Lecture 8 (biol3600) dna damage and repair - winter 2012

DNA mutation and repair
Assigned reading: Chapter 18
http://www.youtube.com/watch?v=bSRC1_OZPIg
Go 1:30min
http://www.youtube.com/watch?v=4fCCVU4y7oE
Must see 1:15min

Mutations
What are they?
• Mutation - Any change made to the DNA sequence or chromosome structure
- Important general properties:
1) Not inherently good or bad
- Can lead to disease/death, but can also create new alleles (evolution)
- Example: HbS, sickle-cell anemia
- Can be w/in junk (usually), coding sequence, promoters
2) They are permanent – can’t be removed or repaired (damage vs mutation)
3) They do not selectively occur (random)
- Do Antibiotics CAUSE Antibioticsr mutations? NO!!
- Mutation occurs randomly  Antibiotic kills all non-mutants  survivors Abicr
• Mutations are classified in different ways:
1) Size
- Mutations can either involve large portions of chromosomes or small regions
a) Chromosomal mutations – Large segments of chromosomes are deleted,
inverted, moved, or duplicated (DISCUSSED LATER)
b) Gene mutations – Smaller changes in the DNA sequence
- One or a few nucleotides

Mutations
Classification
18.1) Mutations are Inherited Alterations in the
DNA Sequence
1) What causes them? (more later)
- Some due to natural biochemical events
- Called spontaneous mutations
- Others helped along by some artificial factor (chemicals, radiation, viral)
- Called induced mutations
2) Type of cell that contains mutated DNA:
- Somatic mutations = Arise in the DNA of somatic cells (normal diploid)
- NEVER passed onto the next generation
- Mutating cells in your arm  Won’t go to kids
- Earlier in development  Worse outcomes (Why?)
- Germ-line mutations = Mutations arise in the DNA of gamete-forming
tissue (those cells that produce sperm and eggs)
- Transmitted to the offspring  Pass onto future generations

Mutations
Classification
3) Other classes of mutations
a) Some are incompatible with life (lethal mutations)
- If lead to prenatal death  embyronic lethal mutations
b) Some only produce an effect under certain environmental conditions
such as hot temperatures (conditional mutations)
- Condition doesn’t cause it, but allows it to be expressed
c) Some reverse (suppress) the effect of a previous
mutation (suppressor mutations)
- Not a direct reversal of the mutation
- Intragenic – 2nd mutation in the same gene
- Mutation 1 alters protein structure, 2 alters it back
- Intergenic – 2nd mutation in totally different gene
- Usually seen in pathways or protein multimers
- Mutant protein 1 is defective, mutant protein 2 does the job of protein 1

Different types of gene mutations
Base-pair substitutions
• Small gene mutations come in 3 main varieties:
1) Base-pair substitutions - One nucleotide is changed to a different nucleotide
- Possible outcomes on the amino acid sequence :
1) No effect– usually see this if the 3rd nucleotide of a
codon is changed
- Called a silent mutation
2) Change causes the wrong amino acid
to be inserted
- Called a missense mutation
- If new type of aa, changes protein shape
- If similar aa, may have little effect (neutral)
- Ex: Sickle-cell, cystic fibrosis, Tay-Sach's
3) Change turns the codon into a stop
codon
- Causes the polypeptide to stop growing
(protein will be truncated/shorter)
- Called a nonsense mutation

Alterations can happen with
Autosomal Chromosomes
Overview and some terms
Cells end up with extra chromosome
Cells end up with extra sets of chromosomes

Alterations can happen with
Autosomal Chromosomes
Overview and some terms
• Cells can acquire larger alterations in chromosome number or structure
- These are more often harmful (as with the sex chromosomes)
• Alterations in chromosome number
- General terms:
1) Aneuploidy – An organism gains or loses 1 or more chromosomes
(but not a full set)
- Monosomy – Individual lacks 1 chromosome (example: XO has 45 chr.)
- Trisomy – Individual has 1 extra chr. (example: XXY has 47 chr.)
- Tetrasomy – 2 extra, penta.....
2) Polyploidy – An organism has 1 or more extra SETS of chromosomes
- Diploidy – normal (2n)
- Triploidy – 1 extra set (3n), tetraploidy (4n)

