19 - Nucleic Acids II

Question 1

What is the replication method for DNA?

Answer 1

Semi-conservative

Question 2

What are the functions (3) of DNA polymerase?

Answer 2

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

Question 3

What is an exonuclease?

Answer 3

Cleave DNA at end of strand

Question 4

What is an endonuclease?

Answer 4

Cleave DNA at middle of strand

Question 5

What is needed to synthesize DNA?

Answer 5

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

Question 6

What are the reactants for DNA synthesis?

Answer 6

Deoxynucleotide triphosphates

Question 7

What are some unresolved questions about DNA synthesis?

Answer 7

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?

Question 8

What factors contribute to replication fidelity?

Answer 8

Selectivity, proofreading, and mismatch repair

Question 9

What is the overall error rate of DNA synthesis?

Answer 9

~10^-9

Question 10

What can defective proofreading lead to?

Answer 10

Cancer in mice

Question 11

How is selectivity achieved in the insertion?

Answer 11

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

Question 12

How is selectivity achieved in the extension?

Answer 12

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)

Question 13

What are some instabilities in DNA?

Answer 13

Oxidation, crosslinking, etc.

Question 14

What are some consequences of DNA damage?

Answer 14

Mutations, cancer, aging, etc.

Question 15

Which nucleotides are easily oxidized?

Answer 15

Guanine and thymine

Question 16

How can altered structures of nucleotides affect function?

Answer 16

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

Question 17

What does 8-oxo-guanine best pair with?

Answer 17

Adenine

Question 18

What are some examples of nucleotide changes?

Answer 18

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

Question 19

What effect can hydrolysis have on nucleotides?

Answer 19

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

Question 20

What effect can UV irridation have on nucleotides?

Answer 20

Create thymine dimers (significant kinks in the DNA)

Question 21

What are some examples of crosslinking agents?

Answer 21

Nitrogen mustard and cisplatin

Question 22

What are the two basic categories of DNA replication?

Answer 22

BER and NER/NMR

Question 23

What does BER stand for?

Answer 23

Base excision repair

Question 24

What is BER used for?

Answer 24

Repairing a single base (smaller changes to DNA structure)

Question 25

What does NER stand for?

Answer 25

Nucleotide excision repair

Question 26

What is NER used for?

Answer 26

Larger, global damage (lots of enzymes involved)

Question 27

What are the steps of BER?

Answer 27

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

Question 28

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

Answer 28

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)

Question 29

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

Answer 29

hOGG1

Question 30

What is chemical DNA synthesis based on?

Answer 30

Phosphate chemistry

Question 31

What is the problem with phosphate chemistry?

Answer 31

They are not the best electrophiles

Question 32

What phosphate was the first gene made with?

Answer 32

Phosphate triester

Question 33

What phosphates are used now for chemical synthesis?

Answer 33

Phosphoramidite

Question 34

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

Answer 34

Helpful for wash (don't need constant purification)

Question 35

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

Answer 35

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

Question 36

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

Answer 36

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

Question 37

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

Answer 37

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

Question 38

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

Answer 38

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)

Question 39

True or false: DNA can be a drug target

Answer 39

True

Question 40

What is intercalation?

Answer 40

Aromatic groups between bases)

Question 41

What are some examples of DNA intercalaters?

Answer 41

Ethidium bromide, doxorubicin

Question 42

What is the structure of an intercalator?

Answer 42

Lots of aromatic rings

Question 43

What does topoisomerase do?

Answer 43

Relaxes supercoiling (makes a nick in DNA)

Question 44

What does campothecin do?

Answer 44

Binds topo-cleavage complex

Question 45

What dies Farrell's phosphate clamp do?

Answer 45

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

Question 46

What governs major and minor groove recognition?

Answer 46

H-bonds

Question 47

What do transcription factors do?

Answer 47

They bind to the major groove of DNA to influence transcription

Question 48

What are some examples of transcription factors?

Answer 48

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

Question 49

What is the significance of the zinc finger sequence?

Answer 49

This can give insight into the pattern that it recognizes

Question 50

How do helix-turn-helix work?

Answer 50

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

Question 51

How do Zinc finger proteins work?

Answer 51

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

Question 52

How do leucine zippers work?

Answer 52

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

Question 53

How come alpha helices are usually used for DNA recognition?

Answer 53

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

Question 54

How does a triple helix form?

Answer 54

Uninterrupted purines on same strand

Question 55

What is needed for reverse Hoogsten base pairing>

Answer 55

A string of pyrimidines

Question 56

How can small molecules recognize DNA?

Answer 56

Through minor groove binding

Question 57

How come alpha helices cannot recognize the minor groove?

Answer 57

Geometric constraint