L 39 DNA Damage and Repair Intro to Cancer
*PROKARYOTIC Nucleotide Excision Repair (NER)* *"NER" repairs lesions that __________ the DNA double helix, such as thymine dimers or DNA crosslinking.* *Remember DNA repair Steps* 1. 2. 3. 4. *Note: There will probably be exam questions on this, ABC basic for prokaryotes*
*"NER" repairs lesions that __________ the DNA double helix, such as thymine dimers or DNA crosslinking.* distort *Remember DNA repair Steps* 1. Recognition: UvrABC excinuclease makes 2 nicks on each side of lesion. 2. Excision: Uvr D helicase excises the damaged DNA leaving a *GAP* 3. Resynthesis: DNA pol I fills the gaps 4. Ligation: DNA ligase seals the nicks
*Single-Strand Annealing Repair (SSA)* *"SSA" repairs _________-_________ breaks in DNA.* -Littered in repetitive elements (direct repeats) -requires long homologous sequences flanking the break. *Remember DNA repair mechanism* 1. 2. 3. 4.
*"SSA" repairs _________-_________ breaks in DNA.* double-strand *Remember DNA repair mechanism* 1. Recognition: Exonuclease digests the broken ends, Single strand annealing protein (SSAP, *RAD52*) binds to the repeat sequences 2. Excision: flaps are degraded by *ERCC1/XPF* endonuclease 3. Resynthesis: No resynthesis (ERROR PRONE) 4. Ligation: DNA ligase seals the nicks
*UV-induced damage* *Ultraviolet radiation (UV radiation) is a common cause of DNA damage.* *-- UV results in the formation of a ____________ ring between adjacent two _____________ rings.* *-- This is especially common for pairs of ___________ forming a __________-dimer. (also called cyclobutane)* *What does the presence of the cyclobutane ring do the the DNA helix?* *This distortion to the helix is the major damage to the DNA and is the cause of skin cancer.* One to guess is that this is going to be on the exam.
*-- UV results in the formation of a ____________ ring between adjacent two _____________ rings.* cyclobutane pyrimidine *-- This is especially common for pairs of ___________ forming a __________-dimer.* thymine thymine *What does the presence of the cyclobutane ring do the the DNA helix?* The cyclobutane ring "kinks" the axis of the DNA helix.
*Ionizing radiation-induced damage* -gamma ray, X-ray, and ultraviolet is the ionizing radiation. (IR) *-Here we can break water into __________ and ___________ ions.* *-This is dangerous because our cells are 98% water.* *The ____________ wavelength (i.e. highest energy) portion of the electromagnetic spectrum possesses SUFFICIENT energy to ionize water, and thus can end up causing DNA damage.* 2H2O - - *Ionization Radiation* - -> H3O+ + OH- *Which of the deamination reactions in DNA bases would be considered the most detrimental to eukaryotes?* a. 5-methylcytosine → thymine b. Cytosine → uracil c. Adenine → hypoxanthine d. Guanine → xanthine e. Guanine → 8-oxoguanine
*-Here we can break water into __________ and ___________ ions.* hydronium and hydroxide *The ____________ wavelength (ie highest energy) portion of the electromagnetic spectrum possesses sufficient energy to ionize water, and can cause DNA damage.* shortest *Which of the deamination reactions in DNA bases would be considered the most detrimental to eukaryotes?* a. 5-methylcytosine → thymine This question requires some critical thinking. Out of the five bases, only uracil is not found in DNA. This allows base excision repair (BER) to easily recognize this mismatch and excise the cytosine to uracil deamination. However, when a 5-met-cytosine deaminates into thymine and the DNA undergoes synthesis, it'll create two double-stranded DNA strands, one with the normal C-G base pairing but the other daughter strand will contain the mutated A-T base pairing, which is near impossible to recognize by DNA repair mechanism because it's a normal base-pair that will possibly result in a completely different polypeptide.
