Biochemistry Exam Iv - Dna Repair

Front 1

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

Back 1

Mechanisms to ensure fidelity

Observed mutation frequency: ~1/10^10 nt

Front 2

1. Point mutations
2. Frameshift mutations

Back 2

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

Back 3

Point mutations (base substitutions)

Front 4

Insertion or deletion of one or more base-pairs

Back 4

Frameshift mutations

Front 5

Bad alteration

Found only in DNA
Thymine will be found across from guanine

Will be removed

Back 5

5-Methylcytosine replacement with thymine

Front 6

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

Back 6

Chemically induced deamination

Front 7

Sodium nitrates + amines + heat ---> N, N Dimethylnitrosamine (DMN)

Back 7

2. Alkylating reagents
Exposing processed foods (bacon, sausage, etc.) containing sodium nitrates/nitrites to high temperatures can generate alkylating reagents.

Front 8

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.

Back 8

Removal of a purine base through the action of alkylation results in an abasic site in the DNA.

Front 9

Chemical reagents are not necessary to cause the loss of bases in DNA

Back 9

Spontaneous loss of bases by hydrolysis

guanine >> adenine > cytosine or thymine

Rate of occurrence for purines:
~10,000 spontaneous events per day per cell

Front 10

1. DNA glycosylases

2. AP endonuclease (AP:apurinic/apyrimidinic)

3. DNA polymerase I (Pol I)

4. DNA ligase (NAD+)

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Base excision-repair (BER) for damaged base

Front 11

Only base-pairing alteration that does not result in a point mutation

Back 11

C - G to C-Xanthine

Xanthine still binds to cytosine

Front 12

Methylation of DNA strands can serve to distinguish ________ strands from newly synthesized strands in E. coli DNA

Back 12

parental strands

a function that is critical to mismatch repair

Front 13

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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6

Front 14

The _____ palindrome occurs numerous times throughout the E. coli genome.

Back 14

GATC

Front 15

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.

Back 15

Methyl-directed mismatch repair (MMR) can repair mismatches (due to misincorporation during replication) up to 1000 bps away from a hemimethylated GATC sequence.

Front 16

DMN

Not directly ingested

Back 16

Occurs when NaNO is aminated with heat

Front 17

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

Back 17

DNA glycosylases

Front 18

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

Back 19

Eukaryotes BER

Front 20

In the transient hemimethylated state, the newly synthesized strand can be distinguished from the parental strand.

Back 20

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

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Mut H

Front 22

Binds to DNA and scans it for damage to structure

When it finds a mismatch

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Mut S

Front 23

Mediates commo between Mut S and Mut H

Mut S directs Mut H to cut the daughter strand on the 5' end of palindrome

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Mut L

Front 24

recognizes mismatch

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Mut S = MSH2

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

Front 25

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

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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

Front 27

Can have crosslinking between any pyrimidines that are located next to each other.

Dimers disrupt replication AND transcription.

Can lead to cancer.

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dimerization of nucleotides

Especially thymine dimers

Front 28

1. UvrA protein: recognition/melting (ATP)

2. UvrB protein: recognition/melting (ATP)

3. UvrC protein: endonuclease activity

4. UvrD protein: helicase (ATP)

Back 28

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.

Front 29

Name the enzyme in prokaryotes

Directly repairs instead of excising DNA
 
Will not work in the dark

Back 29

Photolyase (photoreactivating enzyme)

There are multiple repair systems in prokaryotes to repair thymine dimers.

Front 30

Name the complex:

Contains XPB and XPD
Both are helicases
Unwind dsDNA using ATP

Back 30

TFIIH

Front 31

Single stranded DNA binding protein

Binds to undamaged DNA molecule

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Replication Protein A (RPA)

Front 32

Binds to damaged dimer

Recruits XPF and XPG

Both are endonucleases

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XPA

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XPG cleaves on __' end

Back 33

3

Front 34

XPF cleaves on __' end

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5

Front 35

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

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Xeroderma pigmentosum (XP)

Hint 35

Rare skin disease
Genetically transmitted

Excinuclease system that repairs thymine dimers is defective