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CHE 541 > 19 - Nucleic Acids II > Flashcards

19 - Nucleic Acids II Flashcards

1
Q

What is the replication method for DNA?

A

Semi-conservative

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2
Q

What are the functions (3) of DNA polymerase?

A
  1. Copy DNA in 5’ to 3’ direction
  2. Remove incorrect bases through 3’ to 5’ exonuclease activity
  3. 5’ to 3’ exonuclease activity to remove Okazaki fragments
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3
Q

What is an exonuclease?

A

Cleave DNA at end of strand

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4
Q

What is an endonuclease?

A

Cleave DNA at middle of strand

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5
Q

What is needed to synthesize DNA?

A

RNA primer, template strand, and Mg2+ cation (for enzyme and electrostatics)

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6
Q

What are the reactants for DNA synthesis?

A

Deoxynucleotide triphosphates

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7
Q

What are some unresolved questions about DNA synthesis?

A
  1. How is chromatin reassembled behind the fork?
  2. How do interior proteins interact with the origin?
  3. How do cells coordinate leading and lagging strand synthesis?
  4. How are helicases activated? How do helicases unwind DNA?
  5. How do cells limit replication to a single round?
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8
Q

What factors contribute to replication fidelity?

A

Selectivity, proofreading, and mismatch repair

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9
Q

What is the overall error rate of DNA synthesis?

A

~10^-9

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10
Q

What can defective proofreading lead to?

A

Cancer in mice

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11
Q

How is selectivity achieved in the insertion?

A

The relative rate of incorporation is highest for the proper nucleotide (eg: A-T) compared to a mismatched on (eg: A-C)

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12
Q

How is selectivity achieved in the extension?

A

The relative rate of incorporation after a correct nucleotide (ex: a CG after an AT) is higher than after an incorrect nucleotide (ex: a CG after an AC)

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13
Q

What are some instabilities in DNA?

A

Oxidation, crosslinking, etc.

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14
Q

What are some consequences of DNA damage?

A

Mutations, cancer, aging, etc.

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15
Q

Which nucleotides are easily oxidized?

A

Guanine and thymine

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16
Q

How can altered structures of nucleotides affect function?

A

Aromaticity can affect stacking, can affect rotation around glycosidic bond, etc.

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17
Q

What does 8-oxo-guanine best pair with?

A

Adenine

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18
Q

What are some examples of nucleotide changes?

A

Oxidation, methylation, hydrolysis, UV irridation, aromatic amides, aromatic hydrocarbons, crosslinking, etc.

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19
Q

What effect can hydrolysis have on nucleotides?

A

Can make cytosine look like uracil (will bind to adenine)

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20
Q

What effect can UV irridation have on nucleotides?

A

Create thymine dimers (significant kinks in the DNA)

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21
Q

What are some examples of crosslinking agents?

A

Nitrogen mustard and cisplatin

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22
Q

What are the two basic categories of DNA replication?

A

BER and NER/NMR

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23
Q

What does BER stand for?

A

Base excision repair

24
Q

What is BER used for?

A

Repairing a single base (smaller changes to DNA structure)

25
Q

What does NER stand for?

A

Nucleotide excision repair

26
Q

What is NER used for?

A

Larger, global damage (lots of enzymes involved)

27
Q

What are the steps of BER?

A
  1. DNA glycosylate produces an AP site
  2. An AP endonuclease removes the base
  3. DNA polymerase and DNA ligase repair the DNA
28
Q

What is special about the 8-oxo-G BER pathway?

A

It is evolved to know the A is the mismatched pair (and not the 8-oxo-G), so a C is needed (not a T)

29
Q

What enzyme is involved in the 8-oxo-G pathway?

A

hOGG1

30
Q

What is chemical DNA synthesis based on?

A

Phosphate chemistry

31
Q

What is the problem with phosphate chemistry?

A

They are not the best electrophiles

32
Q

What phosphate was the first gene made with?

A

Phosphate triester

33
Q

What phosphates are used now for chemical synthesis?

A

Phosphoramidite

34
Q

What is the purpose of a solid phase in DNA synthesis?

A

Helpful for wash (don’t need constant purification)

35
Q

What are the steps of the phosphoamirite method of DNA synthesis?

A
  1. Deprotect DMT group to reveal 5’ OH
  2. Couple with activated 3’ phosphoamirite
  3. 5: Acetylate any unreacted 5’ OH (wash)
  4. Oxidize phosphite to phosphate
  5. Add base to cleave from solid phase and remove protecting groups on nucleobases
36
Q

What are the key point (7) of the phosphoamirite method?

A
  1. Synthesis of ssDNA
  2. Synthesis occurs in 3’ to 5’ direction
  3. Need protecting groups on exocyclic amines
  4. Limited to ~75-100-mer
  5. Final product has 3’ and 5’ OH (enzymatic phosphorylation needed)
  6. Purify product by HPLC or gel (big hydrophobic groups)
  7. Nanomoles of pure product
37
Q

What is the significance of water molecules in the Dickerson dodecamer?

A

They are found in the same spots, so DNA has good H-bonds for protein recognition

38
Q

What is molecular recognition of DNA based on (6)?

A
  1. Watson/Crick Binding (nucleotide invasion)
  2. Major groove binding (H-bonds)
  3. Minor groove binding (H-bonds)
  4. Sugar-phosphate backbone binding (electrostatics)
  5. Intercalation (pi stacking)
  6. Covalent/coordinate binding (metals)
39
Q

True or false: DNA can be a drug target

A

True

40
Q

What is intercalation?

A

Aromatic groups between bases)

41
Q

What are some examples of DNA intercalaters?

A

Ethidium bromide, doxorubicin

42
Q

What is the structure of an intercalator?

A

Lots of aromatic rings

43
Q

What does topoisomerase do?

A

Relaxes supercoiling (makes a nick in DNA)

44
Q

What does campothecin do?

A

Binds topo-cleavage complex

45
Q

What dies Farrell’s phosphate clamp do?

A

It is a polymetal (polycation) complex that binds to the sugar-phosphate backbone

46
Q

What governs major and minor groove recognition?

A

H-bonds

47
Q

What do transcription factors do?

A

They bind to the major groove of DNA to influence transcription

48
Q

What are some examples of transcription factors?

A

Zinc finger proteins, helix-turn-helix, leucine zippers

49
Q

What is the significance of the zinc finger sequence?

A

This can give insight into the pattern that it recognizes

50
Q

How do helix-turn-helix work?

A

There is a DNA recognition helix, and positioning helices to properly orient for major groove

51
Q

How do Zinc finger proteins work?

A

They have certain amino acids coordinated so that they can interact with the DNA

52
Q

How do leucine zippers work?

A

They have a coiled coil to orient the recognition amino acids properly

53
Q

How come alpha helices are usually used for DNA recognition?

A

They go in the same direction, and have a good geometric complement

54
Q

How does a triple helix form?

A

Uninterrupted purines on same strand

55
Q

What is needed for reverse Hoogsten base pairing>

A

A string of pyrimidines

56
Q

How can small molecules recognize DNA?

A

Through minor groove binding

57
Q

How come alpha helices cannot recognize the minor groove?

A

Geometric constraint

CHE 541 (20 decks)

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