Podcast
Questions and Answers
What must occur before mutations become locked into the DNA sequence?
What must occur before mutations become locked into the DNA sequence?
Which type of repair mechanism involves directly converting damaged DNA back to the original nucleotide?
Which type of repair mechanism involves directly converting damaged DNA back to the original nucleotide?
What role does the p53 tumor suppressor protein play in response to DNA damage?
What role does the p53 tumor suppressor protein play in response to DNA damage?
Which of the following is NOT a type of sequence repair mechanism?
Which of the following is NOT a type of sequence repair mechanism?
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What is one way that direct reversal of DNA damage can occur?
What is one way that direct reversal of DNA damage can occur?
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What happens when Tyr147 is mutated to Ala?
What happens when Tyr147 is mutated to Ala?
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What is the role of Uracil-N-glycosylase in base excision repair?
What is the role of Uracil-N-glycosylase in base excision repair?
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Which repair mechanism is triggered by changes in the physical structure of the DNA duplex?
Which repair mechanism is triggered by changes in the physical structure of the DNA duplex?
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What is the primary function of MutH in methyl-directed mismatch repair?
What is the primary function of MutH in methyl-directed mismatch repair?
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Which enzyme is primarily responsible for recognizing and hydrolyzing 8-oxo-GTP in E. coli?
Which enzyme is primarily responsible for recognizing and hydrolyzing 8-oxo-GTP in E. coli?
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What is the primary role of DNA ligase in the mismatch repair process?
What is the primary role of DNA ligase in the mismatch repair process?
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What does the UvrABC exinuclease complex do in nucleotide excision repair?
What does the UvrABC exinuclease complex do in nucleotide excision repair?
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Which factor is essential for determining the correct strand in methyl-directed mismatch repair in E. coli?
Which factor is essential for determining the correct strand in methyl-directed mismatch repair in E. coli?
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Which group of proteins in eukaryotes homologous to MutS and MutL are involved in mismatch repair?
Which group of proteins in eukaryotes homologous to MutS and MutL are involved in mismatch repair?
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What is the approximate error rate of DNA repair systems compared to uncorrected polymerase activity?
What is the approximate error rate of DNA repair systems compared to uncorrected polymerase activity?
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What is the outcome of a mismatched base pair during mismatch repair?
What is the outcome of a mismatched base pair during mismatch repair?
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Individuals with Xeroderma Pigmentosum (XP) have increased risk of which type of cancer?
Individuals with Xeroderma Pigmentosum (XP) have increased risk of which type of cancer?
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What size of DNA fragment is typically excised during nucleotide excision repair?
What size of DNA fragment is typically excised during nucleotide excision repair?
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Translesion synthesis in E. coli is primarily performed by which of the following polymerases?
Translesion synthesis in E. coli is primarily performed by which of the following polymerases?
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What is the defect in the hereditary non-polyposis colon cancer (HNPCC) associated with?
What is the defect in the hereditary non-polyposis colon cancer (HNPCC) associated with?
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What does the presence of 8-oxo-G in DNA indicate?
What does the presence of 8-oxo-G in DNA indicate?
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What is the primary function of O6-methylguanine-DNA methyltransferase (MGMT)?
What is the primary function of O6-methylguanine-DNA methyltransferase (MGMT)?
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Which process allows mammals to repair thymine dimers?
Which process allows mammals to repair thymine dimers?
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In base excision repair, what is the role of DNA glycosylases?
In base excision repair, what is the role of DNA glycosylases?
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How does uracil-N-glycosylase (UDGase) distinguish between uracil and thymine?
How does uracil-N-glycosylase (UDGase) distinguish between uracil and thymine?
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What is created when DNA glycosylases remove bases from DNA?
What is created when DNA glycosylases remove bases from DNA?
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What role does FADH play in the photoreactivation process?
What role does FADH play in the photoreactivation process?
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Which of the following types of damage can be repaired by base excision repair (BER)?
Which of the following types of damage can be repaired by base excision repair (BER)?
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Which enzyme is responsible for handling GT pairs during base excision repair?
Which enzyme is responsible for handling GT pairs during base excision repair?
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Study Notes
DNA Repair Lecture 9 Overview
- Lecture covers sources and types of DNA damage, and repair mechanisms.
Repair Mechanisms
- Errors in DNA sequence must be detected.
- Checks to see if the nucleotide is not standard or if the wrong nucleotide is paired.
- Specialized proteins directly reverse errors or edit the DNA to restore the correct sequence.
- Repair ideally occurs before replication to prevent mutations from being locked in.
Repair Mechanisms (Specific Types)
- Deamination, Alkylation, Oxidative damage, UV light, Replication errors, and X-rays cause DNA damage.
- O6-MeG, 3-MeA, U, 8-oxo-G, inosine, Pyrimidine dimers, Mismatches, and Double-strand breaks are types of DNA damage.
- Direct reversal, BER, NER, MMR, Non-homologous end joining are repair mechanisms.
p53 Tumor Suppressor
- Responds to and detects DNA damage.
- Arrests the cell cycle at the end of G1, allowing time for repair.
- Activates DNA repair proteins.
- If damage is too severe, triggers apoptosis or senescence.
