Download DNA Replication

DNA:
The Carrier of Genetic
Information
Chapter 12
Learning Objective 1
•
What evidence was accumulated during
the 1940s and early 1950s demonstrating
that DNA is the genetic material?
The Mystery of Genes
•
Many early geneticists thought genes were
proteins
•
•
Proteins are complex and variable
Nucleic acids are simple molecules
Evidence for DNA
•
•
DNA (deoxyribonucleic acid)
Transformation experiments
•
DNA of one strain of bacteria can transfer
genetic characteristics to related bacteria
Bacteriophage Experiments
•
Bacteriophage (virus) infects bacterium
•
•
only DNA from virus enters the cell
virus reproduces and forms new viral particles
from DNA alone
KEY CONCEPTS
•
Beginning in the 1920s, evidence began to
accumulate that DNA is the hereditary
material
Learning Objective 2
•
What questions did these classic
experiments address?
•
•
•
Griffith’s transformation experiment
Avery’s contribution to Griffith’s work
Hershey–Chase experiments
Griffith’s Transformation Experiment
•
•
Can a genetic trait be transmitted from one
bacterial strain to another?
Answer: Yes
Griffith’s Transformation Experiment
Experiment 1
Experiment 2
Experiment 3
Experiment 4
R cells
injected
S cells
injected
Heat-killed
S cells injected
R cells and heatkilled S cells injected
Mouse lives
Mouse dies
Mouse lives
Mouse dies
Fig. 12-1, p. 261
Animation: Griffith’s Experiment
CLICK
TO PLAY
Avery’s Experiments
•
•
What molecule is responsible for bacterial
transformation?
Answer: DNA
Hershey–Chase Experiments
•
•
Is DNA or protein the genetic material in
bacterial viruses (phages)?
Answer: DNA
Hershey–Chase
Experiments
35S
1
32 P
Bacterial viruses
grown in 35S to
label protein coat
or 32P to label DNA
2
Viruses infect bacteria
Fig. 12-2, p. 262
3
Agitate cells
in blender
Agitate cells
in blender
4
Separate by
centrifugation
Separate by
centrifugation
32 P
35S
5
Bacteria in pellet
contain 32Plabeled DNA
35S-labeled
protein in
supernatant
Fig. 12-2, p. 262
6
Viral reproduction
inside bacterial cells
from pellet
7
Cell lysis
32P
5
6
7
Fig. 12-2, p. 262
Learning Objective 3
•
How do nucleotide subunits link to form a
single DNA strand?
Watson and Crick
•
•
DNA Model
Demonstrated
•
•
how information is stored in molecule’s
structure
how DNA molecules are templates for their
own replication
Nucleotides
•
•
DNA is a polymer of nucleotides
Each nucleotide subunit contains
•
a nitrogenous base
• purines (adenine or guanine)
• pyrimidines (thymine or cytosine)
•
•
a pentose sugar (deoxyribose)
a phosphate group
Forming DNA Chains
•
Backbone
•
•
•
alternating sugar and phosphate groups
joined by covalent phosphodiester linkages
Phosphate group attaches to
•
•
5′ carbon of one deoxyribose
3′ carbon of the next deoxyribose
DNA
Nucleotides
Thymine
Adenine
Phosphate
group
Nucleotide
Cytosine
Guanine
Phosphodiester
linkage
Deoxyribose
(sugar)
Fig. 12-3, p. 264
Animation: Subunits of DNA
CLICK
TO PLAY
KEY CONCEPTS
•
The DNA building blocks consist of four
nucleotide subunits: T, C, A, and G
Learning Objective 4
•
How are the two strands of DNA oriented
with respect to each other?
DNA Molecule
•
2 polynucleotide chains
•
associated as double helix
DNA Molecule
Sugar–phosphate backbone
Minor groove
Major groove
3.4 nm
0.34 nm
2.0 nm
= hydrogen
= atoms in base pairs
= carbon
= oxygen
= phosphorus
Fig. 12-5, p. 266
Double Helix
•
Antiparallel
•
•
5′ end
•
•
chains run in opposite directions
phosphate attached to 5′ deoxyribose carbon
3′ end
•
hydroxyl attached to 3′ deoxyribose carbon
KEY CONCEPTS
•
•
The DNA molecule consists of two strands
that wrap around each other to form a
double helix
The order of its building blocks stores
genetic information
Animation: DNA Close Up
CLICK
TO PLAY
Learning Objective 5
•
•
What are the base-pairing rules for DNA?