Alterations in chromosome number
Polyploidy
• Polyploidy - Not very common in the animal world (some fish and amphibians),
but plants commonly have extra sets of chromosomes
• Organisms acquire an extra set of chromosomes in 2 major ways:
1) Autopolyploidy – Occurs when an organism
acquires an extra copy of its own chromosomes
- AA  AAA (tri), AAAA (tetra), etc....
2) Allopolyploidy – Occurs when chromosome
sets from 2 different species are combined
- AA + BB  AABB
• Autopolyploidy
- Autotriploids (AAA) can be generated in 3 major ways:
1) During meiosis, one gamete (of a diploid) receives both copies of every chr (AA).
If that gamete then gets fertilized by a haploid gamete (A)  result AAA
2) One egg is fertilized by 2 sperms
3) If a (normal) tetraploid organism mates with a (normal) diploid individual
- Tetraploid gametes would be AA, diploid gametes A  result AAA

• Autopolyploidy
- Autotetraploids (AAAA) arise from the premature stoppage of mitosis
(in early embryos)
 A cell will end up with twice the normal
number of chromosomes
- Most autopolyploids are usually sterile
- Produce bad gametes b/c of abnormal chr. separation during meiosis
- Examples: Seedless watermelons
• Allopolyploidy
- This occurs when 2 haploid gametes of 2 closely related species accidentally fuse
- A + B  AB
- These initial individuals would be sterile because A and B chr. can not synapse
during prophase (can not separate properly)
- Occasionally, these organisms will undergo a chromosomal doubling at early stage
- AB  AABB (called allotetraploid)
Polyploidy

• Allopolyploidy
- Can see hybrid allotetraploid plants in nature
(never in animals – mating problems)
- They are fertile b/c they have 2 good copies of
each chromosome (meiosis occurs normally)
- These plants often have traits from both species
- Can be very economically important if the resulting
allotetraploid express good traits from each species
- Scientists attempt to engineer allopolyploids with the
hopes of making lots of $$$$$
Polyploidy

• Trisomy is also a very bad situation for animals and plants
- Animals that have an extra sex chromosome are usually viable, but have
various developmental defects
- Animals that have an extra autosome are usually not viable
- Only a few examples of viable trisomic conditions involving autosomes
- The smaller the chromosome, the likelier that the organism can tolerate
having an extra one
- Plants are a little more tolerant of extra autosomes
as far as viability goes
- Having extra autosomes usually alters the
phenotype
• Examples of autosomal trisomic conditions in humans
1) Down syndrome (trisomy 21)
- Most common trisomic condition (almost all others are embryonic lethal)
- Although individual phenotypes vary, most are short and have mental
impairment, heart/lung malformations, and abnormal skeletal development
Aneuploidy  Trisomy

• Examples of autosomal trisomic conditions in humans
1) Down syndrome (trisomy 21)
- Most cases are due to nondisjunction during meiosis I
- 95% of the cases are due to nondisjunction in the mom's
egg
- Incidence of Down syndrome directly correlates with
mom's age (no one really knows why)
2) Patau syndrome (trisomy 13)
- Infants have severe developmental problems in almost all organ systems
and usually, when born alive, only survive a few months.
- Older the mom and dad, greater chance of having a baby with PS
3) Edward syndrome (trisomy 18)
- Same as in PS, except most infants that have this syndrome are female
http://www.youtube.com/user/paulawaziry?feature=mhee#p/c/C038F6E6BFE2738A/49/lP6aLp-4zg0
Aneuploidy  Trisomy

• Types of Chromosomal Mutations:
1) Change in the number of chromosomes
(what we have seen so far)
2) Rearrangement of genes
- Deletions
- Inversions
- Duplications
- Translocations
Reciprocal
Non-reciprocal

Alterations in chromosome structure
• Changes in chromosomal structure and arrangement are usually due to the
introduction of a chromosomal break (spontaneous or induced by chemicals/
radiation), followed by the loss or rearrangement of the chr. pieces
• When chromosomes break, the free ends are "sticky" and will rejoin with
other free ends in a nonspecific way
• The major types of chromosomal rearrangements include:

