Mutagenesis

What makes mismatch repair different form the other forms of excision repair?

1. Mismatch repair occurs during DNA replication. They are coupled processes. 2. It has to be able to determine which strand is the newly synthesized DNA and which strand is not. There has to be a memory. How? MutS protein has that memory because it can remember which strand it traveled on. In bacteria, the parent strand is methylated whereas the daughter is not.

Describe neer versions of the Ames test

1. Nature of initial mutation can be varied- not just which compound is the mutagen but clue as to how it might act. 2. Test compounds can be pre0incubtaed with liver extract prior to testing on bacteria- some compounds are not mutagenic until activated by body.

Describe the general steps involved in the excision repair pathway

1. Recognize the damaged site 2. Cleave/ the single strand where the damage is 3. Excise out that single strand creating abasic site 4. DNA polymerase enters and synthesizes 5. DNA ligase seals

What types of mutations exist?

1. Substition: Switch from one base to another; can be reverted. 2. Deletion: remove bases ; hard to revert 3. Insertion: add bases usually through transposable elements

Base excision repair process

1. The single-stranded mutation distorts the DNA and is recognized by machinery (DNA glycolyase) 2. AP endonuclease cleaves out the mutated base leaving an abasic site behind (basic pyridine) 3. DNA polymerase fills in gap using the template strand 4. DNA ligase seals it

Describe the process of homologous repair

1. dsDNA break is recognized 2. Resection of both dsDNA 3. Strand invasion in search for homology 4. DNA synthesis and ligation 5. Resolve the holiday junction

Describe the process of non-homologous end joining

1. recognition 2. resection 3. DNA synthesis and ligation 4. stick together the ends.

silent mutation

A mutation that changes a single nucleotide, but does not change the amino acid created. note- although the amino acid is the same, the secondary structure of the protein can change. also called synonymous mutations

nonsense mutation

A mutation that changes an amino acid codon to one of the three stop codons, resulting in a shorter and usually nonfunctional protein.

What is an auxotroph?

An auxotroph is a mutant organism

Define mutagen

Any agent (chemical or radiation) that causes mutations

Define mutation

Any inherited alteration in the genome (either DNA or RNA)

Compare and contrast reversion and suppression

Both are methods that restore the wt phenotype. the difference is that in a true reversion you change the exact mutation back to the original base. in suppression you have a second mutation that compensates for the defect of the first mutation.

Compare and contrast transition and transvehrsion mutations

Both are types of substitution mutations where one base is being switched for another. In a transition, you swap a pyrimidine for a pyramidine or a purine for a purine. In transversion, you swap a pyramidine for a purine or vice versa. pyrimidines are C,T, and U. purines are A and G.

Hereditary non-polyposis colon cancer

Caused by defects in this mismatch repair machinery. As a result of the defect, there is no way to clear out the repeats created by the polymerase slippage.

What are the three general DNA repair strategies?

Direct repair, excision repair, and other (double-stranded break repair and transleson synthesis bypasses)

How does the polymerase deal/repair double stranded DNA breaks?

Double stranded DNA breaks are a unique problem in that you no longer have a "template" you can use to synthesize. Repair comes in one of two forms- either end-joining or recombination.

True or False. The location of a mutation along a genome is random.

False! With genes, there are "hot spots" for mutations. A hot spot is defined as a sequence within the genome where it is 100X more likely that you will have a mutation occur. This is because they are heavily filled with nucleotide repeats which confuses the polymerase causing it to slip off and then reattach leading to either an insertion or deletion based on location of reattachment.

Describe the process of screening for a mutant of interest

Have a plate with your cells. Replica plate- literally copy your colonies once they have grown onto another plate with identical media. expose the replica plate to UV radiation. This will kill your mutants that are hypersensitive to the UV radiation. compare the replica plate to the original plate identify which colonies died- those are your mutants of interest. pick them from the original plate. in selection, your mutants of interest die.

Describe the anti-viral use of APOBEC

If a virus infects a cell with APOBEC, the viral genome acquires so many mutations that the virus dies.

Compare end-joining and homologous recombination

In homologous recombination, the homologous chromosome is used as a template. You base pair with that second chromosome and it serves as the repair copy. Problems with homologous recombination technique are that you can have chromosomal arm swaps which can lead to loss of heterozygosity. Another problem is if you use the wrong copy and now you get repeats. In end-joining, you literally just stick together the two ends like glue. Often leads to mutations and loss of DNA bur bringing them back together is better than not doing anything at all. Another problem you can have is if there are more than two strands, you can glue together the wrong strands.

When you have an insertion or deletion, usually how many base pairs is it?

It varies. It can range from 1 base pair to mega bases. If one or two you get a frameshift mutation.

