Download DNA Replication

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
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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
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Griffith’s Rats
1. First he injected living Type S
bacteria into rats:
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• 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.
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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
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•Two hydrogen bonds are
required to bond Adenine &
Thymine
T
A
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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
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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
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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’
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