Rearrangement of genes

• There is an example of partial monosomy in humans
- These individuals are missing a significant portion of the
small arm of chr. 5
- Individuals with this deletion suffer from a condition called
Cri-du-chat syndrome ("cry of the cat")
• Cri-du-chat symptoms
- Individuals are usually mentally handicapped and usually suffer some physical
abnormalities
- Heart and GI tract are usually malformed
- Malformation of the larynx and glottis leads infants to have a characteristic
cry that sounds like a cat's meow
http://www.youtube.com/user/paulawaziry?feature=mhee#p/c/C038F6E6BFE2738A/19/Bf3O_Q31ZUg

Rearrangement of genes:
Duplication

Deletions
• A deletion is the loss of a portion of a chromosome
- Usually bad (cri-du-chat), but can be good
• Breaks can occur at different places within the
chromosome
- A single break can give rise to a terminal deletion
- Two internal breaks can give rise to a intercalary
deletion
• When a chromosome breaks and the pieces remain
apart, what determines which piece will be lost?
 Whatever piece has the centromere will be
retained by the cell (mitosis)
• Chromosomes containing deletions will usually have normal counterpart
- How do they synapse properly during meiosis (remember that chr. match up
perfectly during prophase I)?
- When one homolog is missing a piece, the other one will loop out the "extra"
sequence so that they can synapse properly

Ex: Huntington disease

Duplications
• A duplication is a when any piece of a chromosome (can be a single gene) is
present more than once in the genome
• How do duplications occur?
1) Improper crossing over between
homologous chromosomes during
prophase I
- Two nonsister chromatids should exchange the exact same genes
- Occasionally there is an error and one chromatid receives too much and
the other receives too little
2) An error occurs during DNA replication that leads to the same piece being
added twice
• Gene duplications can be beneficial for organisms
1) In some cases, cells containing chr. duplications will be able to grow better
than their normal counterpart
- Example: Most organisms have multiple copies of each type of rRNA gene
- More copies of rRNA gene, more rRNA produced, more ribosomes, more protein

mutation: any heritable change in the genetic material
(excludes changes caused by normal recombination events)
C--T happens
The integrity of genomic DNA is constantly under threat, even in
perfectly healthy cells. DNA damage can result from the action of
endogenous reactive oxygen species, or from stochastic errors in
replication or recombination, as well as from environmental and
therapeutic genotoxins.

Spontaneous mutations: a mutation that occurs in
the absence of known mutagens
 uncorrected errors that occur during DNA
replication, repair or recombination
spontaneous lesions that occur to the DNA
molecule under normal physiological conditions and
that are not repaired by the cell’s DNA excision repair
processes

Duplications
• Gene duplications can be beneficial for organisms
2) Encourage the creation of new genes  Evolution
- Evolution generally requires:
a) Formation of new alleles of existing genes
(protein still has the same general function) OR
b) Formation of brand new genes that encode proteins with novel functions
- Suggestion: Take that essential gene, duplicate it, and mutate the duplicate
until it is a brand new gene (that encodes a different protein)
- You now have the essential "old" gene and a brand new gene
 These gene duplication events are thought to be a major source of
new genes and a driving force in evolutionary change
- Examples: Hemoglobin and myoglobin Trypsin and chymotrypsin

Inversions
• Inversion – A piece of the chromosome gets inverted 180º within the chromosome
• How does an inversion take place?
- A double chromosomal break occurs
- Based on where the pieces are
positioned with respect to one another,
they may be ligated in the wrong place
• Inversions can be classified based on the appearance of the arms after the event
- Paracentric – Both breaks occur within 1 arm.
Centromere is not involved in the inversion.
Arm ratios remain unchanged
- Pericentric – Breaks occur in each arm. Arm
ratios will be changed following ligation
• Inversions are a problem for gamete formation and new positions may be bad! WHY?

