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35 Cards in this Set

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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
1. Point mutations
2. Frameshift mutations
Two major types of mutations
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)
Insertion or deletion of one or more base-pairs
Frameshift mutations
Bad alteration

Found only in DNA
Thymine will be found across from guanine

Will be removed
5-Methylcytosine replacement with thymine
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
Sodium nitrates + amines + heat ---> N, N Dimethylnitrosamine (DMN)
2. Alkylating reagents
Exposing processed foods (bacon, sausage, etc.) containing sodium nitrates/nitrites to high temperatures can generate alkylating reagents.
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.
Chemical reagents are not necessary to cause the loss of bases in DNA
Spontaneous loss of bases by hydrolysis

guanine >> adenine > cytosine or thymine

Rate of occurrence for purines:
~10,000 spontaneous events per day per cell
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
Only base-pairing alteration that does not result in a point mutation
C - G to C-Xanthine

Xanthine still binds to cytosine
Methylation of DNA strands can serve to distinguish ________ strands from newly synthesized strands in E. coli DNA
parental strands

a function that is critical to mismatch repair
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.
6
The _____ palindrome occurs numerous times throughout the E. coli genome.
GATC
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.
DMN

Not directly ingested
Occurs when NaNO is aminated with heat
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
Cleaves the DNA molecule backbone in the region of the damaged base
AP endonuclease (AP: apurinic/apyrimidinic)
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
In the transient hemimethylated state, the newly synthesized strand can be distinguished from the parental strand.
Mismatched DNA can be spotted at this state.

Once methylated, cannot fix mismatch mutation
Recognizes palindrome

Binds to it

Has nuclease activity
Mut H
Binds to DNA and scans it for damage to structure

When it finds a mismatch
Mut S
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
recognizes mismatch
Mut S = MSH2

Key concept: Genetic defects in the human MMR can give rise to a diseased state.
binds to mutS
Mut L = MLH1

Key concept: Genetic defects in the human MMR can give rise to a diseased state.
• 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
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
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.
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.
Name the complex:

Contains XPB and XPD
Both are helicases
Unwind dsDNA using ATP
TFIIH
Single stranded DNA binding protein

Binds to undamaged DNA molecule
Replication Protein A (RPA)
Binds to damaged dimer

Recruits XPF and XPG

Both are endonucleases
XPA
XPG cleaves on __' end
3
XPF cleaves on __' end
5
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)
Rare skin disease
Genetically transmitted

Excinuclease system that repairs thymine dimers is defective