*Alkylators: alkylate DNA. The addition of carbon base moieties on the DNA.* *Two broad buckets:* *1. Mono-functional Alkylators: methyl nitrosourea, streptozotocin, temozolomide.* *-they add a methyl group and form a single ___________bond to DNA.* *2. Bi-functional Alkylators: chlorambucil, melphalan, cyclophosphamide* *-these can form two __________ bonds with DNA* -Leads to inter and intra strand cross linking. *Lecture:* Hit cells with these drugs, it will kill the cell because these get stuck on the DNA.
*-they add a methyl group and form a single ___________bond to DNA.* covalent *-these can form two __________ bonds with DNA* covalent
*Base Excision Repair Mismatch (BER) * *Base excision repair "BER" repairs mismatches resulting from ___________ bases, _____________ and ________-_______ _________ in DNA.* -- In mammals, a BER system repairs T:G base pairs to C:G. *Remember DNA Repair steps* 1/2 3. 4. *________ _______ arise from activity of a DNA glycosylase or depurination.*
*Base excision repair "BER" repairs mismatches resulting from ___________ bases, _____________ and ________-_______ _________ in DNA.* damaged depurination single-strand breaks *Remember DNA Repair steps* 1/2. DNA glycosylase will cleave the mismatch and creates an AP site. AP endonuclease will cleave the phosphodiester backbone at the AP site. 3. DNA pol 1 (B in eukaryotes) will resynthesize the new base. 4. DNA ligase will seal the nick *________ _______ arise from activity of a DNA glycosylase or depurination.* abasic sites
*IR Damages Bases* Think about it, this stuff can ionize water! IR can directly and indirectly start to goof up the bases! *How?* *Due to ______ __________ and ______ _________ crosslinking. * Urea half of the chemical structure is ripped away.
*Due to ______ __________ and ______ _________ crosslinking. * intra strand and inter strand crosslinking
*Homologous Recombination DNA Repair* -higher fidelity repair mechanism. *Homologous recombination (HR) repair can also repair a doublestrand break in DNA.* -we won't talk about this today, he will go over it tommorow in lecture. *HR provides a high fidelity ___________ _________ _________ repair because the "UNDAMAGED" sister chromatid is used as a __________ for resynthesis of DNA.*
*HR provides a high fidelity ___________ _________ _________ repair because the ___________ sister chromatid is used as a __________ for resynthesis of DNA.* double strand break template
*IR causes DNA strand breaks* *IR exposure can damage the ______________backbone resulting in _______ ________ ________ (SSB) and if two SSB are closely spaced a _______ ________ __________ (DSB) is formed.* *A key aspect of IR induced SSB and DSB to bear in mind is that these breaks in the DNA strands are highly _________, thus the strands "cannot" be easily re-ligated.* *Which type are considered the "MOST LETHAL" form of DNA damage?* *Key: Ionization radiation causes strand breaks.* -oxidation of the Ribose moieties.
*IR exposure can damage the ______________backbone resulting in _______ ________ ________ (SSB) and if two SSB are closely spaced a _______ ________ __________ (DSB) is formed.* phosphodiester single strand breaks double strand break *A key aspect of IR induced SSB and DSB to bear in mind is that these breaks the DNA strands are highly _________, thus the strands cannot be easily re-ligated.* oxidized *Which type are considered the "MOST LETHAL" form of DNA damage?* DSB
*NHEJ (non-homologous end joining) is the predominant mechanism for repair of ______-________ _______ in mammalian cells.* *What makes NHEJ an error prone mechanism?* *Remember DNA repair mechanism* 1. 2. a. 3. 4.
*NHEJ (non-homologous end joining) is the predominant mechanism for repair of ______-________ _______ in mammalian cells.* Double-strand breaks *What makes NHEJ an error prone mechanism?* Because NHEJ synthesizes nucleic acids without the use of a template. *Remember DNA repair mechanism* 1. Recognition: Ku70/80 complex binds to loose ends of DNA 2. Excision: artemis exonuclease results in loss of DNA a. artemis endonuclease removes overhanging "flaps" 3. Resynthesis: there is none. . . . (ERROR PRONE) 4. Ligation: DNA ligase seals the nicks.