Translocation involves 2 chromosomes
Here is a non-reciprocal translocation
- involves non-homologous pairs

Translocations
• Translocation – A segment of a chromosome is transferred onto a
nonhomologous chromosome
- If 2 nonhomologous chr. trade random pieces  reciprocal translocation
- If 1 chr. just takes a piece from another  nonreciprocal translocation
• How does this occur?
- It they switch end segments, just need
2 chr. to randomly be close together
and each have a break

Translocations
• Translocations are like inversions in that
no genetic info is lost in the process
- The info is just put in a different place
- Often has no real effect on organism viability
• When do translocations (and inversions) create problems:
1) The region of the chromosome near the centromere is transcriptionally
inactive
- If a translocation ends up moving a highly transcribed gene near the
centromere, it will now be shut down
2) Promoters tightly regulate the rate of transcription for each specific gene
- If translocation ends up moving a gene to a new location so that it is under
the control of a different promoter, that can be very bad
- What if a cell death gene that is only activated when the cell is in trouble
is moved downstream of a constitutive promoter?
- translocations can lead to cancer

2) Insertions/deletions – An extra nucleotide gets added or removed
- VERY BAD b/c it causes a frameshift (shift in the reading frame)
- All amino acids after ins/del will be wrong!!
Insertions/deletions
ARGININE GLYCINE TYROSINE TRYPTOPHAN ASPARAGINE
ARGININE GLYCINE LEUCINE GLUTAMATE
LEUCINE
mRNA
PARENTAL DNA
amino acid sequence
altered mRNA
BASE INSERTION
altered amino acid sequence
THE DOG BIT THE MAN
THE DOB ITT HEM AN
delete the G
THE DOG C BIT THE MAN.
THE DOG CBI TTH EMA N
add an extra C

Phenotypic Effects of Mutations
• Forward mutation: wild type  mutant type
• Reverse mutation: mutant type  wild type
• Missense mutation: amino aciddifferent amino acid
• Nonsense mutation: sense codon nonsense codon
• Silent mutation: codonsynonymous codon
• Neutral mutation: no change in function

3) Expansion of trinucleotide repeats (TNRE)
- Some loci contain a series of trinucleotide repeats (e.g. CAGCAGCAG...)
next to a gene or inside the gene
- Everyone has them – usually stable copy number
http://www.youtube.com/watch?v=Symw0nU7Hys
- Abnormal event can occur  Copy number increases
- Ex: Normally have 10 copies of CAG on chr. 8  inc. to 200 copies
- What causes the increase? NO ONE REALLY KNOWS
- Abnormal DNA structure causes DNA pol to slip and copy section 2x
- Such expansion often leads to disease
- If in a gene, expansion increases # of a.a.
- If next to a gene, can trigger methylation of gene
- TNRE disorders usually get worse each generation
- Expansion grows  worse symptoms
- Called anticipation
Expansion of repeats

Causes of mutations
Damage becomes mutation
• How does DNA damage get converted into permanent mutations?
Common theme :
1) A change occurs in the structure of a nt (lesion/damage)
2) DNA replication occurs – DNA pol puts "wrong" nt across from the lesion
3) 2nd DNA rep occurs – Wrong nt serves as a template for complimentary wrong nt
RESULT: DNA now contains a completely wrong PAIR  cell sees as normal!!
lesion

Causes of DNA damage
Spontaneous damage
• Causes of spontaneous damage include:
1) Errors of DNA polymerase
- Polymerases and proofreading/repair enzymes are not perfect
- Some major causes of spontaneous errors during replication include:
a) Strand slippage (see TNRE)
- Repeats cause abnormal loop  DNA pol copies same thing 2x
b) Defective proofreading

Spontaneous damage
2) Tautomeric shifts
- Nitrogenous bases can exist in different chemical
forms called structural isomers
- "Normal" forms  A-T, C-G bonding
- "Rare" isomers  Abnormal base pairing
- Ex: Abnormal T prefers to H bonds w/ G
- Conversion between normal and abnormal
isomers occurs naturally at some
low rate
- VERY BAD if it occurs right
before DNA replication
- DNA pol will read rare form and
insert the wrong base across
(common theme discussed)
- Not a major source of mutations

Concept Check 1
Which of the following changes is a transition base substitution?
a. Adenine is replaced by thymine.
b. Cytosine is replaced by adenine.
c. Guanine is replaced by adenine.
d. Three nucleotide pairs are inserted into DNA.

