19 - Nucleic Acids II Flashcards
What is the replication method for DNA?
Semi-conservative
What are the functions (3) of DNA polymerase?
- Copy DNA in 5’ to 3’ direction
- Remove incorrect bases through 3’ to 5’ exonuclease activity
- 5’ to 3’ exonuclease activity to remove Okazaki fragments
What is an exonuclease?
Cleave DNA at end of strand
What is an endonuclease?
Cleave DNA at middle of strand
What is needed to synthesize DNA?
RNA primer, template strand, and Mg2+ cation (for enzyme and electrostatics)
What are the reactants for DNA synthesis?
Deoxynucleotide triphosphates
What are some unresolved questions about DNA synthesis?
- How is chromatin reassembled behind the fork?
- How do interior proteins interact with the origin?
- How do cells coordinate leading and lagging strand synthesis?
- How are helicases activated? How do helicases unwind DNA?
- How do cells limit replication to a single round?
What factors contribute to replication fidelity?
Selectivity, proofreading, and mismatch repair
What is the overall error rate of DNA synthesis?
~10^-9
What can defective proofreading lead to?
Cancer in mice
How is selectivity achieved in the insertion?
The relative rate of incorporation is highest for the proper nucleotide (eg: A-T) compared to a mismatched on (eg: A-C)
How is selectivity achieved in the extension?
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)
What are some instabilities in DNA?
Oxidation, crosslinking, etc.
What are some consequences of DNA damage?
Mutations, cancer, aging, etc.
Which nucleotides are easily oxidized?
Guanine and thymine
How can altered structures of nucleotides affect function?
Aromaticity can affect stacking, can affect rotation around glycosidic bond, etc.
What does 8-oxo-guanine best pair with?
Adenine
What are some examples of nucleotide changes?
Oxidation, methylation, hydrolysis, UV irridation, aromatic amides, aromatic hydrocarbons, crosslinking, etc.
What effect can hydrolysis have on nucleotides?
Can make cytosine look like uracil (will bind to adenine)
What effect can UV irridation have on nucleotides?
Create thymine dimers (significant kinks in the DNA)
What are some examples of crosslinking agents?
Nitrogen mustard and cisplatin
What are the two basic categories of DNA replication?
BER and NER/NMR
What does BER stand for?
Base excision repair
What is BER used for?
Repairing a single base (smaller changes to DNA structure)
What does NER stand for?
Nucleotide excision repair
What is NER used for?
Larger, global damage (lots of enzymes involved)
What are the steps of BER?
- DNA glycosylate produces an AP site
- An AP endonuclease removes the base
- DNA polymerase and DNA ligase repair the DNA
What is special about the 8-oxo-G BER pathway?
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)
What enzyme is involved in the 8-oxo-G pathway?
hOGG1
What is chemical DNA synthesis based on?
Phosphate chemistry
What is the problem with phosphate chemistry?
They are not the best electrophiles
What phosphate was the first gene made with?
Phosphate triester
What phosphates are used now for chemical synthesis?
Phosphoramidite
What is the purpose of a solid phase in DNA synthesis?
Helpful for wash (don’t need constant purification)
What are the steps of the phosphoamirite method of DNA synthesis?
- Deprotect DMT group to reveal 5’ OH
- Couple with activated 3’ phosphoamirite
- 5: Acetylate any unreacted 5’ OH (wash)
- Oxidize phosphite to phosphate
- Add base to cleave from solid phase and remove protecting groups on nucleobases
What are the key point (7) of the phosphoamirite method?
- Synthesis of ssDNA
- Synthesis occurs in 3’ to 5’ direction
- Need protecting groups on exocyclic amines
- Limited to ~75-100-mer
- Final product has 3’ and 5’ OH (enzymatic phosphorylation needed)
- Purify product by HPLC or gel (big hydrophobic groups)
- Nanomoles of pure product
What is the significance of water molecules in the Dickerson dodecamer?
They are found in the same spots, so DNA has good H-bonds for protein recognition
What is molecular recognition of DNA based on (6)?
- Watson/Crick Binding (nucleotide invasion)
- Major groove binding (H-bonds)
- Minor groove binding (H-bonds)
- Sugar-phosphate backbone binding (electrostatics)
- Intercalation (pi stacking)
- Covalent/coordinate binding (metals)
True or false: DNA can be a drug target
True
What is intercalation?
Aromatic groups between bases)
What are some examples of DNA intercalaters?
Ethidium bromide, doxorubicin
What is the structure of an intercalator?
Lots of aromatic rings
What does topoisomerase do?
Relaxes supercoiling (makes a nick in DNA)
What does campothecin do?
Binds topo-cleavage complex
What dies Farrell’s phosphate clamp do?
It is a polymetal (polycation) complex that binds to the sugar-phosphate backbone
What governs major and minor groove recognition?
H-bonds
What do transcription factors do?
They bind to the major groove of DNA to influence transcription
What are some examples of transcription factors?
Zinc finger proteins, helix-turn-helix, leucine zippers
What is the significance of the zinc finger sequence?
This can give insight into the pattern that it recognizes
How do helix-turn-helix work?
There is a DNA recognition helix, and positioning helices to properly orient for major groove
How do Zinc finger proteins work?
They have certain amino acids coordinated so that they can interact with the DNA
How do leucine zippers work?
They have a coiled coil to orient the recognition amino acids properly
How come alpha helices are usually used for DNA recognition?
They go in the same direction, and have a good geometric complement
How does a triple helix form?
Uninterrupted purines on same strand
What is needed for reverse Hoogsten base pairing>
A string of pyrimidines
How can small molecules recognize DNA?
Through minor groove binding
How come alpha helices cannot recognize the minor groove?
Geometric constraint
