1. DNA Structure and Eukaryotic Genome Organization
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copy deck
- What is the Central Dogma?
- DNA ->/<- RNA -> Protein
- What copies DNA?
- Replication
- What allows DNA and RNA to interchange?
- Transcription and Reverse Transcription
- What takes RNA into Protein?
- Translation
- What is the DNA structure like From microscopic to macroscopic?
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-DNA helix
-Chromatin - beads on string
-Chromatin makes up fibers
-Fibers form loops (75000 bp)
-6 loops make up one rosette
-30 rosettes stacked = 1 coil
-10 coils like a phone cord = 1 chromatid - What are the fundamental building blocks of DNA?
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-Nitrogenous bases (2 flavors)
-Sugars
-Phosphate backbone - What are the pyrimidines?
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Cytosine
Uracil
Thymine - What are the purines?
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Guanine
Adenine - What other molecules are nucleotides important building blocks of?
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-NAD+
-Coenzyme A
-cAMP/cGMP
-ATP/GTP
-ppGpp - What is ppGpp?
- Guanosine tetraphosphate
- What is ppGpp important in?
- Regulating transcription in bacteria.
- What kind of bond joins the nucleotides together in DNA?
- Phosphodiester bond between the 5'-3' hydroxy groups on the phosphate backbone.
- In what direction are bases sequences written?
- 5' -> 3'
- What direction are phosphodiester bonds given in?
- They're called 3'->5' phosphodiester bonds b/c the 3' OH attacks the alpha phosphate of the incoming nucleotide.
- What are the features of B-form DNA?
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-Antiparallel strands
-Bases inside, backbone out
-Bases stacked, perpendicular to helical axis.
-10.5 nt / turn
-Hydrogen bp hold structure together
-Base sequence is unrestricted -
Where in DNA are there:
-COVALENT linkages?
-NONCOVALENT linkages? -
Covalent: in the phosphodiester backbone.
Noncovalent: in the base pairs - What are the 3 noncovalent forces holding together the DNA double strand?
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-Hydrogen bonding btwn base pairs
-Hydrophobic effect (all hydrophobic uncharged bases hide from water)
-Electrostatic force arising from base stacking and Van der Waal interactions. - Why are their major and minor grooves in DNA?
- Because the glycosidic bonds of the base pairs are not diametrically opposed.
- Why are grooves in DNA useful?
- It makes the base pairs accessible for recognition and binding of other proteins.
- What is a certain cancer drug that intercalates the minor groove to exert its effect?
- Actinomycin D
- What is Actinomycin D's effect?
- Interferes w/ RNA/DNA synthesis
-
So what part of DNA would bind
-Base-Specific proteins?
-Nonspecific proteins? -
Base spcfc: major/minor groove
Nonspecfic: phosphate backbone - What is A form DNA?
- A tighter, dehydrated structure. Still right-handed like B form.
- What is the most dramatically different form of DNA?
- Z form - left handed
- What is the significance of different DNA forms?
- Regulation of the DNA within specific regions of the cell.
- What causes bent DNA?
- A string of 4-6 A with a spacer of 10 nt.
- What is the result of bent DNA?
- Distorted spacing of the major and minor grooves - which interferes with protein binding of the grooves.
- What drug induces bent DNA?
- Cisplatin
- How does Cisplatin work?
- Binds both DNA strands; induces intrastrand cross bridging.
- So what is the result of Cisplatin?
- Inhibition of cell growth
- In what type of nucleic acid is non-Watson Crick bpairing more commonly found? Why?
- RNA - because it is singled stranded and more capable of forming alternative H-bonds with non-normal bases.
- How does DNA Triple Helix formation play a role in Hereditary Persistance of fetal Hgb?
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-Naturally forming triplex in fetal Hb promotor necess to downregulate fetal Hgb;
-Mutation destabilizes helix and y-globin persists. - What name denotes the base pair formed in DNA triplexes?
- Hoogsteen base pairs
- What 2 variables can be used to disrupt H-bonds and denature DNA?
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1. Changes in pH
2. Changes in temp - What is the Tm?
- The temperature at which half the helical structure is lost.
- What increases the bond strength of a DNA helix?
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-Increased GC content - b/c it contains 3 H bonds per GC pair
-Increased salt content of solvent - How is DNA melting monitored?