Spontaneous damage
2) Tautomeric shifts (common theme again)
a) A nitrogenous base shifts from the common
tautomer to the rare version (called tautomeric
shift) – let’s use a rare “A” tautomer
b) DNA replication begins
- One strand is normal
- When DNA pol sees the rare “A” tautomer in the
template , it will insert a C into the new strand
 Assume the tautomer goes back to the normal
form
c) DNA replication begins again
- "Wrong" C serves as a template for a G
- A permanent (unrepairable) mutation
has occurred
 Shift, DNA rep I (wrong base put in), DNA rep II (wrong base is template)

Spontaneous damage
3) Depurination and deamination
- Depurination – Sugar-base bond is spontaneously
broken
- Base is lost (usually purines) and nucleotide
is left empty (called apurinic site)
- What would happen to apurinic site during
DNA replication? ______________________
 Happens very often (10k a day)
- See original common theme slide
- Deamination - An amino group of C or A is
spontaneously lost
- C or A w/o amino groups won't hydrogen
bond with normal G and T
- DNA pol sees a deaminated C (or A) and
puts in the wrong base
- Same common theme

Your DNA is under constant assault:
Science Dec. 23, 1994
Every second that you read this, the DNA in each cell of your
body is being damaged
Chemical bonds are breaking
DNA strands are snapping
Nucleotide bases are flying off
Each cell loses more than 10,000 bases per day just from
spontaneous breakdown of DNA at body temperature
Meanwhile many cells are dividing and therefore copying
DNA and each copy introduces the possibility of error
Exposure to carcinogens adds to the injury and causes
strange new forms to sprout from the double helix

Causes of damage
Spontaneous damage
4) Oxidative damage
- Normal process of aerobic cellular
respiration creates extremely reactive
atoms called a free radicals
- Free radicals – An atom or group of
atoms that has an unpaired electron
- Free radicals will steal an electron from wherever it can get it
- Proteins, lipids, DNA
- Removal of electrons from DNA bases  alters their structure
- If happens right before DNA replication  Common theme again!!
- Thought to be major mutagen in our cells  Cancer and aging!!
- Some agents can lead to increased free radical production (induced)
H
H O H O
O H
O
+

Causes of damage
Spontaneous damage
5) Transposons (aka jumping genes)
- Mobile pieces of DNA abundantly found in all living things
- Nearly 45% of human genome
- Cut or copy themselves and then insert
randomly in the host genome
- Replicative vs nonreplicative transposons
- They encode enzyme transposase
- Insertion near genes or within genes can disrupt
host gene expression
- Can also lead to larger chromosomal alterations
- DNA is being cut/pasted – can go wrong
- Control transposase  Control movement
- Methylation and mRNA destruction
http://highered.mcgraw-hill.com/sites/0072835125/student_view0/animations.html#

Causes of damage
Induced damage
• Some external agents (chemical and physical) can induce DNA damage:
1) Base analogs
- Chemicals that resemble normal nucleotides and can substitute
for them during DNA replication
- However, they exhibit abnormal base-pairing properties
- Example: 5-bromouracil resembles thymine
- DNA pol will incorporate 5BU instead of thymine during DNA rep
- 2nd round of rep – DNA pol puts a "G" across from 5BU
- 3rd round of rep – wrong "G" serves as template for wrong "C"

• Causes of induced damage include:
2) Alkylating agents
- These chemicals add an alkyl group (CH3 or CH3CH2) to
amino or ketone groups in nucleotides
- Alkylated nucleotides exhibit abnormal base pairing
- Ex: Ethyl guanine pairs with T
- Two rounds of DNA rep  Mutation (same theme)
- Mustard gas (structure above) – Alkylating agent used as a weapon in WWI
- Soldiers came down with severe burns, blindness, and tumors
Current chemical attacks on unsuspecting populations:
Causes of damage
Induced damage
G C GC G C
T G
+
T
T G
+
A
http://www.youtube.com/watch?v=bwJKYHNGT98&feature=related

