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Flashcards in DNA Mutation and Repair Overview Deck (29):
1

How did we start the lecture

There is a constant battle going on with DNA repair mechanisms and the natural environment where the DNA gets damaged due to ultraviolet rays and other factors. Some DNA damage is required for evolution but that is counter to the goals of a physician which is to maintain a healthy population

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How is damage to the DNA caused

Damage is routine. It is caused by physical, chemical factors as well as the innate errors made by the DNA replication machinery.

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Define mutation

Mutation is defined as the unrepaired damage to the DNA that becomes part of the genome.

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Effect of mutation on somatic cells and germ line cells

It is important here to differentiate between somatic cells and germline cells. Mutations in somatic cells only affects those cells individually whereas mutation in germline cells affect every offspring that arises directly or indirectly from that specific germline cell.

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Comments on DNA repair

DNA repair mechanism are required by all species. Some mutation can be repaired by different routes, by multiple repair mechanisms whereas other mutation can be repaired by only 1 specific mechanism

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Important questions discussed in this lecture

What damages the DNA, what types of damages occur, how are different types of damaged repaired, how failure in the repair mechanism can contribute to a disease and how can repair mechanisms be exploited to treat a disease (of course).

7

Define DNA adduct

A DNA adduct is the part of DNA that is bound to a cancerous agent or to a mutagen. This can initiate carcinogenesis. Many DNA adducts are reactive species that can either break the double stranded DNA at that location or modify the DNA that can cause errors in base pair readings.

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How does ROS affect the DNA

ROS that are naturally present in a cell can also cause damage to the DNA and can result in various forms of damages to the cell. They can break the strands or lead to changes in bases

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Methyl sulfonate

Adds an alkyl group to the CG pair which leads to a conversion such that now this part of the DNA is read as an AT pair.

The side effect of this cancer drug is that it can lead to secondary cancers especially in pediatric patients

Methyl SulfonaTe makes it an AT pair. Rememba dat!

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Cisplatin as an anti cancer drug

Cisplatin is another common cancer drug that has platinum as the active agent. Pt binds with the base pairs either on the same strand or across the strand. DNA replication machinery cannot bypass this bond so the cell is unable to divide

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Physical agents that damage the DNA

Physical agents can cause significant DNA damage such as UA light. These are called UVA and UVB which are in the range of absorbance for DNA, leading to the production of ROS. UVB is more dangerous than UVA since UVB does not only make ROS but also directly damages the DNA by making covalent bonds.

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Further details about UVB

Chromophores in skin (melanin) are the first line in defense.
Remember UVB, B is for bad.
Remember that UV light leads to several DNA mutation. We only talk about thymidine dimers that can be formed (note that a structure of the dimer looks like inside the DNA strand as it is 4 carbons, 2 carbons on each thymidine making a box). When encountered in DNA replication, this can be repaired. Sometimes the DNA pol can jump over it without repairing this site which can lead to point mutation in the daughter strand.

Most common type of DNA damage is from UV light.

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Effects of ionizing radiation on DNA

Ionizing radiation can lead to single stranded and double stranded breaks. This leads to most damaging form of breaks in the DNA as there is great difficulty for the cell to repair it.

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Unique aspect about DNA pol

DNA pol exhibits 3' to 5' exonuclease ability where it can reverse and correct these mistakes. But still some mistakes happen.

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Summary of DNA damage

Summary of DNA damage is that chemicals such as adducts, cross linkers (such as ones used in cancer drugs) and ROS can lead to DNA damage as well as physical agents such as UV light and ionizing radiation. Lastly DNA pol can make mistakes and incorporate the wrong base pairs

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DNA Lesion Repair

There's only 3 things going on, recognize the lesion, remove the lesion and then replace it with the correct base.

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Types of DNA Lesion Repair

There are 2 types of simple DNA lesions repair:
1. Mismatch repair: mismatch of a single nucleotide, it can be due to mismatch or deletion. This triggers the following events: there is insertion in the single strand, nuclease cuts the DNA, DNA pol fills in with the correct nucleotide and then DNA ligase connect the correct nucleotide with the rest of the DNA backbone.
2. Base Excision repair: In here we simply remove the base. An enzyme called DNA glycosylase cuts out the base from the nucleotide and then we have the same events take place, nuclease, DNA pol and then DNA ligase.

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Details on Mismatch Repair

He then showed us a basic schematic diagram of mismatch repair. It involved recognition, removing the lesion, that part of the DNA and then DNA pol theta coming in to join the correct nucelotides.
However, it is important to know if there are mutations in the part of the proteins that are associated with DNA repair mechanisms, then that patient is said to have lynch syndrome or hereditary nonpolyposis colorectal cancer. This is autosomal dominant and the individuals have about 60 to 80% higher risk of colon cancer. This disease is associated with congenital absence of any one of the 4 proteins that are associated with mismatch repair.

