Transcript Creation/Evolution - Geoscience Research Institute

Matthew 5:18
18 For verily I say unto you,
Till heaven and earth pass,
one jot or one tittle shall in
no wise pass from the law,
till all be fulfilled.
©2000 Timothy G. Standish
Restriction
and Repair:
Maintaining the integrity of DNA
Timothy G. Standish, Ph. D.
©2000 Timothy G. Standish
DNA Modification
Maintaining DNA integrity is vital to its function
A number of mechanisms exist to ensure that the
sequence of nucleotides is maintained in DNA
Some of these mechanisms involve the chemical
modification of DNA after replication
The most common modification is methylation, in
which a methyl group is added to bases on DNA
Methylation functions in:
– Distinguishing between a cell’s DNA and foreign DNA
– Distinguishing between old and new DNA strands
– Controlling Gene Expression
©2000 Timothy G. Standish
Methylation
5-Methylcytosine is the most commonly methylated
nulceotide in E. coli.
5
6
NH2
Cytidine
4
3N
1
N
Methylation
CH3
NH2
5-Methyl
cytidine
N
2
O
N
O
4-Methylcytosine is less common, but is also known.
©2000 Timothy G. Standish
Methylation
6-Methyladenine is another common methylated
nulceotide.
H3C
NH2
Adenine
N7 5 6 1 N
8
9
N
4
3
N
2
Methylation
N6-Methyl NH
adenine
N
N
N
N
©2000 Timothy G. Standish
E. coli Methylation Systems
Three methylation systems are known in E. coli:
1 dcm system - Methylates cytosine - Function is
unknown
2 dam system - Methylates adenine - Functions in
distinguishing new strands of DNA is involved in
control of replication, marks DNA strands for
repair and influences transposon activity
3 hsd system - Methylates adenine (cytosine in some
bacteria) - Creates specific methylation patterns
marking a bacteria’s own DNA and distinguishing
it from other species or pathogens’ DNA
©2000 Timothy G. Standish
Destroying Foreign DNA
Methylase enzymes methylate specific bases in specific
sequences of DNA
Only the cell’s own DNA is methylated at a given
sequence
Thus it is possible to differentiate between the cell’s DNA
and DNA that has been introduced into a cell by a virus or
from some other source
In bacteria, restriction enzymes are paired with methylases
that recognize the same sequences
Restriction enzymes will not cut methylated DNA
Thus restriction endonucleases cut up foreign DNA, but
not the cell’s DNA
Working with methylases, REs restrict bacteriophages to
©2000 Timothy G. Standish
Bacteriophage Attack
Infection
Destruction of
the bacteria’s
DNA
Production of
viral parts
Lysis
Packaging
Replication of
the viral
genome
©2000 Timothy G. Standish
Repelling Bacteriophage
Attack
Methylation sites
Methylase
M
©2000 Timothy G. Standish
Repelling Bacteriophage
Attack
Methylation sites
Unmethylated
methylation
sites
R
Munch! Munch!
Munch . . .
©2000 Timothy G. Standish
Repelling Bacteriophage
Attack
Methylation sites
Take that you
wicked virus!
©2000 Timothy G. Standish
Repelling Bacteriophage
Attack
Methylase and restriction endonucleases must
recognize the same sequences if they are to function
as an effective system
Take that you
wicked virus!
©2000 Timothy G. Standish
Restriction Endonucleases
There are a number of different subclasses of
restriction endonucleases
Type I - Recognize specific sequences and cut
DNA a nonspecific site > than 1,000 bp away
Type II - Recognize palindromic sequences and cut
within the palindrome
Type III - Recognize specific 5-7 bp sequences and
cut 24-27 bp downstream of the site.
Type II restriction endonucleases are the most
useful class as they recognize specific
palindromic sequences in DNA and cut the sugar
phosphate backbone within the palindrome
©2000 Timothy G. Standish
What is a Palindrome?
A palindrome is anything that reads the same
forwards and backwards:
English palindromes:
Mom
Dad
Tarzan raized Desi Arnaz rat.
Able was I ere I saw Elba (supposedly said by
Napoleon)
Doc note I dissent, a fast never prevents a fatness,
I diet on cod.
©2000 Timothy G. Standish
DNA Palindromes
Because DNA is double stranded and the strands
run antiparallel, palindromes are defined as any
double-stranded DNA in which reading 5’ to 3’
both are the same
Some examples:
The EcoRI cutting site:
– 5'-GAATTC-3'
– 3'-CTTAAG-5'
The HindIII cutting site:
– 5'-AAGCTT-3'
– 3'-TTCGAA-5'
©2000 Timothy G. Standish
Uses of Type II Restriction
Endonucleases
Because restriction endonucleases cut specific
sequences they can be used to make “DNA
fingerprints” of different samples of DNA. As
long as the cutting site changes on the DNA or
the distance between cutting sites changes,
fragments of different sizes will be made.