3) Intercalating agents
- Flat, multiple-ringed molecules that tightly wedge themselves between
the bases of DNA  distorts its 3-D structure
- DNA pol gets confused  adds or removes a nucleotide
- Cause insertions or deletions in the DNA (unlike all others discussed)
- Examples include acridine orange
and ethidium bromide
- They are common used to visualize
DNA during centrifugation or gel
electrophoresis
Causes of damage
Induced damage

4) UV light and low energy radiation
- All electromagnetic radiation having wavelengths shorter than
visible light (~380 nm) are very energetic
- Disrupt DNA and other macromolecules
- UV light  λ≈260 nm and is very mutagenic
- UV light causes adjacent pyrimidine bases to fuse with one another
- Called pyrimidine dimers (usually two thymines)
- Distort DNA 3-D structure
- Pyrimidine dimers prevent DNA pol
from replicating normally
- Insert wrong, too many, too few
- Cells containing too many of these
dimers will kill themselves via cell suicide (apoptosis)
Causes of damage
Induced damage

5) High-energy radiation (ionizing radiation)
- Electromagnetic radiation with shorter wavelengths
even worse:
- X-rays, gamma rays, cosmic rays
- Mutates DNA in different ways:
1) It cause electrons to be released from various
molecules in the cell producing free radicals
- This is called ionization
- Free radicals mutate DNA as described
2) It directly breaks phosphodiester bonds in the DNA
strands (causes double- stranded breaks)
- Can produce deletions, translocations, inversions
3) Creates thymine dimers
- Why do we treat tumors with X-rays?
Causes of damage
Induced damage

6) Viruses
- Retroviruses have the ability to randomly insert themselves into our genome
- Usually go into junk (no issue)
- If go into a promoter or coding sequence  gene expression disrupted
- Similar to transposons (gigantic insertion)
- EXAMPLE: Retroviral gene therapy and leukemia
- Other viruses produce proteins that directly inhibit
DNA replication, monitoring, or repair mechanisms
- Indirectly encourages mutations to be introduced
- Once they are in, they can't be removed (like
transposons)
- Not really "damage" (damage can be fixed)
Causes of damage
Induced damage

Assessing the mutagenicity of compounds
Ames test
• Ames test
- Used to test if a new chemical has ability to mutate DNA (cause cancer)
- Successfully identified carcinogens in hair dye (1975)
- Set-up
- Uses bacterial strain that can't make its
own histidine (won't grow w/o it)
- Mix bacteria w/ either chemical or H2O
and add to Petri dish lacking histidine
- No bacteria should grow
- Mutations can occur to allow the bacteria
to make histidine  regain ability to grow
- Results
- H2O control  Very few colonies (spontan)
- Mutagenic chemical  lots of colonies (BAD!!)
+ H2O + chemical
to be tested
+ LIVER
ENZYMES

Repairing DNA damage
• Most types of DNA damage can be fixed by the cell
- Must be fixed PRIOR TO DNA REPLICATION
- Remember the common theme
- Exceptions: Transposons and retroviruses (can't be removed)
• Different types of damage exists
- Altered individual bases (alkylated, base analogs, etc...)
- Altered 3-D DNA structure (thymine dimers, intercalating agents)
- Double-strand DNA breaks
 Different repair mechanisms must exist to detect and fix
• DNA repair themes
- How is it detected?
- How is lesion removed/repaired?
- Is any DNA cut out in the process? If so, how much?
- Bacterial vs eukaryotic

Direct repair
• Direct DNA repair – Reverses the alteration w/o cutting out or replacing any nt
- Used primarily for thymine dimers and alkylated bases
• Direct repair of thymine dimers
- Both bacteria and eukaryotic cells use light-dependent pathways
- Eukaryotic cells – use an enzyme called photolyase to cut abnormal covalent
bonds between the two thymines
- Bacteria – use an enzyme called photoreactivation enzyme (PRE) to do same
- PRE is activated by blue light
• Direct repair of alkylated bases
- Methylguanine DNA methyltransferase
enzymes directly cuts off extra CH3
from guanine

