Nov 5/11 – Lecture 6
1. Good mutations
Immunity to HIV - 10% copies of Chemokine receptor #5 in human genome have a deletion due to a
- 1% of population homozygous for CCR5 deletion
- Having this deletion makes people resistant to HIV
EeroMantyranta - Received 7 gold medals
- Had 15% more red blood cells than normal which helped him increase O2 capacity
- He had a mutation in erythropoietin receptor that cause increased blood production.
2. Induced mutations
mutagenesis - Mutagenesis: production of mutations in the laboratory through exposure to
Incorporation of base analogs - Base analogs: chemical compounds sufficiently similar to normal nitrogen bases of
DNA that they’re occasionally incorporated into DNA in place of normal bases.
- Once in place, these analogs have different pairing properties that causes a wrong
nucleotide to be inserted oppositely
- Chemicals that resemble normal nitrogen bases get incorporated into DNA
o Ex. 5-bromouracil, normally paired with adenine, it becomes ionized and
pairs with guanine instead = transition (AG)
o 2- aminopurine2-AP normally paired with T, now pairs with C. (AT GC
o 5 bromodeoxyuridine is a derivative of 5-bromouracil that destroys the
outside of tumours but not the core.
- Alkylating agents alter bases, it’s not incorporated into DNA but alters it in a way
that it’ll form a specific misrepair.
EMS - EMS (ethyl methane sulfonate) add ethyl group
o Ex. GC AT transition due to elimination of the 3 H bond G can make
o TA CG transition
NG - NG (nitrosoguanidine) methyl group
Intercalating agents - Planar molecules that mimic base pairs
- Slips between bases and causes intersertion or deletion (frameshift) of a single
o Ex. Acridine orange
o ICR 191
- Aflatoxin B1 forms a bulky addition product that is produced by Aspergillus fungus
found in peanut butter
- It breaks the glycosidic bond and causes base to get removed
- This results in a replication block because DNA synthesis won’t proceed past a base
that can’t specify its complementary partner.
Physical mutagens – Ionized - Includes X rays and gamma rays
radiation - Has short wavelength and high energy, thus penetrates deeply into molecules
- Causes reactive free radicals that can oxidize DNA
- Results in base deletions, single nicks in DNA strands, cross-linking, and
Non ionizing radiation - Includes UV light
Q: Is 6,4 photoproduct and - Has less energy and can’t penetrate deeply into biological molecules
cyclobutane pyrimidine dimer
both thymine dimer? - Causes formation of cross linked thymine dimers and 6,4-photoproducts
- When DNA strand is replicated, this may cause more mutations or DNA breakage.
A: They’re both thymine dimers
but just more specific names
based on where the bonds are.
3. Testing methods for
mutagens Ames test - A rat’s liver was ground and centrifuged and liver enzymes were extracted
- Liver is used to mimic the mammalian metabolic conditions so that the mutagenic
Q: Why would you test the speed potential of metabolites formed by a parent molecule in the hepatic system can be
at which mutations revert back, assessed
why can’t you test the speed that - A strain of Salmonella typhimurium that can’t synthesize the amino acid histidine
mutations occur? Unless you want due to a point mutation is placed in the liver enzymes
to find which specific gene is - A carcinogen X is mixed in as well
mutated? - The mixture for strain 1, strain 2 and control (no X) is plated and monitored to see
when the second mutation for His- His+ takes place.
- Some salmonella strains revert through base-pair substitution or frameshift.
4. DNA repair - Since most mutatiosn are deleterious, DNA repair systems are vital to the survival of
- In most cases DNA repair is a multi-step process:
1. Irregularity in DNA structure is detected (i.e. DNA too wide or missing)
2. Abnormal DNA is removed
3. Normal DNA is synthesized
- The proofreading function of DNA polymerase 1 and 3 are first defense and most
Direct Repair - An enzyme recognizes an incorrect alteration in DNA structure and directly converts
it back to a correct structure.
Photolyase o Ex. Photolyaserepairs Thymine dimers by splitting them through a light-
required process called photoreactivation
O-6 alkylguaninealkyltransferase o O6-alkylguanine alkyltransferase repairs alkylated bases by transferring
the methyl or ethyl group from the G base to a cysteine side change within
the alkyltransferase protein which also inactivates alkyltransferase.
Base excision repair - A minor base damage is found and DNA glycosylasecleaves base-sugar bond
DNA glycosylase - This creates a site that’s missing a purine or pyridine (apurinic/apyrimidimic)
AP endonuclease - AP endonuclease makes a cut upstream of AP site
dRpase - dRpase (deoxyribophosphodiesterase) removes stretch of DNA
Polymerase - Polymerase synthesizes new DNA
Ligase - Ligase seals nick.
- This requires both strands to be homologous b/c this system exploits the antiparallel
complementarity to restore damaged DNA segments to initial undamaged state.
- GGR can correct lesions anywhere in the genome and is activated by stalled
Global genomic repair (GGR) replication forks
Q: Does the complex falls off - NER normally activated when replication fork stalls or is blocked
when TFIIH goes on? - A heterodimeric complex recognizes damaged base and recruits TF IIH
Diagram seems to show them - TFIIH is a transcription factor with XPB and XPD helicase activity
staying on - This 10 protein subunit unwinds DNa, stabilizes it using RPA (Single stranded
binding protein) and excises it 2-8 nucleotides from lesion
- Poly 5 comes in and synthesizes new strand, and ligase 1 fills in gap
o Ex. XPB and XPD are mutated in Xerodermapigmentosum so mutations accumulate
in cells and increases risk of skin cancer.
Transcription –coupled NER - Repairs transcribed regions
- When RNA polymerase is stalled, CSA and CSB recognize the sequence and recruit
- RNA polymerase, CSA, and CSB falls off and lets TFIIH conduct repair
o Ex. Cockayne syndrome has a stalled transcription complex with CSA and CSB
mutated and TFIIH can’t bind so the cells undergo apoptosis premature aging
Nov 8/11 – Lecture 7
Mismatch repair - Used to repair mismatches missed by the 3’-5’ exonuclease of DNA polymerase III
- Lack of this repair system associated with some hereditary forms of colon cancer
1. Mispairing causes a distortion
2. MutS recognizes a mismatched pair 3. MutH recognizes methylated parent strand and nicks GATC methylation site on
daughter strand (not methylated yet)
4. UVRa (helicase) binds to nick and unwind DNA and excise it
5. New strand is replaced
Q: I’m guessing the replacement
is by Pol V or pol 1?
Translesion synthesis (SOS - Bypasses lesions at stalled replication forks
mechanism) - Pol 3 is stalled at site of damage but DNA continues to be unwound ahead of
- RecA joins single stranded binding proteins to form stiff filament, acts as a signal to
several genes to encode Pol 5.
- Pol 3 is released from its clamp when a ubiquitin molecule or PCNA (proliferating
cell nuclear antigen) is added to change conformation to let Pol 5 enter because it
has a larger active site.
- Pol 5 adds a few bases (can’t add too many – it’s error prone) before falling off and
being replaced by Pol 3.
Double stranded break repair - Caused by X-rays or free radicals
1. Error-prone method:
- KU80 and KU70 are part of a protein complex that recognize one end of the double
strand, they serve to prevent further damage and recruit other proteins that rim the
strand to generate 5’ P and 3’ OH ends required for ligation.
- These proteins recognize both ends, trim and bring together and ligates along with
DNA ligase 4
2. Error-free method:
- Occurs during mitosis
- Proteins bind to broken end and 5’ ends are trimmed by endonuclease to expose a
single stranded region
- RecA/Rad 51 forms long filaments with ssDNA and searches for sister chromatid
for homologous sequence
- The single strand invades its homolog and uses its complemetntarity to synthesize
- Ligation fixes any gaps
1. Cancer cells
- Cancer cells have a faster division, metabolic rate, can invade new territories, and
doesn’t stop growing in response to neighbouring cells (that’s why they pile up on
- Gain of function dominant mutations
- Proteins encoded by oncogenes are usually activated in tumor cells
- Mutation needs to be in only 1 allele and it’ll be dominant and suppress the other
- Proto-oncogenes are the unmutated form.
o Ex. A wt protein that promotes cell cycle progression will be cancerous if an
oncogene is there for upregulation
o A wt protein that stops apoptosis will never let cells die if oncogenes affect it.