Describe the relationship between APOBEC and cancer

Many cancers overexposes APOBECs or have mutations that result in APOBEC-like activity.

How does radiation damage the DNA?

Radiation can either directly transfer the energy to DNA causing a chemical reaction or can indirectly generate reactive molecules called radicals that then damage the DNA. NOte- visible light is not energetic enough to cause this damage. This damage requires X rays or gamma rays. The rays will either cause break in DNA or they form radicals in the water.

Mismatch repair process

Same as base excision and nucleotide excision. It will recognize the distortion, cleave, synthesize, ligate.

Compare induced and spontaneous mutations

Spontaneous mutations refer to mutations that arise due to errors made during the process of DNA replication that are not fixed by the DNA polymerase. Induced mutations refer to mutations that result from the introduction of an environmental mutagen. With induced mutation, you are treating the cell with an environmental mutagen.

Describe APOBEC

Stands for apolipoprotein B mRNA editing enzyme, catalytic polypeptide Converts C to U so you get GU mismatch.

Why do we study mutants?

Studying mutants allows us to better understand human disease (look at the disease and try to identify the genetic component behind it) and cellular processes (figure out how things work in a cell).

Describe the various types of suppressions

Suppression is considered to be intragenic if the second mutation occurs on a site within the same gene. Suppression is considered to be extragenic if the second mutation occurs on a site at a distinct gene. with intragenic you may have a second mutation that occurs in order to enhance the stability of the now very unstable protein. by enhancing its stability, the protein can regain function-restore phenotype. extragenic may occur because those two gene products normally associate with each other- restore phenotype because maybe the second mutation allows the protein to associate with the mutant protein despite the mutation.

Describe the SOS response

There are enzymes in the body that are responsible for repairing damaged DNA. These DNA repair enzymes are not constitutively active. When there is damage, they are activated. They are always active but at a low level. When there is damage - SOS signal - huge increase in activity. The reason they are always active but at a low level is because of just mistakes from DNA replication. They are always active to fix spontaneous mutations. You want to spike that activity if you now have a induced mutation. rate of spontaneous mutation is 1 per second so that is very high. the reason we don't have crazy mutation rates is because these DNA damage repair enzymes are working at that low constitutive level. Our body repairs these lesions 10^17 per hour.

Describe the various classes of mutagens

There are four classes of chemical mutagens and then there are also non-chemical mutagens (aka radiation)

List the various types of excision repair pathways

There are three basic types: 1. Base excision repair (short) 2. Nucleotide excision repair (longer) 3. Mismatch Repair

Intercalating agents

These are flat/planar hydrophobic compounds that stack in between bases in DNA. Leads to errors by DNA polymerase because you insert and now need another pair on the other side so insert ambiguously. This leads to framshifts of one base.

Base analogs mispair

This is a type of chemical mutagen that involves a base analog which is a chemical variant of a neural base that is incorporated into the DNA and causes misfiring. Example- 5-bromouracil which is an analog of thymine but can convert to an enol (electron withdrawing) and base pair with guanine. You get ambigiousity in base pairing.

What is the Ames test used for?

To test is a compound is a mutagen. He tested whether a compound could reverse the histidine synthase inactivating mutation in salmonella enteric typhimurium. works by treating the bacteria with the mutagen and they grow you know that they reverted back- a mutation has occurred. The mutagenic properties of a test sample are measured by its ability to induce his + revertants of a salmonella enteric typhimuirm his-strain. can identify compounds that induce high frequency reversion and those that are mutagenic and carcinogenic.

Identify some environmental alkylating agents

Turns out the the majority of chemical carcinogens are likely to be alkylating agents. Some of these, however, alone are not toxic. They have to be activated by a compound in the body to become active. For example, aflatoxin B1 is a carcinogenic compound made by Aspergillus fungus that is found in cereals and in peanuts but is not activated until reaches the liver. The liver turns it into -oxide so that it can exit with the bile. problem is that as a hypoxide it also interest with DNA. when it interacts with DNA- promotes cancer development

Deaminating agents

Type of chemical mutagen Promotes loss of an amine group from DNA base - that base is now subject to mismatch. For example, you can convert C to U which then converts CAA to UAA- stop codon- no longer make LDL. Uracil also binds to Adenine so convert CG base pair to an AT base pair. You can also reanimate A which turns it into inosine. I binds to G so change AT to GC. Deanimate 5-methyl cytosine and get thymine.

Alkylating agents

Type of chemical mutagen that causes the addition of groups onto electron-rich centers of bases or phosphates. These electron rich centers are nucleiophils on the nitrogenous bases. YOU ARE ADDING AN ADDUCT. Creates a phosphotriester. example of one- EMS (ethyl methane sulfonate), can cause mispairing, base loss (dispurination), or strand crosslink formation.