Sequence Repair 1: Direct Reversal/Repair
- If damaged nucleotides (A, C, G, T) are converted to something else, they can sometimes be converted back to the original nucleotide.
- Examples include:
- Demethylation following alkylation.
- Removal of crosslinks following UV light damage.
- Specific reversal proteins:
- O6-methylguanine-DNA methyltransferase (MGMT)
- Transfers methyl/ethyl group from G to a Cys residue, restoring G.
- O6-methylguanine-DNA methyltransferase (MGMT)
Sequence Repair 1: Photolysis of Dimers (Photoreactivation)
- DNA photolyase absorbs blue light to break T-T internucleotide bonds.
- Restores 2 Ts to the original position using FADH.
- Mammals use a different system to repair thymine dimers because they lack this enzyme.
Sequence Repair 2: Base Excision Repair (BER)
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Removes individual bases (local correction).
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DNA glycosylases recognize and cleave abnormal bases from deoxyribose, forming abasic sites.
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Examples of abnormal bases:
- Deaminated A
- Deaminated C (now U)
- Deaminated methyl-C (now T)
- Oxidized
- Alkylated bases
- Opened ring structures
- Double bond loss
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Uracil N-glycosylase (UDGase)
- Flips out bases for closer checking.
- If the base is incorrect, the sugar–base bond is cleaved, but UDGase remains attached to DNA.
- Also responsible for leading strand fragments.
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How UDGase distinguishes between U and T:
- Steric clash of methyl group on T with Tyr residue.
- A separate enzyme (TDGase) is for GT pairs.
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Base removed by Uracil-N-glycosylase.
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Baseless nucleotide recognized and phosphodiester backbone cleaved by AP (apyrimidinic) endonuclease
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Nicked DNA restored by Pol I, nick translation; followed by DNA ligase sealing the nick.
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Also works following depurination events, Pol β in eukaryotes.
Sequence Repair 3: Nucleotide Excision Repair (NER)
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Removes nucleotide fragments caused by physical changes in the DNA duplex as a result of damage.
- Example is the removal of thymine dimers.
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Protein complex called Uvr complex in E.coli
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Similar systems exist in eukaryotes.
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UvrB and UvrA form a heterodimer, binding to the thymine dimer.
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UvrA subunits dissociate, and UvrC binds.
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UvrC and UvrB migrate 5 nucleotides away and cleave the DNA.
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UvrC and UvrB dissociate, and UvrD helicase displaces the damaged DNA (a fragment of ~12 nt).
Sequence Repair 4: Mismatch Repair (MMR)
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Detects and removes incorrect base pairs.
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A mismatched pair distorts the helix, which is detected by specialist proteins.
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The incorrect base is removed.
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Needs to identify the incorrect base
- Method in bacteria: Hemimethylation.
- Hemimethylation provides the information which strand is parent (correct) and daughter.
- Method in bacteria: Hemimethylation.
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Methyl-directed MMR (bacteria):
- MutH binds to unmethylated GATC at OriC, identifying the daughter strand.
- MutS binds to a distorted site on the duplex.
- MutL binds to MutS.
- MutL/MutS complex travels to the origin and activates MutH.
- MutH cleaves the daughter strand.
- Specialized helicase (UvrD) and exonucleases remove nucleotides until past the distortion.
- Pol III fills in the missing nucleotide; DNA ligase seals the nick.
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Eukaryotes have homologues of MutL and MutS (like MLH1-5 and MSH1-6).
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Lack homologues of MutH; do not use hemimethylation.
Multi-pronged Approach (Bacteria)
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Additional proteins for 8-oxo-G damage (e.g. MutT, MutM, MutY)
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MutT: Recognizes 8-oxo-GTP and hydrolyzes it.
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MutM: Recognizes 8-oxo-G in DNA and removes it via BER.
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MutY: Recognizes 8-oxo-G opposite A in DNA and removes A via BER.
Repair System Summary
- Polymerase error rate reduced by 1000x due to repair systems.
- Polymerase error rate is 10-5 (without proofreading), 10-7 (with proofreading), and 10-10 (with repair).
- Sometimes a risk of mutation is preferable over the alternative, like translesion synthesis. (e.g. Pols IV & V in bacteria, and more in eukaryotes) - where there is a blockage, the polymerase bypasses the damaged base, but the added base may not be correct.
Insights from Disease
- Xeroderma pigmentosum (XP): Inherited defect in one of eight NER genes causes dry, parchment-like skin, many freckles, and a 1000-fold increased risk of skin cancer due to increased UV sensitivity.
- Hereditary non-polyposis colon cancer (HNPCC): Defects in the MutS/L MMR system (like MSH2 and MLH1) which leads to predisposition to colon cancer (2–3% of all colon cancer cases) and accumulation of mutations in the genome.
Lung Cancer Genome
- Comparisons of lung cancer genomes to normal genomes shows rearrangements (58) and point mutations (22,910).
- Approximately one mutation occurs per 15 cigarettes smoked.
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Description
This lecture covers the various sources and types of DNA damage along with the mechanisms of repair. Key topics include the role of specialized proteins in detecting errors and the importance of repairing DNA before replication to avoid mutations. Specific types of damage and repair strategies are also outlined.
