If you haven`t done so already, REGISTER TO VOTE!

advertisement
If you haven’t done so already,
REGISTER TO VOTE!
You live in a free country; make your voice
heard!
This website will take you through the process to register
by mail in any of the 50 states:
http://www.fec.gov/votregis/pdf/nvra.pdf
States have different deadlines, so don’t delay
Tests will be returned on
Thursday, Sept. 23 in class
Test answers will be posted on the
website Thursday afternoon
Gene Mutation and DNA Repair
What is a mutation?
A mutation is a change in the chemical
composition of DNA
Mutations
Mutations are usually the source for genetic variation
within populations.
 Mutations may be detrimental, beneficial or
neutral.
 Mutations provide the basis for genetic studies.
Types of Mutations
1. Chromosome abnormalities
A. variation in number
B. variation in arrangement
2. Alterations in base sequence at discrete locations
Gametic vs. Somatic Mutations
Somatic: expressed only in the mutated population;
not passed on to subsequent generations
Gametic: mutations that become part of the genetic
makeup of the gametes and therefore can be passed
on to subsequent generations
Types of Mutations
Dominant autosomal mutations: expressed in the first
generation
Recessive autosomal mutations: may not be
expressed for many generations. Are usually only
expressed in the absence of the dominant trait, and
may be the result of crossing parents who each
carry the recessive gene.
Types of Mutations
X-linked recessive: mutation associated with the Xchromosome. If the dam is homozygous (both
chromatids have the same, recessive gene), the
gene will be expressed in all male offspring (males
inherit only one X-chromosome and receive it
from the dam).
Types of Mutations
Lethal mutation: the organism cannot survive
Conditional mutation: a mutation only expressed
under certain environmental conditions e.g.
temperature
Causes of Mutations
1.
2.
Spontaneous (random): arise independently of an
identifiable external force.
Induced: arise in response to a factor or force
from or within the environment in which a cell or
organism exists.
Point Mutations
Any change that disrupts a gene sequence or the
coded information qualifies the change as a
mutation.
THE CAT SAW THE DOG
Base Substitutions
THE CAT SAW THE DOG
THE CAT SAW THE HOG
THE CAT SAQ THE DOG
Frameshift: Deletion
THE CAT SAW THE DOG
THE ATS AWT HED OG
C
Frameshift: Insertion
THE CAT SAW THE DOG
M
THE CMA TSA WTH EDO G
How do mutations arise?
Tautomers
Structural isomers of nitrogenous bases (A,T, G and
C)
Shifts of a single proton
Alter the bonding structure (base pairing) and
therefore can induce changes in sequence during
replication.
Tautomers
Note that the only change is a move of a hydrogen
(proton) from one place on the ring to the next.
The shifts are temporary, and the tautomers are much
less stable than the normal forms, but while they
exist, the tautomers can form abnormal base pairs
Tautomers
Base pairing between a tautomer and normal base
will still be between a purine and pyrimidine, but
will not preserve the information content.
Thus, T will pair with G (three hydrogen bonds)
A will pair with C (two hydrogen bonds)
Tautomers
Tautomers are transient because they are less stable,
but if a base exists in its tautomeric form during
replication, the wrong base can be incorporated
into the replicated strand.
Alkylating Agents
Chemical agents that donate a –CH3 or CH2CH3
group to the keto groups (=O) of thymine and
guanine
Alter the base pairing properties
Intercalating Agents
Acridine orange, ethidium bromide
Wedge between the bases and contort the DNA,
increasing the probability of a slip during
replication such that frameshift mutations are
generated
UV Radiation
UV radiation causes the formation of thymine dimers
Adjacent thymine residues on a strand of DNA
become attached to one another
1. Unavailable to base pair with complement strand
2. Contorts the DNA strand
3. Inhibits normal replication
Thymine Dimers
High-Energy Radiation
Shorter wavelength than UV
Includes X-rays, gamma rays and cosmic radiation
(ionizing radiation)
Create free radicals that initiate chemical reactions
that can damage DNA
High-Energy Radiation
1.
2.
Point mutations
Break phosphodiester bonds of the DNA
backbone (can induce chromosomal aberrations)
For X-rays, the relationship between dose and
mutation is linear
High-Energy Radiation
Cells in M (vs G1, S, or G2) are more susceptible
because DNA is condensed into chromosomes and
more accessible.
(Cancer cells divide more frequently than normal
cells, so this is why radiation can be an effective
cancer treatment)
Examples of conditions resulting from
mutation or change in DNA sequence
Duchenne Muscular Dystrophy arises most often
from point mutations that alter the reading frame
of the dystrophin gene.
