Download Chapter 16

Chapter 16 – Replication of DNA
We’ve spent the last few chapters taking about a cell
dividing, and saying that first the DNA is copied.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Overview: Life’s Operating Instructions
• In 1953, James Watson and Francis Crick
shook the world
– With an elegant double-helical model for the
structure of deoxyribonucleic acid, or DNA
Figure 16.1
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• DNA, the substance of inheritance
– Is the most celebrated molecule of our time
• Hereditary information
– Is encoded in the chemical language of DNA
and reproduced in all the cells of your body
• It is the DNA program
– That directs the development of many different
types of traits
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Additional Evidence That DNA Is the Genetic Materia
• Prior to the 1950s, it was
already known that DNA
Sugar-phosphate
backbone
5 end
O–
O P
5
CH2
O
CH3
O
H
O
4 H
H
3
O–
– Is a polymer of
nucleotides, each
consisting of three
components:
Nitrogenous
bases
1
H
2
H
O
H
Thymine (T)
O
O
P
O
CH2
O–
H
H
N
O
H
N
H
N
H
H
H
N
N
H
H
Adenine (A)
O
P
H
H
O
1) a nitrogenous base,
N
N
H
O
CH2
O–
H
N
H
O
H
N
H
N
H
H
O
H
2) a sugar
Cytosine (C)
O
O
3) a phosphate group
Figure 16.5
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
P
5
O CH2
4
O–
Phosphate
H
O
H
H
3
OH
2
H
H
Sugar (deoxyribose)
3 end
N
O
1
N
H
N
H
N
N
H
H
Guanine (G)
DNA nucleotide
• Watson and Crick deduced that DNA was a
double helix
– Through observations of the X-ray
crystallographic images of DNA
G
C
A
T
T
A
1 nm
C
G
C
A
T
G
C
T
A
T
A
A
T
T
A
G
A
Figure 16.7a, c
3.4 nm
G
C
0.34 nm
T
(a) Key features of DNA structure
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
(c) Space-filling model
• It was concluded that DNA
– Was composed of two antiparallel sugarphosphate backbones, with the nitrogenous
bases paired in the molecule’s interior
• The nitrogenous bases
– Are paired in specific combinations: adenine
with thymine, and cytosine with guanine
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Small section of DNA
5 end
O
OH
Hydrogen bond
P
–O
3 end
OH
O
O
A
T
O
O
O
CH2
P
–O
O
H2C
O
–O
P
O
O
G
O
C
O
O
CH2
P
O
O–
O
P
H2C
O
O
C
O
G
O
O
O
CH2
P
–O
O–
O
O
O–
O
P
H2C
O
O
A
O
T
O
CH2
OH
3 end
O
O–
P
O
Figure 16.7b
(b) Partial chemical structure
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
O
5 end
H
N
N
N
N
Sugar
O
H
H
CH3
N
N
N
O
Sugar
Thymine (T)
Adenine (A)
H
O
N
N
Sugar
N
H
N
N
N
N
N
Figure 16.8
H
H
Guanine (G)
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
H
O
Sugar
Cytosine (C)
So how does DNA copy itself?
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Concept 16.2: Many proteins work together in
DNA replication and repair
• The relationship between structure and
function
– Is manifest in the double helix
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
The Basic Principle: Base Pairing to a Template Strand
• Since the two strands of DNA are
complementary
– Each strand acts as a template for building a
new strand in replication
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• In DNA replication
– The parent molecule unwinds, and two new
daughter strands are built based on basepairing rules
T
A
T
A
T
A
C
G
C
G
C
T
A
T
A
T
A
A
T
A
T
A
T
G
C
G
C
G
C
G
A
T
A
T
A
T
C
G
C
G
C
G
T
A
T
A
T
A
T
A
T
A
T
C
G
C
G
C
A
G
(a) The parent molecule has two
complementary strands of DNA.
Each base is paired by hydrogen
bonding with its specific partner,
A with T and G with C.
(b) The first step in replication is
separation of the two DNA
strands.
Figure 16.9 a–d
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
(c) Each parental strand now
serves as a template that
determines the order of
nucleotides along a new,
complementary strand.
(d) The nucleotides are connected
to form the sugar-phosphate
backbones of the new strands.
Each “daughter” DNA
molecule consists of one parental
strand and one new strand.