Indirect repair - excision repair
• Excision repair – Removal of altered base/nucleotide and replacement with
good DNA
1. Recognition of the lesion by 1 or more proteins and the subsequent excision
of that error by a nuclease enzyme
- Sometimes extra "good" sequence also removed
2. A DNA polymerase fills in the space with proper nucleotides
- What enzyme would you predict does this in prokaryotic cells?
3. DNA ligase seals the final nick (the last
phosphodiester bond) between the new and
existing strands
• Cells have 2 types of excision repair systems
- Base excision repair
- Nucleotide excision repair

• Base excision repair - used for correction of minor alterations
to individual bases (free radical, alkylated, base analog)
• Mechanism (described in E.coli, but all cells have it)
1) DNA glycosylase enzymes recognize altered bases
- Different glycosylases recognize different types of altered
bases
2) Glycosylase then cuts out the base only (breaking the
sugar/base bond)
3) AP endonuclease enzyme recognizes the nucleotide
missing the base and makes a cut in the sugar/
phosphate backbone at that site
4) DNA pol I/ligase finish the job (and repair the damage)
 Eukaryotic glycosylases have yet to be identified
Indirect repair – base excision repair

Indirect repair - NER
• Nucleotide excision repair fixes larger lesions that
distort the actual DNA structure and block replication
- Examples: Intercalated agents, thymine dimers
• NER (E.coli NER is described below)
1. DNA is damaged and a lesion forms
2. Proteins called Uvr (UvrA, B, C, D)
recognize the lesion and cut it out
- A-B complex recognizes the lesion
- A comes off and is replaced with C
- B-C together cut the DNA on either side of
the lesion
- Cut out extra "good" DNA on both sides
- D is a helicase that liberates the cut piece
3. DNA pol I fills in the gap/ ligase seals
http://highered.mcgraw-hill.com/sites/0072835125/student_view0/animations.html#

Nucleotide excision repair - Disorders
• Several human disorders exist in which the NER system is defective
- The best characterized of these disorders is called xeroderma pigmentosum
• Xeroderma pigmentosum (XP)
- Contain one of several rare mutations in some part of the NER pathway
- They have severe skin abnormalities when exposed to the sun
- UV light exposure Induces freckling, ulceration, and skin cancer
- Why is the sun so damaging?
- Produce 1000s of TT a day
- No repair  mutation  cells die or become cancerous
- Scientists isolated DNA from XP patients and attempted to find which gene
was mutated
- Found that mutations in any of 7 different genes all lead to XP
 NER in eukaryotic cells involves as many as 20 different proteins

Indirect repair - mismatch repair
G
T
Which strand
is wrong?
• Mismatch repair  fixes mismatches (DNA may
look okay otherwise)
• Problem: If the cell has a G-T mismatch, how does
it know which one is correct? ( the G or the T)
- Hint #1: Mismatches usually appear following DNA
replication
- Common theme review
 "Wrong" nucleotide is always on the new strand
- Hint #2: Newly-made DNA strands stay
unmethylated for a little while
- New and old DNA strands look different for a
short time (hemi-methylated)
- If wait too long, both become methylated
 Wrong nucleotide is always on the new,
unmethylated strand!!!

Indirect repair - mismatch repair
• Mismatch repair
- DNA commonly contains methylated adenines
- No effect on transcription (cytosine CH3)
- Adenine methylase add CH3 when seeing GATC
- Mechanism (E. coli)
1) MutS protein locates mismatches
- Forms complex with MutL afterward (linker)
2) MutL binds to MutH, which is bound to a nearby
hemi-methylated site
- DNA must loop out to allow L-H interaction
3) MutH makes a cut in the unmethylated strand
4) MutU acts as a helicase to release the
unmethylated strand before an exonuclease
destroys it
5) DNA pol III fills in with proper sequence, ligase seals
http://www.youtube.com/watch?v=ESBL6Qxsi90

