Lecture 10 – DNA Replication

New p
part: Molecular g
genetics
 Hereditary material must fulfill three
requirements
i t
• Replication: It must be able to replicate
precisely, so that copies can be transmitted
from pparents to offspring.
p g
• Information: It must contain information to
guide the development
g p and function of the
organism.
• Mutation: It must be able to change, on rare
occasions, to explain the existence of variant
alleles and the evolution of new forms.
Deoxyribonucleic acid (DNA) fulfills
all three requirements
• R
Replication:
li ti P i semiconservative
Precise i ti
replication of the DNA double helix.
• Information: Encoding of specific
polypeptides by the nucleotide sequence of
DNA.
• Mutation: Rare changes in the nucleotide
sequence
q of DNA that change
g either the
polypeptide it encodes or the time and place
that the encoded protein is produced.
 DNA is a polymer: a molecule
composed of repeating subunits. The
subunits of DNA are nucleotides
Each nucleotide has three parts

Nitrogenous
Phosphate
base

Deoxyribose sugar
 DNA is made of four different nucleotides
Two have pyrimidine bases Two have purine bases

Thymine (T) Adenine (A)

Cytosine ( C) Guanine (G)

 Nucleotides link together to form a chain
The chain consists of
5’ a sugar-phosphate
backbone with
nitrogenous bases
sticking out the side.

The chain
Th h i is
i di
directional:
ti l it
runs from a phosphate
group at the 5’ end to a
hydroxyl group at the 3’ end.
3’
Each DNA molecule consists of two strands
tthat
at are
a e co
complementary
p e e ta y a
and
daantiparallel
t pa a e

5’ 3’

3’ 5’
The DNA strands are held together by hydrogen bonds
that link base pairs in opposite strands.

Hydrogen bonds
(d h d lilines)) lilink
(dashed k
negatively charged
molecules ((O,, N))
in one base to
positively charged
molecules (H) in a
base on the
opposite strand.
Hydrogen bonds join bases in complementary pairs
Each pair consists of one purine and one pyrimidine
A thymine in one strand is always bound
to an adenine in the other strand.

A cytosine in one strand is always bound to

a guanine in the other strand.
 1) When the base composition of all the DNA
from the bacterium Myxobacterium tuberculosis
was determined, 18% of the bases were found to
be adenine
adenine. Based on this information
information, what
percentage of the bases are cytosine?

95%

A. 18%
A
B. 32%
C. 36%
D 64%
D.
1% 3% 1% 1%
E. 72% A. B. C. D. E.
Rules of DNA base composition
• The total amount of purines in a DNA
y equal
double helix must exactly q the total
amount of pyrimidines.
• The total amount of adenine (A) must
equal the total amount of thymine (T).
• The total amount of cytosine (C) must
equal the total amount of guanine (G).
 The double-stranded DNA molecule is in the
form of a double helix
Major groove:
thi is
this i the
th site
it
of most
interactions
Each turn of between DNA
the helix is 3.4 and proteins
nanometers
Minor groove
long and
contains 10
base pairs
 DNA is very
y stable
• Very strong covalent bonds connect the
nucleotides
l tid alongl each
h strand.
t d
• Large numbers of hydrogen bonds join
th ttwo strands.
the t d
• The base pairs are hydrophobic, and
th f
therefore poorly
l soluble
l bl iin water.
t Th The
double helix structure keeps them away
from the surrounding water of the cell
interior. This makes the helix more
stable.
The mechanism of DNA replication
 The mechanism of DNA replication is
semiconservative: each strand of the double
helix serves as a template to make a new strand
Identical daughter
double helices

Parent double helix

In E. coli, replication starts at a unique
origin of replication called oriC
oriC E. coli has a
E
single circular
chromosome

The double
Th d bl liline
represents the two
complementary
strands of DNA
Viewed in detail, oriC consists of five identical 13-
base ppair regions
g called DnaA boxes
AT-rich DNA DnaA boxes
DnaA p proteins bind
to the boxes and
nearby DNA,
causing the AT
AT-rich
rich
region to unwind.

Helicase enzymes
then bind to the
open replication
bubble and
proceed to unwind
the DNA at each
end.
Whyy is the origin
g of replication
p AT-rich?
Adenine and thymine Cytosine and guanine
are joined by only two are joined by three
hydrogen bonds. hydrogen bonds.

Therefore, AT-rich regions are bound less strongly than

CG-rich regions. This makes them easier to unwind.
 Once the replication bubble is opened,
p
replication p
proceeds in two directions
Origin of
replication

Two replication forks travel in opposite directions

from the origin. New DNA is synthesized at each fork.
2)) One strand of a DNA double helix is 5’-
TCGGTCA-3’. What is the sequence of
the complementary strand?