• DNA replication is
semiconservative
– Each of the two
new daughter
molecules will
have one old
strand, derived
from the parent
molecule, and
one newly made
strand
Parent cell
Conservative
model. The two
parental strands
reassociate
after acting as
templates for
new strands,
thus restoring
the parental
double helix.
(a)
(b)
(c)
Figure 16.10 a–c
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Semiconservative
model. The two
strands of the
parental molecule
separate,
and each functions
as a template
for synthesis of
a new, complementary strand.
Dispersive
model. Each
strand of both
daughter molecules contains
a mixture of
old and newly
synthesized
DNA.
First
replication
Second
replication
DNA Replication: A Closer Look
• The copying of DNA
– Is remarkable in its speed and accuracy
• More than a dozen enzymes and other proteins
– Participate in DNA replication
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Getting Started: Origins of Replication
• The replication of a DNA molecule
– Begins at special sites called origins of
replication, where the two strands are
separated
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• A eukaryotic chromosome
– May have hundreds or even thousands of
replication origins
Origin of replication
1 Replication begins at specific sites
where the two parental strands
separate and form replication
bubbles.
Bubble
Parental (template) strand
Daughter (new) strand
0.25 µm
Replication fork
2 The bubbles expand laterally, as
DNA replication proceeds in both
directions.
3 Eventually, the replication
bubbles fuse, and synthesis of
the daughter strands is
complete.
Two daughter DNA molecules
(a) In eukaryotes, DNA replication begins at many sites along the giant
DNA molecule of each chromosome.
Figure 16.12 a, b
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
(b) In this micrograph, three replication
bubbles are visible along the DNA of
a cultured Chinese hamster cell (TEM).
• At the end of each replication bubble is a
replication fork where the new strands of DNA
are elongating
– Replication occurs in both directions from the
origin of replication
– Eventually the replication fork of one bubble
meets one of another bubble
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Elongating a New DNA Strand
• Elongation of new DNA at a replication fork
– Is catalyzed by enzymes called DNA
polymerases, which add nucleotides to the
3 end of a growing strand
New strand
5 end
Sugar
Template strand
3 end
A
Base
Phosphate
T
A
T
C
G
C
G
G
C
G
C
A
T
A
OH
Pyrophosphate 3 end
P
OH
Figure 16.13
3 end
5 end
Nucleoside
triphosphate
C
P
C
2 P
5 end
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
5 end
Remember that DNA polymerase
can ONLY add new nucleotides to
the 3’ end!
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Antiparallel Elongation
• How does the antiparallel structure of the
double helix affect replication?
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• DNA polymerases add nucleotides
– Only to the free 3end of a growing strand
• Along one template strand of DNA, the leading
strand
– DNA polymerase III can synthesize a
complementary strand continuously, moving
toward the replication fork
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• To elongate the other new strand of DNA, the
lagging strand
– DNA polymerase III must work in the direction
away from the replication fork
• The lagging strand
– Is synthesized as a series of segments called
Okazaki fragments, which are then joined
together by DNA ligase
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Synthesis of leading and lagging strands during
DNA replication
1 DNA pol Ill elongates
DNA strands only in the
5
3 direction. 3
5
Parental DNA
5
3
Okazaki
fragments
2
1
3
5
DNA pol III
2 One new strand, the leading strand,
can elongate continuously 5
3
as the replication fork progresses.
3 The other new strand, the
lagging strand must grow in an overall
3
5 direction by addition of short
segments, Okazaki fragments, that grow
5
3 (numbered here in the order
they were made).
Template
strand
3
Leading strand
Lagging strand
2
Template
strand
Figure 16.14
1
DNA ligase
Overall direction of replication
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
4 DNA ligase joins Okazaki
fragments by forming a bond between
their free ends. This results in a
continuous strand.
Priming DNA Synthesis
• DNA polymerases cannot initiate the
synthesis of a polynucleotide
– They can only add nucleotides to the 3 end
• The initial nucleotide strand
– Is an RNA primer (may occasionally be a
primer made of DNA)
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Only one primer is needed for synthesis of the
leading strand
– But for synthesis of the lagging strand, each
Okazaki fragment must be primed separately
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
1
Primase joins RNA nucleotides
into a primer.
3
5
5
3
Template
strand
RNA primer
3
5
3
DNA pol III adds DNA nucleotides to the
primer, forming an Okazaki fragment.
2
5
3
1
After reaching the next
RNA primer (not shown),
DNA pol III falls off.