Describe a common example of how UV impacts DNA

UV radiation causes cross linking between tow adjacent thymines who form a cyclobutane. (pyrimidine dimer). the dimer then blocks replication and transcription. Can lead to error prone repair and/or DNA breaks. This dimer can be photo reversed.Treatment with visible light fixes and takes it back.

Nucleotide excision repair process

Unlike with base excision repair, here you are removing many bases (12 or so). Uses the uvrABC nuclease system. 1. distorted DNA is recognized. 2. DNA polymerase 1 fixes 3. Ligase seals Overall, same thing as base excision repair but larger number of bases Another difference is tat there is transcription-coupled nucleotide excision repair where this pathway is coupled with transcriptionally active genes through specific transcription factor recruitment.

Describe the specificity of excision repair pathway

Unlike with the direct reversal pathways, in this case, it is a general path that can be used for a variety of mistakes.

Describe the process of selection for a mutant of interest

Use antibiotic resistance. You have a plate of cells. You treat with your antibiotic such that only the colonies that are reisstant will survive. Those that survive have your mutation of interest. In selection, the mutants survive.

Describe how the Ames test works.

Use bacteria than generalize to humans since mutagens of bacterial DNA are also mutagens of human DNA. - use bacteria as model organism. effects of the mutagen are similar across species. in the original study, he had bacteria that were mutated of the histidine synthase enzyme so they could not synthesize histidine. he found that even int he absence of histidine (non-supplemented media) some colonies grew- those colonies had reverted to the WT genotype. the idea is that you add mutagen- colonies that grow are those that revert back to WT genotype. H He has developed a series of mutagens that are now available to be used. Test to see if a compound is a mutagen Use a bacteria (either salmonella or E. coli) that carry a mutation. If using salmonella the mutation is histidine. treat with mutagen and that causes reversion- that is how you know that the chemical causes mutation- if it can grow - then the chemical is in fact a mutant! YOU IDENTIFY MUTAGENS THAT CAUSE REVERSION. THIS IS HOW YOU CAN DETERMINE THE MUTAGENIC CAPABILITY OF THE COMPOUND.

In order to study mutants, we have to first obtain them. How can we?

We can obtain spontaneous mutants from patients or disease isolates. This is very slow and rare. Alternatively, we can induce the mutation in a lab on an experimental system with identified/known mutagens.

You have treated cells with mutagen, how can you now isolate the mutagen?

You can use screening or selection using either

Describe the process of complementation analysis

You get two cells. One cell has good copy of gene A and bad gene B. Other cell has bad gene A and good gene B. Fuse the two together- heterokaryon. Fusion- mutant phenotype- same gene Fusion - rescue phenotype - different genes

When do you use complementation analysis?

You have two mutations that yield the same phenotype. Want to know whether or not those two mutations are on the same or different gene. Also used to develop recombination maps.

List the four classes of chemical mutagens

base analogs, deaminating, alkylating agents, intercalating agents.

How does the cell determine whether to use non-homologous end joining or homologous recombination?

depends on the stage of cell cycle it is in. If it is G1, homologous recombination is suppressed so it would have to go through non-homologous end joining.

What is a deleterious mutation? How does the body deal with these types of mutations?

individual who carries the mutation has a lower fitness (produces lower successful descendants) The body deals with this by removing them- the body is very good at ensuring accurate copying of genome.

frameshift mutation

mutation that shifts the "reading" frame of the genetic message by inserting or deleting a nucleotide. this is when you add or delete one or two bases so the reading frame changes.

What impact does the mismatch repair machinery have on polymerase slippage?

polymerase slippage often leads to the insertion/ production of repeats. Mismatch repair machinery can detect that repeat and clear it.

Describe direct repair

this is a direct reversal of the damage. 1. UV radiation causes the thymine dimer to form (cross link) but you use photo reversal of the thymine dimer and break it bro the original two mono thymines. This is the photo reactivation of UV damage (photolyase) 2. Ada protein can carry out dealkylation- ada protein will capture the methyl group that was added during the alkylation. Interestingly, unlike other enzymes, this is not an enzyme because it cannot turnover. When the ada protein captures the methyl group, it is never used again. 3. Ligation of a single stranded DNA break using DNA ligase

Describe how we can use genetics to understand cellular/metabolic pathways

we can use auxotrophs! Lets say you have pathway X: A - B - C E1: A -B E2: B-C If you have a mutant of E1 you have to insert B to restore product. If you insert A, no product will form. If you have a mutant of E2, need to insert C. Inserting B will still yield no product. You can determine order of these cellular pathways. Additionally/ alternatively, You can add intermediates and determine which enzyme is mutated. will only rescue the phenotype if the distal intermediate is added.

Missence mutation

when a point mutation results in substituting one amino acid for another