You track these patients very closely and they need a much higher levels of screening.
DNA glycosylase removes the base (it also recognized the mismatch), then AP endonuclease breaks the phosphodiester bond of this base and then DNA polymerase and DNA ligase come in to correctly fill in the space.

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Congenital defects in BER

Congenital defects in Base Excision Repair are rare, there is only 1 documented case so far.

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Complex lesion types and how are they repaired

Complex lesions consists of pyrimidine dimers (NER), Intrastrand cross links (NER) and Interstrand cross links (ICL repair, associated with Fanconi's Anemia Proteins).

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Explain NER

NER involves recognizing distorting lesions in the helix, removing about 20 to 30 nts, and then pol and ligase come in to add in the correct nts. Loss of pathway results in a variety of syndromes.

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NER disease

Here we have to know about 1 disease that is called Xeroderma Pigmentosum. In XP disease the NER mechanism does not function properly due to the absence or defective in one of the involved proteins.

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FA

 There are 13 of these proteins, if any of these proteins are lost, then the individual gets Fanconi's Anemia
FA is extremely rare, prevalence is 5000 worldwide.
Bone marorw failure is the biggest challenge here. They often end up with various types of leukemia. They have a high sensitivity to cross-linking chemotherapeutic agents.

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SSB repair

We will leave SSB at that since it is a simpler process and it is not involved with a lot of pathology and diseases

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DSB repair

DSB are important and there are 2 ways to repair them, homologous recombination repair and non homologous end joining
HRR is also known as the error free mechanism. This is restricted to S phase or G2 phase. In this mechanism the broken DNA or the partially single stranded DNA invade the other perfectly normal DNA and forms bonds with the nucleotide (where it was previously missing nucleotides). DNA repair machinery then kicks in and repairs both of the DNAs. Proteins involved in this repair are BRCA1, BRCA2 and FANCD1, last one is also involved in Fanconi's Anemia.
Defects in HR repair can lead to chromosome instability syndrome. We are gonna learn more about these in Gen/Neo module.
NHEJ is the default pathway for DNA repair in people who have a defective HRR. Proteins identify loose ends on the DNA, KU proteins binds to the end of the DNA, Kinase recruites or activates end joining enzymes and then the joins are filled in. This is potentially harmful to the individual as the randomly added nucleotides can either activate or suppress downstream genes or they can introduce an entirely new gene.

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Significance of BRACA 1 and 2

It is important to know that BRCA1 and BRCA2 are involved in preparing the ends of the DNA for strand invasion HRR. If BRCA 1 and 2 are faulty then the DNA will just be repaired by HEJR.
Loss of DNA repair pathway leads to breast cancer.

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He asked the question that can we some how exploit the same underlying mechanisms that lead to breast cancer to cure breast cancer.

Poly (ADP-ribose) polymerase 1, PARP1, this is a DNA binding protein that modifies the nearby histones, flags rest of the repair mechanisms. So if we inhibit this enzyme then the DNA eventually will get double stranded breaks due to these distortions. Cancer cells are particularly susceptible to that since they can only repair their DNA be NHEJ, which randomly adds in the nucleotides and the cancer cells eventually dies because there are too many errors in the DNA.

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Flow chart at the end of lecture

Cancer cells lack BRCA 1 and 2.
It is important to know the flow chart that he showed us at the end of the slide. When there is a distortion in the DNA then PARP1 recognizes them and leads to the repair of the DNA, this maybe done by NER, the DNA is repaired and the cell survives as a normal functioning cell. If PARP1 is inhibited by PARP1 inhibitor then the damage to the DNA can lead to double strand break. This can be repaired by BRCA1 and 2 by HRR, which if done the cell survives. If BRCA 1 and 2 are missing like they are in cancer cells then there will be genomic instability and the cancer cells will die.
Cancer cells lack 1 or more of the repair mechanism due to which they become cancerous. If the existing repair mechanisms of cancer cells are also inhibited then this gives us a specific way of killing these cancer cells.

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Steps of ICL

Steps in ICL:
1. Helicase opens up the DNA,
2. single stranded protein RPA binds to the DNA,
3. exonucleases cut the DNA (XPG and XPA) 4. then we have DNA pol that comes in to fill in with the correct nucleotides.

In ICL, the distortions stall the replication fork. 3 steps then take place associated with ICL:
1. Recognize stalled replication forks.
2. Necessary factors are recruited and activated
3. Homologous recombination occurs