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35 Cards in this Set
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- 3rd side (hint)
1. Base pairing (A:T & G:C)
Mispairing: ~1/10^4 times 2. Proofreading (3'-5' epsilon nuclease activity) Fails: ~1/10^3 times 3. Postreplicative repair Fails: ~1/10^3 times |
Mechanisms to ensure fidelity
Observed mutation frequency: ~1/10^10 nt |
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1. Point mutations
2. Frameshift mutations |
Two major types of mutations
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a. Transition
Replacement of one purine:pyrimidine bp with another purine:pyrimidine bp b. Transversion bp with a pyrimidine:purine bp |
Point mutations (base substitutions)
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Insertion or deletion of one or more base-pairs
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Frameshift mutations
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Bad alteration
Found only in DNA Thymine will be found across from guanine Will be removed |
5-Methylcytosine replacement with thymine
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a. Nitrous acid: HNO2
b. Spontaneous deamination by hydrolysis I. Cytosine >> adenine, guanine II. Rate of occurrence for cytosine: ~100 spontaneous events per day per cell |
Chemically induced deamination
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Sodium nitrates + amines + heat ---> N, N Dimethylnitrosamine (DMN)
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2. Alkylating reagents
Exposing processed foods (bacon, sausage, etc.) containing sodium nitrates/nitrites to high temperatures can generate alkylating reagents. |
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Position 7 of purines is most common site of modification by alkylating reagents
Bond between sugar moiety and base (i.e. the N-glycosidic bond) is more readily hydrolyzed. |
Removal of a purine base through the action of alkylation results in an abasic site in the DNA.
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Chemical reagents are not necessary to cause the loss of bases in DNA
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Spontaneous loss of bases by hydrolysis
guanine >> adenine > cytosine or thymine Rate of occurrence for purines: ~10,000 spontaneous events per day per cell |
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1. DNA glycosylases
2. AP endonuclease (AP:apurinic/apyrimidinic) 3. DNA polymerase I (Pol I) 4. DNA ligase (NAD+) |
Base excision-repair (BER) for damaged base
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Only base-pairing alteration that does not result in a point mutation
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C - G to C-Xanthine
Xanthine still binds to cytosine |
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Methylation of DNA strands can serve to distinguish ________ strands from newly synthesized strands in E. coli DNA
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parental strands
a function that is critical to mismatch repair |
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The methylation occurs at N ___ of adenines in GATC sequences.
This sequence is a palindrome and thus is present in opposite orientations on both strands. |
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The _____ palindrome occurs numerous times throughout the E. coli genome.
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GATC
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1. mutH cleaves on the 5' side of the palindrome in the defective strand.
2. Helicase II (ATP) unwinds the DNA duplex starting from the nick. 3. Exonuclease I degrades a portion of the newly synthesized strand. 4. SSB binds to and protects the parental single-stranded region. 5. Pol III fills in the gap. 6. DNA ligase ligates the DNA fragments. |
Methyl-directed mismatch repair (MMR) can repair mismatches (due to misincorporation during replication) up to 1000 bps away from a hemimethylated GATC sequence.
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DMN
Not directly ingested |
Occurs when NaNO is aminated with heat
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Has specificity for damaged bases.
Recognizes damaged base. Cleaves the bond between base and sugar moiety Excises space between DNA molecule Leads to abasic site |
DNA glycosylases
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Cleaves the DNA molecule backbone in the region of the damaged base
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AP endonuclease (AP: apurinic/apyrimidinic)
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1. DNA Glycosylases
2. AP Endonucleases 3. Nucleases i. Remove segment of DNA molecule 4. Pol beta or epsilon i. Fills in missing DNA 5. DNA ligase i. Requires ATP |
Eukaryotes BER
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In the transient hemimethylated state, the newly synthesized strand can be distinguished from the parental strand.
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Mismatched DNA can be spotted at this state.
Once methylated, cannot fix mismatch mutation |
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Recognizes palindrome
Binds to it Has nuclease activity |
Mut H
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Binds to DNA and scans it for damage to structure
When it finds a mismatch |
Mut S
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Mediates commo between Mut S and Mut H
Mut S directs Mut H to cut the daughter strand on the 5' end of palindrome |
Mut L
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recognizes mismatch
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Mut S = MSH2
Key concept: Genetic defects in the human MMR can give rise to a diseased state. |
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binds to mutS
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Mut L = MLH1
Key concept: Genetic defects in the human MMR can give rise to a diseased state. |
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• Autosomal dominant disease
• Patients have mutations mostly in MSH2 (some in MLH1) • 2-15% of all colon cancers |
Hereditary Non-Polyposis Colon Cancer (HNPCC) (Lynch syndrome)
Women (mutations in MSH2 or MLH1) • Increased risk of endomentrial (~60%) and ovarian (12%) cancers General • Increased risk of liver, brain & skin cancers |
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Can have crosslinking between any pyrimidines that are located next to each other.
Dimers disrupt replication AND transcription. Can lead to cancer. |
dimerization of nucleotides
Especially thymine dimers |
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1. UvrA protein: recognition/melting (ATP)
2. UvrB protein: recognition/melting (ATP) 3. UvrC protein: endonuclease activity 4. UvrD protein: helicase (ATP) |
Nucleotide excision-repair (NER) of thymine dimers (prokaryotes)
Key concept: Through the action of key enzymes in the nucleotide excision-repair (NER) system, thymine dimers can be removed from the prokaryotic genome. |
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Name the enzyme in prokaryotes
Directly repairs instead of excising DNA Will not work in the dark |
Photolyase (photoreactivating enzyme)
There are multiple repair systems in prokaryotes to repair thymine dimers. |
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Name the complex:
Contains XPB and XPD Both are helicases Unwind dsDNA using ATP |
TFIIH
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Single stranded DNA binding protein
Binds to undamaged DNA molecule |
Replication Protein A (RPA)
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Binds to damaged dimer
Recruits XPF and XPG Both are endonucleases |
XPA
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XPG cleaves on __' end
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3
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XPF cleaves on __' end
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5
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Skin is extremely sensitive to sunlight (homozygous state)
Skin lesions that usually become sites of cancer development UV light produces thymine dimers in the DNA |
Xeroderma pigmentosum (XP)
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Rare skin disease
Genetically transmitted Excinuclease system that repairs thymine dimers is defective |