Examples of conditions resulting from
mutation or change in DNA sequence
ABO Blood groups
Arise from differences in activity of the enzyme
glycosyltransferase that modify substance H, a
protein on the surface of red blood cells.
A, B are different modifications of substance H
O is the lack of modification of substance H
Examples of conditions resulting from
mutation or change in DNA sequence
The O phenotype is due to a frameshift mutation
(deletion) in the glycosyltransferase gene such that
a non-functional protein is made
The result is that substance H cannot be modified
Ames test for mutagenicity
Developed by Bruce Ames of the University of Iowa
Asssess the ability of a compound or chemical to
induce mutation
Ames test for mutagenicity
The test looks at the return mutation rate, that is, for
the ability to grow in the absence of histidine
(return to normal or wild type phenotype).
Ames test for mutagenicity
Uses strains of Salmonella bacteria that are sensitive
to mutation (Protective coat and DNA repair
mechanisms have been impaired)
These strains are his -, that is, they cannot synthesize
the amino acid histidine, and must be grown in the
presence of histidine to survive.
Ames test for mutagenicity
The test compound is either incubated directly with
bacteria,
or
Injected first into a mouse such that it passes
through the liver to be modified in whatever way
the compound would be metabolically processed if
it entered the body (many compounds become
more chemically reactive after passing through the
liver)
Ames test for mutagenicity
The bacteria are then incubated on plates in medium
that does not contain histidine.
After 24 hours, the plates are removed and the
number of bacterial colonies are counted
Ames test for mutagenicity
+ histidine
-histidine,
Non-mutagenic
compound
-histidine,
Mutagenic compound
DNA Repair
Numerous mechanisms exist to identify and
repair mismatches and mutations
Excision Repair: cut and paste
1.
2.
3.
4.
Distortion or error is detected
Nuclease clips out the error and several
nucleotides on either side
DNA polymerase fills in the gap with
nucleotides, based on the nucleotide sequence of
the complementary strand
DNA ligase seals the nicks between the new
DNA and the existing DNA strand at the 3’ end
Excision Repair
Xeroderma Pigmentosum (XP)
People with XP (usually) lack the excision repair
system and as such are very susceptible to skin
damage
Very susceptible to skin cancer
Must be protected from UV radiation
Proofreading and Mismatch Repair
The normal error rate of DNA polymerase III
(responsible for DNA replication in E coli) is 1
base per 100,000 bases replicated or 10-5.
The E coli genome is 4 x 106 bases, so in the absence
of proofreading, there would be 40 mutations
every generation.
Proofreading
DNA pol III (and its eukaryotic compatriot) have
proofreading functions, which means that the
enzyme detects errors, reverses direction, clips out
the wrong nucleotide and inserts the correct one.
This reduces the error rate to 10-7, or one in ten
million.
Mismatch Repair
10-7 is still pretty high, so a second mechanism exists
to further reduce errors in replicated DNA.
The alteration is detected, the mismatch is clipped
out and replaced with the correct nucleotide.
Mismatch Repair
Hmm. How does the enzyme know which is the
strand with the error??
5’AGGTCTAGATC3’
3’TCTAGATCTAG5’
Mismatch Repair
Adenine residues are methylated (CH3 group added)
periodically
The enzyme adenine methylase recognizes this
sequence:
5’GATC3’
3’CTAG5’
and adds the methyl group to each of the A residues:
Mismatch Repair
5’GAmTC3’
3’CTAmG5’
This is a stable modification that persists throughout
the cell cycle.
In the case of a mismatch, the repair enzyme uses
this modification to differentiate between the
strands.
Mismatch Repair
Newly replicated DNA is not yet methylated, so the
repair enzyme chooses the methylated (old) strand
as the template strand and fixes the error
5’AGGTCTAGAmTC3’
3’TCTAGATCTAG5’
5’AGGTCTAGAmTC3’
3’TCCAGATCTAG5’
Double Strand Break Repair
Ionizing radiation, in particular, causes breaks in
both DNA strands.
Also, several immune deficiency diseases and
familial breast and ovarian cancers may be a result
of double strand DNA breaks.
Double Strand Break Repair
Homologous recombinational repair:
No template strand is available within the DNA
strand (both are damaged), so the genetic
information in the homologous sister chromatid is
used. The undamaged region is recombined into
the damaged region
Usually occurs during late S/G2 phase of the cell
cycle,.
Double Strand Break Repair
Nonhomologous recombinational repair (end
joining):
Does not involve homologous recombination
Two protein complexes are involved in bringing
damaged ends together and rejoining DNA strands.
Download