Because Type II restriction endonucleases cut at
palindromes, they may leave “sticky ends” that
will base pair with any other fragment of DNA
cut with the same enzyme. This is useful in
cloning.
©2000 Timothy G. Standish
R. E.s and DNA Ligase
Can be used to make recombinant DNA
EcoRI
EcoRI
GAATTC
CTTAAG
GAATTC
CTTAAG
G
CTTAA
1 Digestion
AATTC
G
2 Annealing of sticky ends
Ligase
G AATTC
CTTAA G
3 Ligation
4 Recombinant DNA
G AATTC
CTTAA G
©2000 Timothy G. Standish
Question
Where did Type II restriction endonucleases and their
associated methylases come from?
In bacteria, restriction enzymes would be lethal in the
absence of the methylase that methylates their
recognition site
Methylation of specific recognition sites would be
pointless in the absence of restriction enzymes
Modification and restriction systems appear to be
irreducibly complex
Restriction enzymes and their associated methylase do
not have significant sequence homology, thus they do
not share the same DNA recognition domain with
different enzyme domains and must have evolved
independently
©2000 Timothy G. Standish
Mutation And Repair
Maintaining the integrity of genetic material is
vital to the survival of organisms
Somatic cell mutations are known to lead to
cancers in multicelled eukaryotes
Mutations in gametes are passed to offspring and
most commonly will result in decreased fitness
Elaborate systems for prevention and repair of
mutations are known in prokaryotes and are
believed to exist in eukaryotes although, in
eukaryotes, these systems have not yet been well
characterized
©2000 Timothy G. Standish
Mutations
Mutation = A random change in the genetic
material of a cell
Two major types of mutations:
1 Macromutations:
– Chromosome number mutations
– Addition or deletion of large chunks of DNA
– Movement of large chunks of DNA
2 Point mutations:
– Changes in only one or two bases in a gene
Not all mutations result in phenotypic change
©2000 Timothy G. Standish
Micro or Point Mutations
Two major types of Micromutations are
recognized:
1 Frame Shift - Loss or addition of one or two
nucleotides
2 Substitutions - Replacement of one nucleotide by
another one. There are a number of different types:
– Transition - Substitution of one purine for another
purine, or one pyrimidine for another pyrimidine (more
common)
– Transversion - Replacement of a purine with a
pyrimidine or vice versa (less common)
©2000 Timothy G. Standish
Frame Shift Mutations
3’AGTTCAG-TAC-TGA-ACA-CCA-TCA-ACT-GATCATC5’
5’AGUC-AUG-ACU-UGU-GGU-AGU-UGA-CUAGAAA3’
Met
Thr
Cys
Gly
Ser
3’AGTTCAG-TAC-TGA-AAC-CAT-CAA-CTG-ATCATC5’
5’AGUC-AUG-ACU-UUG-GUA-GUU-GAC-UAG-AAA3’
Met
Thr
Leu
Val
Val
Val
Frame shift mutations tend to have a dramatic effect on proteins as
all codons downstream from the mutation are changed and thus
code for different amino acids. As a result of the frame shift, the
length of the polypeptide may also be changed as a stop codon will
probably come at a different spot than the original stop codon.
©2000 Timothy G. Standish
Substitution Mutations
3’AGTTCAG-TAC-TGA-ACA-CCA-TCA-ACT-GATCATC5’
5’AGUC-AUG-ACU-UGU-GGU-AGU-UGA-CUAGAAA3’
Transition
Met
Thr
Cys
Gly
Ser
3’AGTTCAG-TAC-TGA-ATA-CCA-TCA-ACT-GATCATC5’
5’AGUC-AUG-ACU-UAU-GGU-AGU-UGA-CUAGAAA3’
Met
Thr
Tyr
Gly
Ser
Pyrimidine to
Pyrimidine
3’AGTTCAG-TAC-TGA-ACA-CCA-TCA-ACT-GATCATC5’
5’AGUC-AUG-ACU-UGU-GGU-AGU-UGA-CUAGAAA3’
Transversion
Met
Thr
Cys
Gly
Ser
3’AGTTCAG-TAC-TGA-AAA-CCA-TCA-ACT-GATCATC5’
5’AGUC-AUG-ACU-UUU-GGU-AGU-UGA-CUAGAAA3’
Met
Thr
Phe
Gly
Ser
Purine to
Pyrimidine
©2000 Timothy G. Standish
Transitions Vs Transversions
Cells have many different mechanisms for
preventing mutations
These mechanisms make mutations very
uncommon
Even when point mutations occur in the DNA,
there may be no change in the protein coded for
Because of the way these mechanisms work,
