Ensherah Mokheemer. Mamoun Ahram. 1 P a g e
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1 6 Ensherah Mokheemer - Mamoun Ahram 1 P a g e
2 The Doctor forgot to mention this topic in lecture 5 in section 2 so he mentioned it in lecture 6. Mutagens and Carcinogens: In food industry a lot of chemicals are being used, some of these chemicals can cause mutations and so they are called Mutagens, and others can cause cancer and they are called Carcinogens. Mutagenicity and carcinogenicity are correlated; 90% of Mutagens are also carcinogens. The question is how do we know if a certain chemical is mutagenic? A bacteria-based test called Ames test can test for mutagenicity. A little History: In previous tests animals (Like rats) were used to test the mutagenicity of chemicals, but in animals it takes a long time to develop cancer, and so Bruce Ames decided to use bacteria in his experiments to test the mutagenicity in a test called Ames Test. The bacteria which he used was: Salmonella typhimurium. But it wasn t the normal Salmonella typhimurium he was smart enough to use a mutant strain of the bacteria. These mutant strains had mutations in: 1-Mutations in the genes responsible for lipopolysaccharide synthesis, making the cell wall of the bacteria more permeable to chemicals. 2- A mutation that inactivates a DNA repair system, this mutation will result in high frequency of spontaneous mutations. So, the addition of chemicals will lead to induced mutations in addition to the spontaneous ones which resulted from the defective DNA repair system. 3-The normal strain of this bacteria can live in the absence of the amino acid Histidine, because it has the necessary system to synthesize Histidine. The mutant strain of the bacteria which is used in Ames test cannot live in the absence of Histidine, that is because a mutation has occurred in a gene that encodes one of the enzymes necessary for histidine biosynthesis. 2 P a g e
3 The Ames Test: To test whether a certain chemical is mutagenic, the chemical is added to the mutated strain (cannot live without Histidine, has defective DNA repair system, does not synthesize lipopolysaccharide so it s cell wall is permeable to chemicals), the idea he used to reach a conclusion was Reversion mutation. What is reversion mutation? -Basically, what occurs in normal mutations is that we have a gene in the normal form and when a mutation occurs it will be converted to the mutated form. But in the reversion mutation when a mutation occurs the mutated form will be converted to the normal form. In the test, if the mutant strain of the bacteria is back to the normal strain after adding the chemical, that means the chemical we used is mutagenic because it was able to mutate the mutated genes back to the normal ones. This means that the bacteria which resulted after adding the chemical will be able to synthesize lipopolysaccharide, can live without histidine and has an active DNA repair system. -This type of mutation is known as reversion, because this second mutation returns the mutant to the normal genotype. - This reversion can also happen spontaneously or as the result of a mutagen. The conclusion: if a chemical is mutagenic, it will cause random mutations all over the place, including, probably, the same mutated sites (the three mentioned above) fixing them so it becomes normal. Let me remind you that we still haven t talked about how the experiment is done. -First, we have 2 groups the control group which is colonies of mutated bacteria but without adding the chemical the control group is for comparison and the experimental group which is the mutated bacteria with the adding of the chemical. We put them in a medium with minimal amount of histidine 3 P a g e
4 Remember that: Mutated strains cannot survive without histidine, so it will not grow on the plate, however the normal strain can survive without histidine, so it will grow on the plate. - What happens is that in the control group there is only spontaneous mutations because of the defective DNA repair system, some of these mutations will result in reversion mutation (low probability).as for the experimental group,if the chemical is mutagenic, spontaneous mutations occur but also there will be many induced mutations because of the addition of the chemical, so there is a higher probability of having reversion mutations because we have both induced and spontaneous mutations. -Because of the minimal amount of histidine only the bacteria which have the normal gene (the mutated bacteria which was reversely mutated back to normal) will grow on the plate, because it has the normal gene which enables it to synthesize histidine. In the control group the number of the growing bacteria will be much less than the number in the experimental group, if a mutagenic chemical was added. 4 P a g e