Fixing double-stranded breaks
• Most repair pathways require 1 good template strand
- What is done when both strands damaged?
• Two repair pathways fix double-stranded breaks:
1) Homologous recombination repair (E.coli)
a) Homologous chromosome first brought in
- Usually the sister chromatid
b) RecBCD recognizes double stranded breaks
- Partially degrades 1 strand on each side
- Creates single-stranded overhangs
c) RecA binds to single-stranded end and
promotes invasion of the homologous chr.
- The good strand loops up (D-loop)
d) RuvABC, DNA polymerase, and ligase help
to recreate the gaps and resolve the structure
 The once damaged chromosome will contain a piece of the homologous chr.
 Very similar to what happens during crossing over

Fixing double-stranded breaks
• Two repair pathways fix double-stranded breaks:
2) Non-homologous end-joining
- The two broken ends are simply glued back
together
- No requirement of sister chromatid
- End-binding proteins bind to each side of the
break (to stabilize)
- Cross-bridging proteins recruited to prevent
drifting of the two pieces
- Ends are processed, filled, and ligated
- Advantage  Can happen any time in cell
cycle (no sister chr. required)
- Disadvantage  Can lead to small deletions
near the break site (result of processing)

Translesion synthesis
• Some lesions (e.g. TT) block normal DNA replication (via DNA pol III)
- If other repair fails, translesion synthesis will initiate to allow DNA replication to
finish
• Translesion synthesis (called SOS repair in E. coli)
- Stalling of normal DNA polymerase by lesion triggers
recruitment of "emergency" polymerases
- Have different binding pocket  more tolerant of
altered DNA structure
- Emergency pols (e.g. DNA pol II, IV, V) replicate
over the lesion
- Problem: They are very error prone
- DNA gets replicate, but with mistakes
- Original lesion remains (not fixed)
- Translesion synthesis enables rep to
continue

GENETICS
A Conceptual Approach
FOURTH EDITION
Benjamin A. Pierce
© 2012 W. H. Freeman and Company
CHAPTER 19
Molecular Genetic Analysis and
Biotechnology

Chapter 19 Outline
19.1 Techniques of Molecular Genetics Have
Revolutionized Biology, 514
19.2 Molecular Techniques Are Used to Isolate,
Recombine, and Amplify Genes, 515
19.3 Molecular Techniques Can Be Used to Find
Genes of Interest, 527

Chapter 19 Outline
19.4 DNA Sequences Can Be Determined and
Analyzed, 533
19.5 Molecular Techniques Are Increasingly Used to
Analyze Gene Function, 541
19.6 Biotechnology Harnesses the Power of
Molecular Genetics, 547

Locating DNA Fragments with
Southern Blotting and Probes
• Probe: DNA or RNA with a base sequence
complementary to a sequence in the gene of
interest

19.3 Molecular Techniques Can Be Used to
Find Genes of Interest
Gene Libraries
In Situ Hybridization

Gene Libraries
DNA library: a collection of clones containing all
the DNA fragments from one source
• Creating a genomic DNA library
• cDNA libraries: consisting only of those DNA
sequences that are transcribed into mRNA

•Gene libraries
finding genes of interest
- Paradoxically, researchers must first clone a
Gene in order to “find” it
- What are the advantages and disadvantages of:
- Creating a genomic library using partial digestion
With restriction endonucleases
- Creating a cDNA library

• Screening Gene libraries:
- Usually done using a probe from a similar
Gene isolated from other species and hybridizing
- Can also deduce DNA sequence from known
Protein sequence
In this case, a mixture of all possible nucleotides
combinations is used as probe.
- May also look for the protein product of a gene
(Western Blot: specific antibodies)

Genomic and cDNA libraries can be screened with a probe to
find the gene of interest.

Site-Directed Mutagenesis
• Oligonucleotide-directed mutagenesis

Transgenic Animals
• Transgene

Knockout Mice
• A normal gene of the mouse
• has been fully disabled.
• Knock-in mice: a mouse carries
• an inserted DNA sequence
• at specific locations

Lecture 8 (biol3600) dna damage and repair - winter 2012

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