transversions are less likely than transitions
Tranversions tend to cause greater change in
proteins than transitions
©2000 Timothy G. Standish
The Sickle Cell Anemia Mutation
Normal b-globin DNA
C
Mutant b-globin DNA
T
T
C
G A
A
G U A
mRNA
mRNA
Normal b-globin
Mutant b-globin
Glu
H2 N
C
C
A T
Val
O
OH
H
CH2
H2C
C OH
O Acid
H2 N
C
C
O
OH
H
CH
CH3
H3C
Neutral
Non-polar
©1998 Timothy G. Stan
Sickle Cell Anemia:
A Pleiotropic Trait
Mutation of base 2 in b globin codon 6 from A to T
causing a change in meaning from Glutamate to Valine
Mutant b globin is produced
Breakdown of
red blood cells
Anemia
Clogging of small
blood vessels
Tower skull
Weakness
Heart failure
Impaired
mental function
Accumulation of sickled
cells in the spleen
Red blood cells sickle
Brain
damage
Paralysis
Pain and
Fever
Damage to
other organs
Rheumatism
Kidney
failure
Spleen
damage
Infections
especially
pneumonia
©2000 Timothy G. Standish
Repair Systems
Direct repair - Uncommon: Direct reversal or removal
of damage
Excision repair - Common: Recognition of damage
followed by cutting out of damaged strand and
replacement with a new strand
Mismatch repair - Detection of mismatched bases
followed by excision and replacement of one, generally
the one on the new strand
Tolerance systems - Important in higher eukaryotes:
Used when DNA is damaged so that replication cannot
proceed normally. May involve many errors
Retrieval systems - Important in prokaryotes
“Recombination repair” damaged sections of DNA are
filled in using recombination
©2000 Timothy G. Standish
Direct Repair
The best characterized system of direct repair is
widespread and found in everything from plants to
E. coli
DNA strongly absorbs ultraviolet light; this energy
may be dissipated by joining adjacent pyrimidines
(i.e., thymine) together to form pyrimidine dimers
Photoreactivation of pyrimidine dimers is achieved
by the detection of the primers by a light-dependant
enzyme that then uses light energy to reverse the
reaction and separate the pyrimidines
In E. coli a single enzyme, photolyase (the phr gene
product), is responsible for this process
©2000 Timothy G. Standish
Thymine Dimers
OH
HO
P
NH2
O
N
O
CH2
OH
Thymine
N
N
O
H
N
O
CH2
O
O
N
O
CH2
N
O
OH
H
Thymine
N
N
O
CH2
H
OH
P
O
OH
O
P
O
HO
NH2
OH
P
O
H
O
H
Thymine Dimers
OH
P
NH2
O
N
O
CH2
N
N
O
H
N
Photolyase
HO
OH
O
Thymine
CH2
O
O
N
O
CH2
N
O
N
N
OH
H
O
Thymine
CH2
H
OH
P
O
OH
O
P
O
HO
NH2
OH
P
O
H
O
H
Thymine Dimers
OH
P
NH2
O
N
O
CH2
N
N
O
H
N
Photolyase
HO
OH
O
Thymine
CH2
O
O
N
O
CH2
N
O
N
N
OH
H
O
Thymine
CH2
H
OH
P
O
OH
O
P
O
HO
NH2
OH
P
O
H
O
H
Thymine Dimers
OH
HO
P
NH2
O
N
O
CH2
OH
Thymine
N
N
O
H
N
O
CH2
O
O
N
O
CH2
N
O
OH
H
Thymine
N
N
O
CH2
H
OH
P
O
OH
O
P
O
HO
NH2
OH
P
O
H
O
H
Mutation
When Mistakes Are Made
5’
DNA
Pol.
5’
5’
DNA
Pol.
3’ to 5’ Exonuclease activity
5’
3’
DNA
Pol.
3’
5’
3’
5’
©2000 Timothy G. Standish
Mutation
Excision Repair
5’
3’
3’
5’
5’
3’
3’
5’
3’
EndoNuclease
5’
Nicks
DNA
Ligase
Pol.
3’
5’
©2000 Timothy G. Standish
©2000 Timothy G. Standish
Macromutations
1
2
3
4
Four major types of Macromutations are
recognized:
Deletions - Loss of chromosome sections
Duplications - Duplication of chromosome
sections
Inversions - Flipping of parts of
chromosomes
Translocations - Movement of one part of
a chromosome to another part
©2000 Timothy G. Standish
Macromutation - Deletion
Chromosome
Centromere
Genes
A
B
C
D
E
F
A
B
C
D
G
H
G
H
E
F
©2000 Timothy G. Standish
Macromutation - Duplication
Chromosome
Centromere
Genes
A
B
C
D
E
F
G
H
A
B
C
D
E
F
EE
FF
G
H
Duplication
©2000 Timothy G. Standish
Macromutation - Inversion
Chromosome
Centromere
Genes
A
B
C
D
E
F
A
B
C
D
F
E
Inversion
G
H
G
H
©2000 Timothy G. Standish
Macromutation - Translocation
Chromosome
Centromere
A
B
C
A
B
E
Genes
D
F
E
C
F
G
H
D
G
H
©2000 Timothy G. Standish