5 What is the ratio of the number of colonies between the control and the experimental groups to consider a chemical a mutagenic? A compound must increase the number of colonies grown in the absence of histidine by double compared to cells grown in the absence of the compound. Let s take this example: (the numbers are from the doctor s imagination do not take them seriously) water Motor oil Alcohol Drug X شيبس أبو 5 قروش Water is the control Group (because there is no chemical added) we have 10 colonies, But why? Due to spontaneous mutations. Motor oil and Alcohol and the chips are mutagenic because the number of colonies increased by more than double (5x,4x,20x) respectively. Drug X is not a mutagenic chemical because the increase in number of colonies was less than a double. -please note that every carcinogenic is mutagenic, but not every mutagenic is carcinogenic. The role of liver enzymes: -In mammals, chemicals are normally detoxified by liver enzymes. -liver enzyme is a factor, present in humans and animals-based experiments but not in the bacteria-based ones. -Ames incorporated mammalian liver enzymes in his bacterial test system to consider the human liver enzymes effect on mutagenicity. -In some cases, liver enzymes can create a toxic or mutagenic compound from a substance that was originally safe. 5 P a g e
6 Example: Condition water Motor Oil Alcohol Drug X شيبس أبو 5 قروش Without liver enzymes With liver enzymes Water is the control group, we noticed that liver enzymes did not affect the mutagenicity. Motor oil was mutagenic (50), but after adding the liver enzyme it has lost its mutagenicity, because the increase has become less than double (we compare 18 to 12). In alcohol liver enzymes did not affect mutagenicity. (from 43 to 50) isn t a big difference Drug X was not mutagenic, but after adding the liver enzymes it became mutagenic (compare 35 to 12) the increase is more than double. As for the chips the mutagenicity has increased as well. NOTE: if we want to compare the mutagenicity, we compare according to the water effect. But if we want to compare the effect of the liver enzymes, we compare according to the condition before adding the liver enzymes. Three parent babies (controversial issue): 6 P a g e
7 Mitochondria is inherited from mothers; so defective mitochondria can be transmitted only from a female to her baby. In 2010 a couple (female has a defective mitochondria) went to scientists to have a healthy child with healthy mitochondria, and they came up with a great idea; all we need to do is to take an egg with healthy mitochondria from a donor, remove its nucleus and put the mother s nucleus instead, then we do IVF (in vitro fertilization) by the father s sperm. The resulting zygote has three types of DNA; nuclear DNA from father, nuclear DNA from mother and mitochondrial DNA from the donor ovum, the born baby was healthy with healthy mitochondria. The first Jordanian couple to try this technique did so in 2016, in Mexico, and they got a healthy baby with three types of DNA. Do you think its ethical? Think about it. Now let s continue with DNA repair mechanism. As we mentioned before there are many mechanisms for DNA repair, we talked about some and we will continue about excision pathways (specific excision pathways, Mismatch repair), and we will talk about Translesion DNA synthesis, Recombinational repair. Specific excision pathways. DNA glycosylase repair pathway. 7 P a g e
8 -Excision pathways: they cut the DNA at the site of lesion, remove this DNA, and then DNA polymerase comes and fills the gap. Then ligase will do its function. -Recall that in bacteria there are 5 types of DNA polymerase (1, 2, 3, 4, and 5). In humans there is polymerase (alpha, beta, lambda ). Some of the DNA polymerases are involved in repairing, and these are the polymerases which fill the gap. -The presence of Uracil in DNA is not normal, so what happens in specific excision pathways is that, an enzyme called DNA glycosylase cleaves the N-glyosidic bond; the bond between the sugar and the base of the damaged bases, producing an apurinic or apyrimidinic site, both are called AP sites, then an endonuclease comes and removes the phosphate and sugar and finally DNA polymerase fills the gap. In the case of uracil in DNA, DNA glycosylase removes the uracil (by cleaving the bond between the sugar and uracil) generating an apyrimidinic site then an endonuclease comes and removes the phosphate and sugar, DNA polymerase comes and puts the right nucleotide which in this case is C (uracil was bound to Guanine) and DNA ligase reforms the phosphodiester bond. 8 P a g e
9 - Uracil residues result from the spontaneous deamination of cytosine and can lead to a C T transition if unrepaired and enters the replication process. Post replication repair: In Prokaryotes, there is a system called Mismatch repair system, it recognizes the mismatched base pairs. In the case of mismatched base pairs, how is it determined which base is incorrect? If you have (G/T pairing) how is it determined whether G is the incorrect one or T? 9 P a g e
