PHAR 303 Lecture Notes - Lecture 8: Polycyclic Aromatic Hydrocarbon, Dna Mismatch Repair, Dna Adduct
11 May 2018
School
Department
Course
Professor
Lecture 8 (Feb. 1st) Genetic Toxicology
The effects of any chemical & physical agents on DNA & on the genetic processes of living cells
DNA Damage due to environmental factors and normal processes inside the cell, occurs at a rate of 10,000
to 1 million molecular lesions per cell per day
• Abasic sites
• Abnormal bases – sometimes bases can be modified
• Base adducts
• ss-breaks, ds – breaks - Some of anticancer drugs cause ds breaks (good)
• Thymine dimers – 2 T linked
• Chemical mutagens
• Interstand crosslinks – crosslinks between 2 strands , chromosomal aberrations
• AG, TC mismatch
• Base insertions, deletions
• Base
Mutagen – induces gene mutations
Polycyclic aromatic hydrocarbon (carcinogenicity) lead to a lot of genetic lesions & cancer
Genetic alterations on different types of cells has different consequences:
• Somatic cells Cell death
Alter cell Senescence/ Aging
Cancer
• Germ cells Cell death (could try to kill the cell)
Effects can be transmitted on Offsprings (transgenerational)
DNA Damage
Physical & chemical agents can cause DNA damage
• Alkylation (covalently bind) by alkylating agents that are usually electron deficient – look for an
electron in the cell & if they covalently bind to DNA, they could cause altered bases, base adducts &
lead to mutations (remove purine & give an apyrimidic, apurinic site). They can go wherever they find
an electron
• Radicals formations ( BrdU + light, X-Rays)
• Single-strand breaks (X-Rays, UV) & ds-breaks (ionizing radiation)
• Phosphotriesters (by alkylating agents) – modify the phosphate backbone & base damage (by X-Ray)
• Adducts of bulky molecule (Benzoapyrene – enters inside the DNA)
• Interstrand cross-links (Bifunctional alkylating agents) &
• Intrastrand cross-links (polyfunctional alkylating agents)
• DNA protein cross-links (X-Rays, polyfunctional alkylating agents
Clastogens – chromosomal aberations
• Possible to have fluorescent probes tagged to chromosomes and give them a different colors & we can
see if there are missing parts or misarrangemements
• Misarrangements could cause problems when cells divide
The effects of mutagens
• A small rearrangement of bp on the DNA strand either due to errors in DNA replication or from outside
damage to DNA
• A mutagen is an agent which induces/increases the frequency of mutations, in some cases this occur at
DNA “hot spots” – spots are not random, specific spots are more likely to be mutagenized
• All kinds of mutations affecting one hot spot in particular, not totally random – there are hot spots more
susceptible to mutations
• Change end product of gene transcribed
• Base damage – may cause mispairing (point mutations)
o GC exposed to EMS (electrophilic) modify the Oxygen (not free anymore) in EMS and gets attached
to G (EMS-G), which cannot pair with C anymore.
o G mispairs with T ( point mutations –bases mispair)&change sequence that will be read
• Insertions & deletions change the reading frame – transcripts produced have different codons & aa.
which will change the end product.
• Mutated DNA (depending on where it is mutated) could lead to different effects:
o No protein
find more resources at oneclass.com
find more resources at oneclass.com
o Too much proteins
o Shortened proteins – stop codons
o Protein with mutation – altered amino acid
o Normal amount of protein
Some amino acids changes do not matter and others changes the whole product
Depending on which bases are in a row (1st, 2nd, etc), will have different consequences bc you will code for
different amino acids.
Trinucleotide repeats (GAA) – could result in Friedreich’s Ataxia
Some deletions could give cystic fibrosis
Usually, diseases when you substitute non-polar for a polar amino acid
DNA Repair
We rely on DNA Repair to remove adducts, mutations and correct lesions either spontaneous or induced by
chemicals.
• Mismatch Repair
o Base mispairs
o Similar to nucleotide excision repair, but not the same factors involved
o Incorrect base detected & nicking by endonucleases, digestion by exonuclease.
o Resynthesis of good base by DNA polymerase & ligation by DNA ligase
Direct Reversal Mismatch Repair: O6 methylguanine DNA methyltransferase (MGMT)
- Guanine G can have a CH3 methyl group attached to it, modifying the base– specific lesion caused by
some kinds of carcinogens
- Enzyme MGMT has an active Cys that removes the methyl group MGMT-Cys-Me (covalently bound) -
do not have to redo the while mismatch repair synthesis and cut a whole bubble.
- Enzyme determine susceptibility to some kind of cancers – liver cancer.
- When enzyme binds CH3 group, it gets degraded and has to be resynthesized.
• Nucleotide Excision Repair – in general it removes bulke adducts
o Base adducts & modifications
o Crosslinks
o Recognition & unwind : Damaged base triggers signals & protein complex forms a denaturation
bubble
o Endonuclease cleaves ~20 nucleotides (ss) from the bubble & exonuclease digests them
o DNA polymerase – re synthesis of complementary strand
o DNA ligase – ligation
o Processing enzymes, TF, XPA,C – a lot of factors involved
o NER deficiency (one factor not present) Xeroderma Pigmentosum
▪ Sensitivity to sunlight & increase risk of cancer.
Preferential repair of the transcribed strand
• XP cells vs. Normal cells – look at how much damage is remaining in transcribed & non transcribed
strand.
• Non transcribed strand – at 6 hours or 10 hours, a lot of damage is still remaining, practically the same
so it proves it wasn’t repaired (NER deficiency)
• Transcribed strand – from 6 to 10 hours, there is a lot less damage remaining, meaning that DNA can be
repaired & NER intact.
Mechanisms to repair more quickly the ones that are more critical.
• Base Excision Repair
o Base modifications
o Single strand breaks
o Types of components involved in DNA repair process :
▪ DNA glycosilase – cleavage of glycosidic bond in order to remove wrong base gives an
empty abasic site
▪ AP endonuclease – phosphodiester bond cleavage (cut backbone)
▪ Addition of new base + phosphate backbone by DNA polymerase
▪ Ligation by DNA ligase
• Recombination Repair
o Single/double strand breaks
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
The effects of any chemical & physical agents on dna & on the genetic processes of living cells. Interstand crosslinks crosslinks between 2 strands , chromosomal aberrations. Polycyclic aromatic hydrocarbon (carcinogenicity) lead to a lot of genetic lesions & cancer. Clastogens chromosomal aberations: possible to have fluorescent probes tagged to chromosomes and give them a different colors & we can. Intrastrand cross-links (polyfunctional alkylating agents) see if there are missing parts or misarrangemements: misarrangements could cause problems when cells divide. Some amino acids changes do not matter and others changes the whole product. Depending on which bases are in a row (1st, 2nd, etc), will have different consequences bc you will code for different amino acids. Trinucleotide repeats (gaa) could result in friedreich"s ataxia. Usually, diseases when you substitute non-polar for a polar amino acid. Direct reversal mismatch repair: o6 methylguanine dna methyltransferase (mgmt)