How do complementary bases bind to
each other?
Base Pairs
•
Hydrogen bonding
•
•
•
Adenine (A) with thymine (T)
•
•
between specific base pairs
binds two chains of helix
forms two hydrogen bonds
Guanine (G) with cytosine (C)
•
forms three hydrogen bonds
Base Pairs and Hydrogen Bonds
Fig. 12-6a, p. 267
Adenine
Deoxyribose
Guanine
Deoxyribose
Thymine
Deoxyribose
Cytosine
Deoxyribose
Fig. 12-6b, p. 267
Chargaff’s Rules
•
Complementary base pairing
•
•
•
between A and T; G and C
therefore A = T; G = C
If base sequence of 1 strand is known
•
base sequence of other strand can be
predicted
KEY CONCEPTS
•
•
Nucleotide subunits pair, based on precise
pairing rules: T pairs with A, and C pairs
with G
Hydrogen bonding between base pairs
holds two strands of DNA together
Learning Objective 6
•
What evidence from Meselson and Stahl’s
experiment enabled scientists to
differentiate between semiconservative
replication of DNA and alternative models?
Models of DNA
Replication
(a) Hypothesis 1: Semiconservative replication
Parental DNA
First generation
Second generation
Fig. 12-7a, p. 268
(b) Hypothesis 2: Conservative replication
Parental DNA
First generation
Second generation
Fig. 12-7b, p. 268
(c) Hypothesis 3: Dispersive replication
Parental DNA
First generation
Second generation
Fig. 12-7c, p. 268
Meselson-Stahl Experiment
•
E. coli
•
•
•
grown in medium containing heavy nitrogen
(15N)
incorporated 15N into DNA
Transferred from 15N to 14N medium
•
after one or two generations, DNA density
supported semiconservative replication
Meselson-Stahl
Experiment
Bacteria are grown in
(heavy) medium. All
DNA is heavy.
15N
Some cells are
transferred to
14N (light)
medium.
Some cells
continue to
grow in 14N
medium.
First generation Second generation
Cesium
chloride
(CsCl)
High
Low
density density
DNA
DNA is mixed with CsCl
solution, placed in an
ultracentrifuge, and
centrifuged at very high
speed for about 48 hours.
The greater concentration
of CsCl at the bottom of
the tube is due to
sedimentation under
centrifigal force.
15N (heavy)
14N (light)
14N – 15N
DNA
DNA
hybrid DNA
DNA molecules move to positions
where their density equals that of
the CsCl solution.
Fig. 12-8a, p. 269
14N
(light)
DNA
– 15N
hybrid DNA
14N
– 15N
hybrid DNA
14N
15N
(heavy)
DNA
Before transfer
to 14N
One cell generation
after transfer to 14N
Two cell generations
after transfer to 14N
The location of DNA molecules within the centrifuge tube can be
determined by UV optics. DNA solutions absorb strongly at 260 nm.
Fig. 12-8b, p. 269
Semiconservative Replication
•
Each daughter double helix consists of
•
•
1 original strand from parent molecule
1 new complementary strand
Learning Objective 7
•
•
How does DNA replicate?
What are some unique features of the
process?
DNA Replication
•
2 strands of double helix unwind
•
•
Replication is initiated
•
•
each is template for complementary strand
DNA primase synthesizes RNA primer
DNA strand grows
•
DNA polymerase adds nucleotide subunits
DNA Replication
Base
Nucleotide joined to
growing chain by
DNA polymerase
Phosphates
released
Fig. 12-10, p. 271
Other Enzymes
•
DNA helicases
•
•
open the double helix
Topoisomerases
•
prevent tangling and knotting
KEY CONCEPTS
•
DNA replication results in two identical
double-stranded DNA molecules
•
molecular mechanism passes genetic
information from one generation to the next
Learning Objective 8
•
What makes DNA replication (a)
bidirectional and (b) continuous in one
strand and discontinuous in the other?
Bidirectional Replication
•
Starting at origin of replication
•
•
proceeding in both directions
Eukaryotic chromosome
•
•
may have multiple origins of replication
may replicate at many points at same time
Bidirectional Replication
DNA polymerase
3’
5’
Origin of replication
on DNA molecule
3’
5’
Fig. 12-11a, p. 272
Twist introduced into the
helix by unwinding
RNA primer
Single-strand
binding proteins
DNA polymerase
3’
5’
3’
DNA
helicase
3’
5’
3’
RNA primer
Direction of
replication
Fig. 12-11b, p. 272
3’
3’
5’
5’
3’
3’
5’
5’
Fig. 12-11c, p. 272
DNA Synthesis
•
•
Always proceeds in 5′ → 3′ direction
Leading strand
•
•
synthesized continuously
Lagging strand
•
•
•
•
synthesized discontinuously
forms short Okazaki fragments
DNA primase synthesizes RNA primers
DNA ligase links Okazaki fragments
DNA Synthesis
3’
5’
Leading strand
DNA helix
RNA primer
DNA polymerase
3’
5’
3’
5’
3’
5’
Replication fork
Lagging strand
(first Okazaki fragment)
Direction of
replication
Fig. 12-12a, p. 273
3’
5’
Leading strand
3’
RNA
primers
3’
5’
5’
5’3’
3’
5’
Two Okazaki
fragments
Fig. 12-12b, p. 273
3’
5’
Leading strand
3’
3’
DNA ligase
5’ 3’
5’
5’
Third Okazaki
fragment
Lagging strand
3’
5’
Fig. 12-12c, p. 273
Replication in Bacteria and
Eukaryotes
Template DNA
(light blue)
New DNA (dark blue)
3’
5’
5’
3’
Fig. 12-13a, p. 274
340 nm
Fig. 12-13b, p. 274
5’
Replication
“bubbles”
3’
Single replication
bubble formed
from two merged
bubbles
3’
5’
Replication fork
Fig. 12-13c, p. 274
Animation: Overview of DNA
replication and base pairing
CLICK
TO PLAY
Learning Objective 9
•
How do enzymes proofread and repair
errors in DNA?
DNA Polymerases
•
Proofread each new nucleotide
•
•
against template nucleotide
Find errors in base pairing
•
•
remove incorrect nucleotide
insert correct one
DNA Mutation
Mutation
Fig. 12-9, p. 270
Mutation
Stepped Art
Fig. 12-9, p. 270
Mismatch Repair
•
•
Enzymes recognize incorrectly paired
nucleotides and remove them
DNA polymerases fill in missing
nucleotides
Nucleotide Excision Repair
•
Repairs DNA lesions
•
•
caused by sun or harmful chemicals
3 enzymes
•
•
•
nuclease cuts out damaged DNA
DNA polymerase adds correct nucleotides
DNA ligase closes breaks in sugar–phosphate
backbone
Nucleotide Excision Repair
Nuclease enzyme
bound to DNA
DNA lesion
5’
3’
3’
5’
5’
3’
3’
5’
DNA polymerase
DNA ligase
5’
3’
New DNA
3’
5’
Fig. 12-14, p. 275
Learning Objective 10
•
•
What is a telomere?
What are the possible connections
between telomerase and cell aging, and
between telomerase and cancer?
Telomeres
•
Eukaryotic chromosome ends
•
•
•
noncoding, repetitive DNA sequences
Shorten slightly with each cell cycle
Can be extended by telomerase
Replication at Telomeres
5’
3’
3’
5’
DNA replication
5’
3’
3’
5’
+
RNA primer
RNA primer
5’
3’
3’
5’
Removal of primer
3’
5’
3’
5’
+
5’
3’
3’
5’
Fig. 12-15a, p. 276
3’
5’
Fig. 12-15b, p. 276
Cell Aging
•
•
May be caused by absence of telomerase
activity
Cells lose ability to divide
•
after a limited number of cell divisions
Cancer Cells
•
Have telomerase
•
•
to maintain telomere length and possibly
resist apoptosis
Including human cancers
•
breast, lung, colon, prostate gland, pancreas