10 The system consists of MUT proteins (S, L, H), these proteins detect a certain lesion after replication, Mut S Binds to the mismatch site and forms a complex with Mut L, the formation of this complex recruits Mut H which binds to a hemi methylated site, The (Mut S, Mut L) complex interacts with Mut H by DNA looping mechanism, then Mut H makes a cut only in the nonmethylated strand, then an exonuclease hydrolyses the nonmethylated strand from the cleavage site to a site a little after the mismatch site, the gap in the daughter strand is filled by DNA polymerase 3 and DNA ligase re-forms the phosphodiester bonds. The interaction between the complex (Mut S and Mut L) with Mut H by DNA looping (the DNA becomes like a loop) 10 P a g e
11 hemi methylated site: is where only one of the two (complementary) strands is methylated. After replication DNA is methylated following replication by the enzyme adenine methylase (pay attention that the old strand is already methylated and the enzyme needs to methylate the new strand only). However, it takes the adenine methylase several minutes to methylate the newly synthesized DNA. The mismatch repair system in bacteria takes advantage of this delay to repair mismatches in the newly synthesized strand. After repairing, the newly synthesised strand is methylated. - The mismatch repair system has also been characterized in humans. It works the same way as the bacteria s. Two of the proteins, hmsh2 and hmlh1, are very similar to their bacterial counterparts, Mut S and Mut L, respectively. -A mutation in the mismatch repair system in humans is related to some types of colon cancer like hereditary nonpolyposis colon cancer (HNPCC) Lynch syndrome. -Individuals with mutations in the mismatch repair system will have lots of colon tumours and these tumours will develop to colon cancer. - 15% of colon cancer cases. -50% caused by mutation in MSH and most of the remaining is caused by mutated MLH. 11 P a g e
12 -It s hereditary so it can be transmitted from parents to children. Now we will talk about another repair system which is called Translesion. -DNA replication in the cell is the decision-making time, if there is a lesion during the replication the cell will have to make a choice either to repair and continue replication or stop and commit suicide. The Translesion system allows the cell to continue DNA synthesis and to bypass the lesion by adding any type of nucleotide at that lesion to continue the replication process. Pay attention that it did not repair the lesion it only allowed the DNA to continue replication even with the presence of that lesion. -This system may produce errors while working because it puts any type of nucleotide at the site of the lesion and it may result in a mutation. 12 P a g e
13 Recombinational repair: -Sometimes UV light can break the phosphodiester bond leading to a double strand break, the whole DNA is broken. The cell has two systems to repair the break which are: 1-Non-homologous end joining: Which fixes DNA but creates mutations. what happens is that the broken ends are directly ligated without the need for a homologous template 2-Homologous repair with the undamaged chromosome. This involves a protein called Rad51. -Remember that our cells are diploid meaning we have 2 copies of the chromosome, so if one chromosome is broken the homologous repair will use the other as a template to fix it and fill the gap. Breast Cancer -It may be hereditary. -BRCA1 and BRCA2 if they are mutated they will increase the risk of breast cancer in both males and females. Mutations in BRCA1 account for 2% of all breast cancers and, at most, 5% of ovarian cancer. - BRCA1 activates homologous recombination repair of DNA doublestranded breaks by recruiting Rad51 to the ssdna. Which means it is involved in DNA repairing so a mutation in this gene will affect Homologous recombination repair system, leading to an increase in the mutation thus increasing the chance of developing breast cancer. 13 P a g e
14 - BRCA1 is also involved in transcription and transcription-coupled DNA repair. Some controversial issues: -Bacteria-like humans- have immunity, when a virus tries to infect bacteria, it activates its immune system and degrades the viral DNA. -CRISPR Cas 9 is the name of the system involved in bacterial immunity, this system degrades the viral DNA, then it takes some of the Viral DNA and incorporates it in the genetic system of the bacteria, making a memory for the viral DNA, in case the bacteria are exposed to it again. -Scientists are trying to use the Cas 9 system to fix mutated genes, by manipulating the system to make it able to remove the mutated DNA sequence and replace it with a normal DNA sequence; exchange the genetic material. -Some scientists suggest using Cas 9 system to replace the genes in a fertilized egg in the future, DO YOU THINNK IT S ETHICAL?? The End Sorry for any mistakes Good Luck 14 P a g e
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