Okazaki
fragment
3
3
5
1
5
4
After the second fragment is
primed. DNA pol III adds DNA
nucleotides until it reaches the
first primer and falls off. 5
3
5
3
2
5
1
DNA pol 1 replaces the
RNA with DNA, adding to
the 3 end of fragment 2.
5
3
6
5
1
DNA ligase forms a bond
between the newest DNA
and the adjacent DNA of
fragment 1.
5
3
Figure 16.15
3
2
7
The lagging strand
in this region is now
complete.
3
2
1
Overall direction of replication
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
5
Other Proteins That Assist DNA Replication
• Helicase, topoisomerase, single-strand binding
protein
– Are all proteins that assist DNA replication
Helicase – untwist the double strand at the
replication fork and opens up the two strands
for access of base pairs
Topoisomerase – relaxes strand ahead of
replication fork
Single-strand binding protein – keeps strands
from closing back up
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Table 16.1
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• A summary of DNA replication
Overall direction of replication
1 Helicase unwinds the
parental double helix.
2 Molecules of singlestrand binding protein
stabilize the unwound
template strands.
3 The leading strand is
synthesized continuously in the
5 3 direction by DNA pol III.
DNA pol III
Lagging
Leading
strand Origin of replication strand
Lagging
strand
OVERVIEW
Leading
strand
Leading
strand
5
3
Parental DNA
4 Primase begins synthesis
of RNA primer for fifth
Okazaki fragment.
5 DNA pol III is completing synthesis of
the fourth fragment, when it reaches the
RNA primer on the third fragment, it will
dissociate, move to the replication fork,
and add DNA nucleotides to the 3 end
of the fifth fragment primer.
Replication fork
Primase
DNA pol III
Primer
4
DNA ligase
DNA pol I
Lagging
strand
3
2
6 DNA pol I removes the primer from the 5 end
of the second fragment, replacing it with DNA
nucleotides that it adds one by one to the 3 end
of the third fragment. The replacement of the
last RNA nucleotide with DNA leaves the sugarphosphate backbone with a free 3 end.
Figure 16.16
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
1
3
5
7 DNA ligase bonds
the 3 end of the
second fragment to
the 5 end of the first
fragment.
The DNA Replication Machine as a Stationary Complex
• The various proteins that participate in DNA
replication
– Form a single large complex, a DNA replication
“machine”
• The DNA replication machine
– Is probably stationary during the replication
process
– It doesn’t move like a train on a track, rather it
probably reels in the DNA
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Proofreading and Repairing DNA
• DNA polymerases proofread newly made DNA
– Replacing any incorrect nucleotides
DNA polymerases are usually very accurate,
mistake do sometimes occur.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• In mismatch repair of DNA
– Seen if an error arises after proof-reading has
occurred
So,
– Repair enzymes correct errors in base pairing
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• In nucleotide excision repair
– Enzymes cut out and replace damaged
stretches of DNA
1 A thymine dimer
distorts the DNA molecule.
2 A nuclease enzyme cuts
the damaged DNA strand
at two points and the
damaged section is
removed.
Nuclease
DNA
polymerase
3 Repair synthesis by
a DNA polymerase
fills in the missing
nucleotides.
DNA
ligase
Figure 16.17
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
4 DNA ligase seals the
Free end of the new DNA
To the old DNA, making the
strand complete.
Replicating the Ends of DNA Molecules
• The ends of eukaryotic chromosomal DNA
– Get shorter with each round of replication
5
End of parental
DNA strands
Leading strand
Lagging strand
3
Last fragment
Previous fragment
RNA primer
Lagging strand
5
3
Primer removed but
cannot be replaced
with DNA because
no 3 end available
for DNA polymerase
Removal of primers and
replacement with DNA
where a 3 end is available
5
3
Second round
of replication
5
New leading strand 3
New lagging strand 5
3
Further rounds
of replication
Figure 16.18
Shorter and shorter
daughter molecules
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
• Eukaryotic chromosomal DNA molecules
– Have at their ends nucleotide sequences,
called telomeres, that postpone the erosion of
genes near the ends of DNA molecules
Figure 16.19
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
1 µm
• If the chromosomes of germ cells became
shorter in every cell cycle
– Essential genes would eventually be missing
from the gametes they produce
• An enzyme called telomerase
– Catalyzes the lengthening of telomeres in
germ cells
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings