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Transcript
DNA and
Replication
1
History
of DNA
2
History of DNA
• Early scientists thought
protein was the cell’s
hereditary material because
it was more complex than
DNA
• Proteins were composed of
20 different amino acids in
long polypeptide chains
3
Genetic facts in 1900:
• Both female and male organisms
have identical chromosomes
except for one pair.
• Genes are located on
chromosomes
• All organisms have two types of
chromosomes:
– Sex chromosomes
– Autosomes
4
Male vs Female
• MALE
• Usually the Y
chromosome.
• Y is usually
smaller
• Male genotype
= XY
• FEMALE
• Usually the X
chromosome.
• Larger than the
Y
• Female
genotype
XX
Except Birds
Male = XX
Female = XY
5
Frederick Griffith
• British bacteriologist
• 1928 = designed and performed
experiment on rats and bacteria
that causes pneumonia.
• 2 strains of the bacteria
• Type S = causes severe
pneumonia
• Type R = relatively harmless
6
Griffith’s Rats
1. First he injected living Type S
bacteria into rats:
7
• Second he injected dead Type S
into the rats.
8
• Next he injected living type R
bacteria
9
• Finally he injected a mixture of
living Type R and dead Type S :
10
Results of experiments:
• Because the dead rat tissue
showed living Type S bacteria,
something “brought the Type S
back to life”
• Actually one bacterial type
incorporated the DNA, or
instructions, from the dead
bacteria into its own DNA
• Known as transformation.
Confirmed by Avery, MacLeod,
and McCarty in 1944
11
Oswald Avery
• Canadian
biologist
(18771955)
• Discovered
DNA in
1944 with a
team of
scientists.
12
Hershey and Chase
• 1952
• Attempted to
solve the
debate on
whether DNA
or proteins are
responsible for
providing the
genetic
13
• They used a
bacteriophage
(a virus which
attacks
bacteria) to
prove that
DNA was
definitely the
genetic
material.
14
Fig. 16-3
Phage
head
Tail
sheath
Tail fiber
100 nm
DNA
Bacterial
cell
15
Fig. 16-4-3
EXPERIMENT
Phage
Empty
protein
Radioactive shell
protein
Radioactivity
(phage
protein)
in liquid
Bacterial cell
Batch 1:
radioactive
sulfur (35S)
DNA
Phage
DNA
Centrifuge
Pellet (bacterial
cells and contents)
Radioactive
DNA
Batch 2:
radioactive
phosphorus (32P)
Centrifuge
Pellet
Radioactivity
(phage DNA)
in pellet
16
17
History of DNA
• Chromosomes are made
of both DNA and
protein
• Experiments on
bacteriophage viruses
by Hershey & Chase
proved that DNA was
the cell’s genetic
material
Radioactive
32P
was injected into bacteria!
18
Phoebus A. Levene
• Russian born; immigrated to
America, moves to Europe.
• 1920’s discovered
nucleotides (building blocks
of DNA)
1. Sugar
2. Phosphate group
3. Nitrogenous base
19
Composition of DNA
20
Components of DNA
• A very long molecule. 4
nitrogenous bases:
21
Chargaff’s rules
• The relative amounts of adenine
and thymine are the same in DNA
• The relative amounts of cytosine
and guanine are the same.
• Named after Erwin Chargaff
22
Chargaff’s Rule
• Adenine must pair with
Thymine
• Guanine must pair with
Cytosine
• The bases form weak
hydrogen bonds
T
A
G
C
23
Discovery of DNA
Structure
• Erwin Chargaff showed the
amounts of the four bases on
DNA ( A,T,C,G)
• In a body or somatic cell:
A = 30.3%
T = 30.3%
G = 19.5%
C = 19.9%
24
Question:
• If there is 30%
Adenine, how much
Cytosine is present?
25
Answer:
• There would be 20%
Cytosine
• Adenine (30%) = Thymine
(30%)
• Guanine (20%) = Cytosine
(20%)
• Therefore, 60% A-T and
40% C-G
26
DNA Structure
• Rosalind Franklin took
diffraction x-ray
photographs of DNA
crystals
• In the 1950’s, Watson &
Crick built the first model
of DNA using Franklin’s
x-rays
27
Rosalind Franklin
28
Rosalind Franklin
• Used X-Ray
diffraction to get
information
about the
structure of
DNA:
29
Fig. 16-6a
(a) Rosalind Franklin
30
Fig. 16-6b
(b) Franklin’s X-ray diffraction
photograph of DNA
31
Structure of DNA
• Discovered in
1953 by two
scientists:
• James Watson
(USA)
• Francis Crick
(GBR)
• Known as the
double-helix
model.
32
Fig. 16-1
33
34
The double-helix
• A twisted ladder with two long
chains of alternating phosphates
and sugars. The nitrogenous
bases act as the “rungs” joining
the two strands.
35
How long is the DNA
molecule?
36
Chromosomes & DNA
replication
• The nucleus of one human cell
contains approximately 1 meter
of DNA.
• Histones = DNA tightly wrapped
around a protein
• Nucleosome:
37
Chromosome
structure:
38
DNA
Structure
39
DNA
• Two strands coiled called
a double helix
• Sides made of a pentose
sugar Deoxyribose bonded
to phosphate (PO4) groups
by phosphodiester bonds
• Center made of nitrogen
bases bonded together by
weak hydrogen bonds
40
DNA Double Helix
“Rungs of ladder”
Nitrogenous
Base (A,T,G or C)
“Legs of ladder”
Phosphate &
Sugar Backbone
41
Helix
• Most DNA has a right-hand
twist with 10 base pairs in a
complete turn
• Left twisted DNA is called
Z-DNA or southpaw DNA
• Hot spots occur where right
and left twisted DNA meet
producing mutations
42
DNA
• Stands for
Deoxyribonucleic acid
• Made up of subunits
called nucleotides
• Nucleotide made of:
1. Phosphate group
2. 5-carbon sugar
3. Nitrogenous base
43
DNA Nucleotide
Phosphate
Group
O
O=P-O
O
5
CH2
O
N
C1
C4
Sugar
(deoxyribose)
C3
C2
Nitrogenous base
(A, G, C, or T)
44
Pentose Sugar
• Carbons are numbered clockwise
1’ to 5’
5
CH2
O
C1
C4
Sugar
(deoxyribose)
C3
C2
45
5
DNA
O
3
3
P
5
O
O
C
G
1
P
5
3
2
4
4
P
5
P
2
3
1
O
T
A
3
O
3
5
O
5
P
P
46
Antiparallel Strands
• One strand of
DNA goes from
5’ to 3’ (sugars)
• The other
strand is
opposite in
direction going
3’ to 5’ (sugars)
47
Nitrogenous Bases
• Double ring PURINES
Adenine (A)
Guanine (G)
A or G
• Single ring PYRIMIDINES
Thymine (T)
Cytosine (C)
T or C
48
Base-Pairings
• Purines only pair with
Pyrimidines
• Three hydrogen bonds
required to bond Guanine
& Cytosine
3 H-bonds
G
C
49
•Two hydrogen bonds are
required to bond Adenine &
Thymine
T
A
50
Fig. 16-7a
5 end
Hydrogen bond
3 end
1 nm
3.4 nm
3 end
0.34 nm
(a) Key features of DNA structure (b) Partial chemical structure
5 end
51
Fig. 16-UN1
Purine + purine: too wide
Pyrimidine + pyrimidine: too narrow
Purine + pyrimidine: width
consistent with X-ray data
52
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
• In DNA replication, the parent
molecule unwinds, and two new
daughter strands are built based on
base-pairing rules
Animation: DNA Replication Overview
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
53
DNA
Replication
54
Fig. 16-9-1
A
T
C
G
T
A
A
T
G
C
(a) Parent molecule
55
Fig. 16-9-2
A
T
A
T
C
G
C
G
T
A
T
A
A
T
A
T
G
C
G
C
(a) Parent molecule
(b) Separation of
strands
56
Fig. 16-9-3
A
T
A
T
A
T
A
T
C
G
C
G
C
G
C
G
T
A
T
A
T
A
T
A
A
T
A
T
A
T
A
T
G
C
G
C
G
C
G
C
(a) Parent molecule
(b) Separation of
strands
(c) “Daughter” DNA molecules,
each consisting of one
parental strand and one
new strand
57
Replication Facts
• DNA has to be copied
before a cell divides
• New cells will need identical
DNA strands
58
DNA replication
Why needed?
• Step 1: DNA
unzips.
• DNA polymerase
helps in unzipping
• Starts at many
different points.
59
DNA Replication
• Step 2:
• Begins at Origins of Replication
• Two strands open forming Replication
Forks (Y-shaped region)
3’
• New strands grow at the forks
5’ Parental DNA Molecule
3’
Replication
Fork
60
5’
DNA Replication
• Step 3:
• As the 2 DNA strands open at
the origin, Replication Bubbles
form
• Human chromosomes have MANY
bubbles but bacteria have one.
Bubbles
Bubbles
61
DNA Replication
• Step 4:
• Enzyme Helicase unwinds
and separates the 2 DNA
strands
• Single-Strand Binding
Proteins keep the 2 DNA
strands separated and
untwisted
62
DNA Replication
•
Step 5: RNA primers start the
addition of new nucleotides
•
Step 6: DNA polymerase can
then add and finish adding the
new nucleotides
63
Fig. 16-13
Primase
Single-strand binding
proteins
3
Topoisomerase
5
RNA
primer
5
3
5
Helicase
64
3
DNA Replication
• DNA polymerase can only add
nucleotides to the 3’ end of the
DNA
• This causes the NEW strand to be
built in a 5’ to 3’ direction
5’
3’
Nucleotide
DNA Polymerase
Direction of Replication
RNA
Primer
65
5’
Remember the Strands are
Antiparallel
5
O
3
3
P
5
O
O
C
G
1
P
5
3
2
4
4
P
5
P
2
3
1
O
T
A
3
O
3
5
O
5
P
P
66
Synthesis of the New DNA
Strands
• Step 7: The Leading Strand is
added in 5 to 3 direction
5’
3’
Nucleotides
DNA Polymerase
5’
RNA
Primer
67
• Step 8: The Lagging Strand is added
discontinuously on 3 to 5 direction.
• Step 9: This strand has many short segments
called OKAZAKI fragments
Leading Strand
5
’
3’
DNA Polymerase
5’
3’
Lagging Strand
RNA Primer
3’
5’
3’
5’
68
Joining of Okazaki Fragments
• Step 10: The enzyme Ligase
joins the Okazaki fragments
together to make one strand
DNA ligase
5’
3’
Okazaki Fragment 1
Okazaki Fragment 2
3’
5’
Lagging Strand
69
Fig. 16-17
Overview
Origin of replication
Lagging strand
Leading strand
Leading strand
Lagging strand
Overall directions
of replication
Single-strand
binding protein
Helicase
5
Leading strand
3
DNA pol III
3
Parental DNA
Primer
5
Primase
3
DNA pol III
Lagging strand
5
4
DNA pol I
3 5
3
DNA ligase
2
1
70
3
5
71
Completing the replication
• After the DNA
molecule
comes apart,
bases of free
nucleotides in
the nucleus
join their
complimentary
bases.
72
Proofreading New DNA
• DNA polymerase initially makes
about 1 in 10,000 base pairing
errors
• Enzymes proofread and correct
these mistakes
• The new error rate for DNA that
has been proofread is 1 in 1 billion
base pairing errors
73
Semiconservative Model of
Replication
• Idea presented by Watson & Crick
• The two strands of the parental
molecule separate, and each acts as a
template for a new complementary
strand
• New DNA consists of 1
PARENTAL (original) and 1 NEW
DNA Template
strand of DNA
Parental DNA
New DNA
74
DNA Damage & Repair
• Chemicals & ultraviolet radiation
damage the DNA in our body cells
• Cells must continuously repair
DAMAGED DNA
• Excision repair occurs when any of
over 50 repair enzymes remove
damaged parts of DNA
• DNA polymerase and DNA ligase
replace and bond the new nucleotides
together
75
Question:
• What would be the
complementary DNA
strand for the following
DNA sequence?
DNA 5’-CGTATG-3’
76
Answer:
DNA 5’-GCGTATG-3’
DNA 3’-CGCATAC-5’
77
78