- By measuring DNA's absorbance at 260 nm.
- How does absorbance change when DNA is melted?
- It increases - ssDNA absorbs more than dsDNA does, b/c the stacked base pairs are shielded and absorb less energy.
- What term denotes the increase in absorbance?
- Hyperchromic shift at 260 nm.
- Why does increased GC content increase the strength of a DNA double strand?
-
2 reasons:
1. 3 H bonds
2. Increased Van der Waals interactions in GC base pairs - What would cause a decreased Tm?
-
-Decreased GC content
-Decreased salt content - What would cause an increased?
- the opposite...
- Why does changing salt concentration change the stregnth of the DNA helix?
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-Higher ionic strength shields the negatively charged phosphate backbone;
-Lower strength allows the charges to predominate and pull charge away from the interior H bonds of the base pairs, decreasing stability. - In what 3 ways can DNA molecules be found? (states)
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-Linear
-Relaxed circular
-Supercoiled circular -
What causes
-Positive supercoiling?
-Negative supercoiling? -
Pos: overwinding
Neg: underwinding - For every untwist of a DNA helic twist, how many supercoils are created?
- 1 neg supercoil per untwist.
- What enzymes relieve the tension caused by supercoiling?
- Topoisomerases
- How does Topoisomerase I work?
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Relieves supercoils by
1. Nicks one DNA strand
2. Passes a DNA segment thru
3. Reseals the nick - What type of supercoiling does Topo I work on?
- Both pos and negative.
- Do topoisomerases need ATP?
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Type one, no: their action is thermodynamically favorable.
Type Two, YES. - How does Topo II work?
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Relieves supercoils by
1. Binds One DNA coil/2 ATP
2. Cleaves one dsDNA coil
3. Passes other strand thru
4. Reseals complete DNA coil - So in general what does Topo II do?
- Relaxes negative and positive supercoils
- What enzyme can introduce negative supercoils?
- Only prokaryotic Type II topoisomerase GYRASE.
- Eukaryotic DNA is not circular, so how do supercoils arise?
- B/c of the fact that eukaryotic DNA is packaged in nucleosomes.
- How does supercoiling affect the way DNA shows on a gel?
- It compacts it, so it moves faster
- What are the medical implications of our knowledge on superhelicity and topoisomerases?
- We can inhibit the bacterial Topo II (gyrase) and kill the bacterial cells, without affecting eukaryotic topo.
- What are two drugs that inhibit prokaryotic gyrase?
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-Novobiocin
-Ciprofloxacin - What is Ciprofloxacin used to treat?
- Bacillus anthracis
- What are 2 drugs that inhibit EUKARYOTIC Topo II? What are they used to treat?
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Doxorubicin + Etoposide
-Treats cancer - How exactly do Topo II inhibitors WORK?
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-Disable the ATPase activity
-Enhance nicking or reduce resealing, leaving permanent nicks in the DNA. - How do eukaryotes get ~1 meter of DNA into 1 tiny cell?
- By packaging it into chromatin.
- What is a 30 nm fiber of chromosome material made of?
- Nucleosomes
- What is a nucleosome?
- Supercoils of DNA wrapped around histones.
- What are histones?
- Positively charged basic proteins that form ionic bonds with the phosphate backbone of DNA.
- Why are histones good for DNA helix stability?
- They neutralize the large negative charge of the backbone.
- What is a nucleosome core made of?
- H2A, H2B, H3, and H4
- What links Nucleosomes?
- Linker DNA
- What stabilizes the nucleosome/linker structure?
- Histone 1 protein
- What type of structure is each part of a nucleosome core?
- a dimer - so that makes the whole nucleosome core an octomer.
- What are HATS?
- Histone Acetyl Transferases
- What do HATS do?
- Add acetyl groups to the terminal NH3+ groups of Lys residues in histone core (H3/H4)
- What does acetylation of histone core subunits do?
- Neutralizes the basicity of histones, which reduces their affinity for DNA - this OPENS UP THE DNA CHROMOSOME.
- What 2 amino acid residues are largely responsible for histone protein's basicity?
- lys and arginine
- What are HDACS?
- Histone deacetylases
- What do HDACS do?
- Repress active (open) regions of chromatin.
- What's an example of a gene that turns off chromatin opening-up?
- Sir 2