*Characteristics of Excision Repair Mechanisms* *Excision repair (MMR, BER and NER) mechanisms can be broken down into four distinct phases:* *What are the 4 phases?* (What is the acronym?) 1. 2. 3. 4. *The excision repair mechanisms all work through the same principles.*
*R*oby *E*ats *R*ice *L*iberally *What are the 4 phases?* 1) Recognition of the lesion. 2) Excision of the lesion. 3) Resynthesis of the DNA. 4) Ligation of DNA strands.
*EUKARYOTIC Nucleotide Excision Repair (NER)* *In humans, NER repair works similarly to prokaryotes.* *Remember DNA repair Steps* 1. 2. 3. 4. *If we do not have the XP genes required for NER, we run into the inability to repair UV-induced DNA damage.* Xeroderma Pigmentosum results.
*Remember DNA repair Steps* 1. Recognition: XP excinuclease (complex) scans the DNA for kinks 2. Excision: TFIIH helicase melts the DNA resulting in a gap 3. Resynthesis: DNA pol e fills the gaps 4. Ligation: DNA ligase seals the nicks
*Bulky compounds* *These compounds can also form covalent bonds to DNA. * *Cisplatin-DNA interaction. This is NOT an alkylation agent. * *The carbons can form ______ strand and _______strand crosslinks. All kinds of mess to kill cells.* *How does nitrogen mustard act as a DNA alkylator?* a. It is a mono-functional alkylator that covalently links a methyl group on the N-7 of adenine, so it can no longer base pair with thymine b. It is a bi-functional alkylator that forms a cyclobutane ring between adjacent pyrimidine rings, usually resulting in a thymine-dimer c. It is a bi-functional alkylator that causes the ribose sugars of purines to become covalently linked on the N-7 of either adenine or guanine d. It is a bi-functional alkylator that crosslinks DNA by covalently linking the N-7 in two guanine bases to form a G-G crosslink e. None of the above
*The carbons can form ______ strand and _______strand crosslinks. All kinds of mess to kill cells.* inter strand and intra strand *How does nitrogen mustard act as a DNA alkylator?* d. It is a bi-functional alkylator that crosslinks DNA by covalently linking the N-7 in two guanine bases to form a G-G crosslink
*DNA intercalators* *They have ___________ structure that allows them to sit in the DNA just so nicely. Can sit in between stacked bases. 2 points of valency to allow them to be covalently bonded inside. * *Benzo[a]pyrene: cancer causing agent in cigarette smoke.* *Which of these processes creates a specific location in DNA without a cytosine or thymine base (AP site) that will later get repaired?* a. Depurination b. Prokaryotic mismatch repair (MMR) c. Non-homologous end joining (NHEJ) d. Direct repair mechanism e. None of the above
*They have ___________ structure that allows them to sit in the DNA just so nicely. Can sit in between stacked bases. 2 points of valency to allow them to be covalently bonded inside. * planar *Which of these processes creates a specific location in DNA without a cytosine or thymine base (AP site) that will later get repaired?* e. None of the above From Lecture 39, Dr. Brown only mentioned depurination and base excision repair as creating abasic (AP) site, which are sites in the DNA without either a purine or pyrimidine base. Depurination is specific to creating AP sites without a purine (guanine or adenine). Base excision repair is not specific to either purines or pyrimidines, but because it's not an option, E is the correct answer.