95%

A. 5
5’-AGCCAGT-3’
AGCCAGT 3
B. 3’-TGACCGA-5’
C 5’-ACTGGCT-3’
C. 5’ ACTGGCT 3’
D. 3’-AGCCAGT-5’
1% 3% 1%
E. 3’-TCGGTCA-5’ 0%

A. B. C. D. E.
At each replication fork, the
DNA is unwound. Each
strand serves as a template
to make a complementary
new strand
DNA polymerase III adds nucleotides
to the 3’
3 end of a growing DNA strand
DNA DNA
primer strand primer strand
 DNA polymerase III has two
key limitations
• It can only add nucleotides to the 3’
end d off a growing
i chain.
h i
• It can onlyy extend a chain that
already exists. It cannot start one
from scratch
scratch.
At each replication fork, there are two growing DNA
strands: the leading
g strand and the lagging
gg g strand

DNA polymerase can only

add nucleotides to the 3’
3 end.
end
 Overview of DNA replication

The lagging strand must be synthesized

discontinuously: it is made in small pieces
that are then joined together.

The leading
Th l di strand
t d iis made
d bby continuously
ti l
extending its 3’ end.
 Overview of DNA replication

The first step is synthesis of a short RNA

primer by the enzyme primase.
primase

This is necessary, because DNA polymerase can only

extend a chain, not start one from scratch.
 Overview of DNA replication

Next, DNA polymerase III extends the primer, 5’ to

3’, using the original DNA strand as a template
3 template.

This creates a short (1000-2000 base pair) DNA chain

called an Okazaki fragment.
 Overview of DNA replication

Next, DNA polymerase I removes the

adjacent RNA primer
primer.
At the same time, it extends the Okazaki
fragment to fill in the gap
gap.
 Overview of DNA replication

Finally,
y, Ligase
g connects the Okazaki
fragment to the adjacent fragment.
http://bcs.whfreeman.com/thelifewire/content/chp11/1102003.html
 3) This drawing shows a replication bubble
in E. coli. What does the arrow point to?

A. The 5’ end of an Okazaki fragment

B. The 3’ end of an Okazaki fragment
C. The 5’ end of the leading strand
D. The 3’end of the leading strand
E
E. Th 5’ end
The d off a ttemplate
l t strand
t d

74%

24%

1% 1% 1%

A. B. C. D. E.
 4) This drawing shows a replication bubble
in E. coli. What does the arrow point to?

A. The 5’ end of an Okazaki fragment

B. The 3’ end of an Okazaki fragment
C. The 5’ end of the leading strand
D. The 3’end of the leading strand
E
E. Th 5’ end
The d off a ttemplate
l t strand
t d

82%
8 %

14%

2% 3%
0%

A. B. C. D. E.
Replication is carried out by a complex
molecular machine called the replisome
• Major components of the replisome
– Helicase
– Gyrase
G
– Primase
– DNA polymerase
l III h
holoenzyme
l
– DNA polymerase I
– Ligase
– Single-strand binding proteins
Helicase catalyzes the unwinding
of the DNA double helix
 As DNA helicase unwinds the double helix, it
introduces supercoils elsewhere in the DNA
molecule

Supercoils are loops in

the DNA introduced when
it is unwound or
overwound.
Gyrase (a kind of topoisomerase) relaxes these
supercoiled
p regions
g by
y cutting
g the DNA ((allowing
g
it to rotate) and then rejoining the strands
 Primase makes the RNA primer necessary to start
each Okazaki fragment

• A primer is about
10 bases long
long.
• Each primer is
complementary to
the DNA template
strand.
 The DNA polymerase III holoenzyme
includes two catalytic cores,
cores one for the
leading strand and one for the lagging strand
The holoenzyme also
contains many
accessory proteins,
t i
including
•A donut-shaped
p beta
clamp that fastens the
catalytic core to the
DNA
•A bridge that ties the
two cores together.
 Primase and DNA polymerase III start
and extend each Okazaki fragment
Previous Okazaki
fragment
g

Template
p strand
Primase builds a new RNA primer

DNA polymerase
l III extends
t d a new
Okazaki fragment from the primer
DNA polymerase I removes the RNA
primer and fills in the g
p gap
p with DNA
Ligase joins the Okazaki fragments with
covalent bonds
Single-strand binding (SSB) proteins stabilize
p
the exposed single-stranded
g DNA
Single stranded
DNA can
hydrogen bond
to itself and
form hairpin
structures

SSB proteins
stabilize the
exposed bases.

DNA pol III removes the SSB proteins as it builds the DNA strand.
Overview of the
(Gyrase)
E coli replisome
E.