** Oncogenes make proteins more active!
Tumor Suppressor genes - Encode proteins whose loss of activity can contribute to a cancerous state
- Loss of function – recessive mutations, so for cancer to develop, mutation must be in
o Ex. BRCA1 and BRCA2 for breast cancer
o Ex. A wt protein that inhibits cell cycle progression will activate progression when
tumor suppressor mutations stop wt from working.
** Tumor suppressor makes inhibitors not inhibit.
Proto-oncogenes - Proteins that induce (+vely control) the cell cycle
o Ex growth factor receptors
o Signal transduction proteins
o Transcription regulators - Can also inhibit (-vely control) the apoptotic pathway
o Ex. Ras mutation can cause a Rasoncoprotein to be blocked so it’s always
on and Ras is constantly produced.
2. Chromosome numerical
Polyploidy - Extra complete sets of chromosomes (e.g. 3n triploidy)
- Found in some spontaneously aborted human foetuses
- Wrong number of chromosomes
- Trisomy (2n + 1) is the presence of an extra copy of one specific chromosome
o Other sex chromosome aneuploidies associated with viability are XXY
(Klinefelter syndrome) and XYY
o Most human autosomal trisomies are embryonic lethals but Ts13 (Patau),
18 (Edwards) and 21 (Down) survive in many cases
- Monosomy (2n -1) the absence of one copy of a specific chromosome
- Origins of aneuploidy most frequently lie in meiotic nondisjunction, during
metaphase as separation occurs or caused by defect in spindle/ centromere.
o There are no viable human monosomies except that of the X chromosome
(X0 = turner)
Q: It makes sense for there to be - Abnormalities and lethality associated with monosomy and trisomy suggest tight
no dosage compensation for
monosomy but why shouldn’t dosage control with no dosage compensation
there be dosage compensation for
XO Turner Syndrome - Short, low hairline, wide neck due to fold of skin, infertile, brown spots (nevi)
- Has undeveloped female characteristics
- Has slightly feminized features such as female type pubic hair, breast development,
poor bear growth, and small testes
Down Syndrome - An extra chromosome 21 arises from translocations which can be transmitted from
parent to child
- Nondisjunction can happen during either mitosis or meiosis
o First division nondisjunction: In the 2 division, one cell is empty and one
cndl is full resulting in 2 (n+1) cells and 2 (n-1) or empty cells at the end
o 2 Cell nondisjunction: will end up with 2 normal cells and a n+1 and n-1
Nov 9/2011 – Lecture 8
1. Chromosomal structural
Chromosome structure - Chromosomes are single dsDNA molecules
- Rearrangements can occur when there are 2 or more double- stranded breaks
- Acentric chromosomes: lacking centromere, it is lost and won’t be dragged to
either pole at anaphase – it is not inherited
- Dicentric : 2 centromeres, pulled in opposite directions and usually break. These
chromosomes will be pulled to opposite poles, forming an anaphase bridge which
will not be incorporated into either progeny cell
Unbalanced rearrangement 1. Deletions
- Unbalanced rearrangements changes gene dosage
- Deletions can be within a gene (intragenic) or multigenic (many genes)
- Can occur through loss or crossing over at repetitive sequences – where a piece
loops out and is lost
- Cause loss of heterozygosity
Balanced Rearrangements 2. Duplications
- Can be within the same chromosome or different chromosome, when a gene is
Q: What is the difference between - Can occur when 2 homologs break and rejoin incorrectly, giving a copy from one crossing over and breakage & homolog to another, creating a duplicate
rejoin for duplications? (ref chart) - Or through crossing over, duplication can occur
- Genes change order but this doesn’t remove or duplicate DNA
- Genes break and flip or loop and crossover at the incorrect and opposite ends.
2. Reciprocal Traslocations
- 2 non homologous chromosomes break and rejoin
- When another chromosome piece is relocated to your chromosome
- When a breakage occurs and the broken strand join