*DNA alkylation* *This is a good example of a __________. This can form covalent bonds with DNA. Effective ways of killing cells.* *What gets crosslinked?* *During prokaryotic mismatch repair, how does MutH identify the parental strand so it can nick the newly synthesized strand?* *a.* After DNA replication, the parental strand has a methylated guanine within its CTAG sequence, while the newly synthesized strand remains unmethylated at a GATC palindrome. MutH then cleaves the unmethylated strand *b. *Directly after DNA replication, the parental strand has a methylated adenine within its GATC sequence, while the newly synthesized strand remains unmethylated at a GATC palindrome. MutH then cleaves the unmethylated strand *c.* After DNA replication, the parental strand has an unmethylated adenine within its CTAG sequence, while the newly synthesized strand remains methylated at a CTAG palindrome. MutH then cleaves the methylated strand *d. *The MutH complex can tell which base from the parent or newly synthesized DNA strand is deaminated, and it'll nick the incorrect base *e.* Directly after DNA replication, the parental strand has a methylated cytosine within its CTAG sequence, while the newly synthesized strand remains unmethylated at a CTAG palindrome. MutH then cleaves the unmethylated strand
*This is a good example of a __________. This can form covalent bonds with DNA. Effective ways of killing cells.* crosslinkage *What gets crosslinked?* G-C at the nitrogens. N7 interstrand crosslinked DNA. *During prokaryotic mismatch repair, how does MutH identify the parental strand so it can nick the newly synthesized strand?* b. Directly after DNA replication, the parental strand has a methylated adenine within its GATC sequence, while the newly synthesized strand remains unmethylated at a GATC palindrome. MutH then cleaves the unmethylated strand
*Depurination* *Draw the reaction of converting GMP to guanine and a sugar phosphate. * *What is the cause of depurination?* -losing a base *Depurination yields an "_______" site. synonym: "AP" site* *Most of the time, this happens in ________. *(Purine specific event) *Normal loss of the glycosidic bond will lead to a loss of a base. You can think of it like a little boy or girl who has lost their tooth, there is no loss in integrity of the backbone. This is an "abasic site". * Deamination, oxidation, and depurinations are the things that happen minute by minute in the cell.
*What is the cause of depurination?* Depurination is caused by hydrolysis of the N-B-glycosyl bond bond linking a purine base to the sugarphosphate backbone. *Depurination yields an "_______" site. synonym: "AP" site* abasic *Most of the time, this happens in ________. *(Purine specific event) guanine
*Deamination of DNA Bases happens every single day.* *What is the most common base that gets deaminated on a minute to minute basis? Also, what is it converted into?* -Deamination of 5-methylCytosine→Thymine occurs spontaneously in a human cell. *Deamination of A and G also occurs spontaneously, but at a _________ rate.* *Some common chemicals can accelerate the rate of deamination.* 1. 2. 3. *Which of the following is NOT a similarity between prokaryotic and eukaryotic nucleotide excision repair (NER)?* a. Both mechanisms repair DNA double helix lesions by excising single-stranded DNA b. An excinuclease recognizes the DNA lesion and nicks the DNA on both sides of the lesion c. When it excises the DNA lesion and creates a gap, it exposes a 3'-OH and 5'-P d. The excinuclease has an embedded helicase activity that melts the DNA lesion e. More than one of the above
*What is the most common base that gets deaminated on a minute to minute basis? Also, what is it converted into?* Cytosine to uracil *Deamination of A and G also occurs spontaneously, but at a _________ rate.* slower *Some common chemicals can accelerate the rate of deamination.* 1. Sodium nitrite 2. sodium nitrate 3. nitrosamine *Which of the following is NOT a similarity between prokaryotic and eukaryotic nucleotide excision repair (NER)?* d. The excinuclease has an embedded helicase activity that melts the DNA lesion
