direction of DNA replication?
3)) Where does DNA replication
p
start?

Is DNA replication conservative or

semiconservative?
i
i ?
Two options:
1) DNA synthesis is
conservative

2) DNA synthesis is
semiconservative

parental
Red strands? parental

Blue strands?
newly synthesized
newly
synthesized
(See also Alberts Figure 5-5)

Initial Questions
1) Is DNA replication conservative or
semiconservative?
2) What is the direction of DNA replication?
3)) Where does DNA replication
p
start?

What is the direction of DNA replication?

Three possible models:
1)

2))

3)

all three are found in nature

Unidirectional growth of single strands from two starting

virus
linear
virus
points.
Example: linear
3

Unidirectional g
growth of two strands from one starting
gp
point.
some plasmids
plasmids
Example: some
3

Bidirectional growth from one starting point.

3
3
5

Example:
eukaryotes and
5 Eukaryotes
5
bacteria
and bacteria
3

In the preceding slide, the newly

synthesized strand is always made...
A. in the 5 to 3 direction
B. in an anti-parallel
p
orientation to the template
p
strand
C. by sequentially adding nucleotides to the 3OH
C
3 OH
of the growing chain
D Answers A and C are correct
D.
E. Answers A,B and C are correct

Initial Questions
1) Is DNA replication conservative or
semiconservative?
2) What is the direction of DNA replication?
3)) Where does DNA replication
p
start?

Where does DNA replication start?

Two possibilities:

test

1)) Always
y start from the same location on DNA
What are some of the characteristics of the
sequences at replication origins?
Easy to open,
A-T rich, TATA
to open,
A-T box
rich
Easy
byinitiator
and proteins
bind initiator proteins
Recognized
recognized by and bind

2) Random start

How many origins of replication?

1) Single
bacteria
Bacteria

2) Multiple

Alberts, Fig. 5-26

Eukaryotes
Eukaryotes

Alberts, Figure 5-34

10

An experiment to identify origins of

replication
li i
What does ARS
stand for?
Autonomously replicating
sequences (function as
origins of replication)

Alberts, Figure 5-33

11

How does DNA replication proceed in

bacteria?

This
s sty
style
eo
of replication
ep cat o
only applies to:
circular genomes
replication fork

Alberts, Figure 5-26

12

What happens at the DNA replication

forks?

most recentlyy
syntheized DNA
"Nick" needs to be
covalently bonded

Alberts, Figure 5-7

13

Overview of DNA replication

Procedure:

Ingredients for synthesis:

1) Separate DNA
strands
2) Synthesize DNA
3)) Proofread newlyy
synthesized DNA

1) Origin
2)) primers
3) dNTPs
4) ATP
5) DNA polymerase
6) Accessory proteins

14

DNA synthesis
Base pairing:
A-T
C-G

Alb t Fi
Alberts,
Figure 5
5-3
3
15

Steps
p in bacterial DNA replication
p
1)
2)
3)
4)
5)
6)
7)
8)

Origin of replication
Binding of initiator proteins
helicase
Unwinding by helicase
Binding of single-strand binding proteins
primase
RNA primers made by primase
DNA polymerase
Sliding clamp clamp holds polymerase onto DNA
Sliding
DNA ligase ligase
Nick sealing by DNA
16

Initiator proteins for replication in E.coli

1)

binds to origin

2))

helps helicase bind

3)

ATP

primosome

17

Alberts, Figure 5-27

Steps
p in bacterial DNA replication
p
1)
2)
3)
4)
5)
6)
7)
8)

Origin of replication
Binding of initiator proteins
Unwinding by helicase
Binding of single-strand binding proteins
RNA primers made by primase
DNA polymerase
Sliding clamp holds polymerase onto DNA
Nick sealing by DNA ligase
18

Unwinding DNA
1) Two types of
helicases exist
exist. The
predominant one
moves in which
direction?
5' to 3' along the lagging strand template
1) How many subunits
does the helicase
have?
6

19

Alberts, Figure 5-14

Helicase structure

Alberts, Figure 5-15

20

Steps
p in bacterial DNA replication
p
1)
2)
3)
4)
5)
6)
7)
8)

Origin of replication
Binding of initiator proteins
Unwinding by helicase
Binding of single-strand binding proteins
RNA primers made by primase
DNA polymerase
Sliding clamp holds polymerase onto DNA
Nick sealing by DNA ligase
21

Following the action of helicase, single strand

binding
gp
proteins keep
p DNA strands separated
p

1) Separates the
strands by:
binding single- ssDNA
Binding
stranded DNA
2) P
Prevents
t strands
t d
from:
H-bonding with
H-bonding
itself

Alberts, Figure 5-16

22

Structure of SSBs
Single strand binding proteins straighten DNA and
prevent formation of:

hairpins & kinks

23

Alberts, Figure 5-17

Steps
p in bacterial DNA replication
p
1)
2)
3)
4)
5)
6)
7)
8)

Origin of replication
Binding of initiator proteins
Unwinding by helicase
Binding of single-strand binding protein
RNA primers made by primase
DNA polymerase
Sliding clamp holds polymerase onto DNA
Nick sealing by DNA ligase
24

RNA primers made by primase

1) In order to begin, DNA
polymerase
p
y
requires:
q

bound primer

2) What is the purpose

of the primase in
replication?
p

synthesize an RNA
primer

3) P
Primase
i
proceeds
d iin
which direction?

5' to 3'

Alberts, Figure 5-11

25

Steps
p in bacterial DNA replication
p
1)
2)
3)
4)
5)
6)
7)
8)

Origin of replication
Binding of initiator proteins
Unwinding by helicase
Binding of single-strand binding protein
RNA primers made by primase
DNA polymerase
Sliding clamp holds polymerase onto DNA
Nick sealing by DNA ligase
26

DNA polymerase

Alberts, Figure 5-4

27

Steps
p in bacterial DNA replication
p
1)
2)
3)
4)
5)
6)
7)
8)

Origin of replication
Binding of initiator proteins
Unwinding by helicase
Binding of single-strand binding protein
RNA primers made by primase
DNA polymerase
Sliding clamp holds polymerase onto DNA
Nick sealing by DNA ligase
28

Sliding clamp holds polymerase onto

DNA

29

Alberts, Figure 5-18

Steps
p in bacterial DNA replication
p
1)
2)
3)
4)
5)
6)
7)
8)

Origin of replication
Binding of initiator proteins
Unwinding by helicase
Binding of single-strand binding protein
RNA primers made by primase
DNA polymerase
Sliding clamp holds polymerase onto DNA
Nick sealing by DNA ligase
30

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
ith a correctly
tl matched
t h d DNA
sequence

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

Summary of bacterial DNA replication

sliding clamp
DNA polymerase on leading
strand

DNA helicase+
DNA primase= primeosome
SSB
clamp loader
DNA polymerase
on lagging strand

32

An active bacterial DNA replication fork

Alberts, Figure 5-19a

33

The End

34