*Prokaryotic Mismatch Repair (MMR) Step 1: Recognition (Roby)* *"MMR" corrects mismatched base pairs.* *Prokaryotic MMR proteins:* *What is the name of the complex that "recognizes" the site of the mismatches in prokaryotes?* *What is the name of the "endonuclease" that identifies the parental GAmeTC strand and newly synthesized strand? Also called a "nicase"* *Key: the bad base is there due to bad DNA replication. The C is in the daughter strand. How does the bacteria know that? The DNA exists in an hemimethylated state. When the DNA undergoes replication, the parental DNA is methylated and the nascent strand is unmethylated. (hemimethylated)* *This shows what strand is correct and which is not correct. The MutH endonuclease (nicase) will only target the _______________strand. *
*What is the name of the complex that recognizes the site of the mismatches?* MutL-MutS *What is the name of the endonuclease that identifies the parental GAmeTC strand and newly synthesized strand?* MutH endonuclease *This shows what strand is correct and which is not correct. The MutH endonuclease (nicase) will only target the _______________strand. * unmethylated
*What is the name of the disease for not being able to repair UV-Induced DNA damage? (XP)* *Defects in XP genes required for NER results in an inability to repair UV-induced DNA damage. As we will learn that mutation is the root cause of cancer, it follows that XP patients have very high rates of skin cancer * -Defencts in XP genes results in an inability to repair UV- induced DNA damage, and causes XP. *Why is this disease so deadly?*
*What is the name of the disease for not being able to repair UV-Induced DNA damage?* Xeroderma Pigmentosum *Why is this disease so deadly?* Can't undergo nucleotide exicision repair. Can not repair UV-induced DNA damage, you will get skin cancer.
*Direct Repair Mechanism* *Why do we need to repair O6-methylguanine? Like why is this dangerous for our DNA or why does it make it highly mutagenic?* *O6=methylguanine is repaired by what enzyme?* *Lecture:* *The protein (not really an enzyme because it doesn't have a way to un-methylate itself) is responsible for taking the methyl group off of the O6-methylguanine and transfer it to the ___________to regenerate a normal guanine.*
*Why do we need to repair O6-methylguanine? Like why is this dangerous for our DNA?* Guanine usually pairs to cytosine (G-C A-T) but when it is methylated, the O6 position becomes inactive to hydrogen bonding. Thus O6-methylguanine can now base pair to thymine which is highly mutagenic. *Therefore O6meG is highly ____________* mutagenic *O6=methylguanine is repaired by what enzyme?* O6-methylguanine-DNA methyltransferase (MGMT) *Lecture:* *The protein (not really an enzyme because it doesn't have a way to un-methylate itself) is responsible for taking the methyl group off of the O6-methylguanine and transfer it to the ___________to regenerate a normal guanine.* cysteine (C)
*Oxidation of DNA* *_________ ____________ ___________ "ROS" are generated by respiration and during inflammation.* *Hydroxide free radical ( OH) "nucleophillically attacks" either ___ or ___.* i.e. Guanine -(⚈OH)-> 8-oxoguanine i.e. Thymine -(⚈OH)-> thymine glycol *____ and ___ are LESS frequently oxidized, and high levels of ROS can result in DNA strand breaks. * We can ring a lot of energy out of oxygen. The downside is that oxygen is an awesome oxidizer. Oxidation of bases occurs! Oxidation of guanine to 8-oxoguanine is very common. This is due to the build up of oxygen species in the body.
*_________ ____________ ___________ "ROS" are generated by respiration and during inflammation.* Reactive oxygen species *Hydroxide free radical (⚈OH) nucleophillically attacks either ___ or ___.* G or T *____ and ___ are less frequently oxidized, and high levels of ROS can result in DNA strand breaks. * C and A
*Prokaryotic Mismatch Repair Step 2: Excision (Eats)* *Remember DNA Repair steps* 1. a. b. 2. 3. 4. Ends with Resynthesis (Rice) and Ligation (Liberally)
1. *Recognize*: Mut S/L recognize mismatch a. Mut H recognizes the GA TC palidrome b. Mut H endonuclease nicks strand 2. *Excision*: Exonuclease digests DNA up to the methylated site 3. *Resynthesis*: DNA pol III 4. *Ligation*: DNA ligase seals nicks