Super Coiling
What is twist and writhe?
Twist: #of revolutions about the Axis
-#of turns for all intents and purposes
Writhe: #of super helical turns
-amount of supercoiling
Does circular DNA replication occur in Eukaryotic cells
Yes >:). The mitochondria has its own DNA, which is circular. So technically yes.
Is Nuclease activity in the 3'->5' direction looking ahead or behind the synthesis
Looking back, its review past work as it's in the opposite direction of synthesis. Proofreading.
What is the Ames test?
A way of testing if a substance is mutagenic.
Done by adding the substance you want to test on a bacteria plate with only a small amount of histidine needed for initial growth.
too many mutations: death
caused mutations: growth
no mutations: no growth
What is the name for a Base + a Sugar in DNA
Bonus: What type of bond do they share
Nucleoside
BA: Glycosidic Bond (Beta glycosidic linkage)
What type of super coil is this image, over or underwound?
Typical human DNA is what kind of supercoil, over or underwound?
Left and Positive, overwound
Typical is right and negative, underwound
Identify each nucleotide
Bonus points: which are purine which are pyrimidines
1. Adenine
2. Guanine
3. Thymine
4. Cytosine
5. Uracil
What is the main contributor to cell cycle control, and how do they work
(broad strokes)
CDKs or cyclin-dependent kinases, and they work via binding cyclin and phosphorylating protein which allows progression through the phases of the cell cycle.
Methodology is different for each phase. I'm hoping we won't need to know specifics as they aren't in the slides or textbook.
Identify which strand is formed from the lagging strand and which is formed by the leading strand. Determine which is the G-rich strand and C rich strand. Which strand does the Telomerase act on?
Top strand: leading, G rich, Telomerase target
Bottom strand: Lagging, C rich
True or False
DNA POL 1 removes the Uracil from the RNA primers and replaces them with Thymine to complete the replacement of the RNA with DNA
False DNA POL 1 removes the entire primer and replaces it with DNA.
Note: don't forget RNA has 2 hydroxyls, the sugars are not the same
What distinguishes Type 1 and type 2 Topoisomerases?
Bonus: What Topoisomerases apply + supercoils, what topoisomerases apply - supercoils
Type 1 perform single stranded breaks while type 2
perform double stranded breaks
Bonus:
+: Type 1b,1a
-: Type 1b,2(gyrase)
Show the mechanism for the polymerization of nucleotides
Answer sheet
Label all parts of the diagram. Enzymes, Lagging strand, leading strand ect.
Answer sheet
If you stain a cell and you see that it has both low Hoechst signaling and low Edu signaling. What phase of the cell cycle is it likely to be in?
G0 or G1. High Edu means it's in S phase, and low Hoechst signaling means it hasn't undergone much replication, and thus it must be G0 or G1
What is a Hoogsteen base pairing?
Bonus points: draw it
When a non-Watson-Crick pairing occurs where the purine rotates 180* with respect to the helical axis and takes a syn conformation
TLDR: it's when the purine flips
How does Camptothecin work and what makes it a good anti-cancer drug?
Hint: Topo 1b
Works on type 1b topoisomerase by binding to the tyrosine residue and preventing re-ligation, halting DNA synthesis
In eukaryotes, what do Alpha, Delta, and Epsilon polymerases do?
Hint: what are their difference? Processive or non-processive? Lagging or leading? Proofreading?
Bonus points: What is the purpose of RPA (Replication protein A)
Alpha: pseudo-primase; adds primer and a section of DNA nucleotides (~20). Moderately processive, Both lagging and leading.
Delta: Lagging-strand polymerase, 3->5 exonuclease, Processive when bound to PCNA (Proliferating cell nuclear Antigen)
Epsilon: leading strand polymerase, 3->5 exonuclease, very processive regardless of PCNA
Bonus points: basically SSB proteins
A Mutant E.coli strain has a mutation that leads to limited expression of the TUS (Terminalization utilization substance) protein. What would you expect from the E.Coli and would this mutation be lethal?
The under-expression of the TUS protein would lead to replisomes passing beyond their termination sites. But when they come into contact, they just bump off each other. Experimentally its shown that these cells would be viable; potential problems could be over-expression of the TER site, potential DNA damage (double/single strand breaks), or misincorporation of DNA. If a replisome encounters a strand break, it could lead to loss of a chromosome, which would be very bad.
How does the cell protect the telomeres from DNA repair enzymes?
(Can you name the complex?)
It forms a T loop, the shelterin complex (because it gives the ends of the DNA strand something to... shelter-in. Very clever, I know).
4 proteins
POT1 and TPP1 bind to the ssDNA over hang of telomeres
TRF1 and TRF2 bind to dsDNA and help form the T loop
Describe the Hayflick limit and why it exists. What famous human cells are the exception to this rule?
The Hayflick limit is the fact that cultured human somatic cells can only be replicated 20-60 times before they fail and cannot be cultured (Senescence). This is due to the Telomeres shortening with each generation.
Henrietta Lacks cells, or HeLa cells, are an exception as the cervical cancer cells have a means to get around this, being Telomeres
Interesting side note: HeLa cells have actually contaminated most cancer research labs. A large amount of research done is unintentionally on HeLa cells rather than the cancers they were supposed to be studying.
2 parts
1. Arrow Pushing: show me the mechanism of type 1 topoisomerase
Hint: Tyrosine residue
2. Conceptually explain how Topoisomerase 2 works. Include how ATP is involved.
1: answer sheet
2. There are 2 segments of the strand, the G and T segments. The G segment enters the Topoisomerase, ATP binds and causes a conformational change that arranges the T segment in place, the g strand is cut, and the t passes through. At which point the T segment leaves and the ATP dissociates from the enzyme as ADP and inorganic phosphate.
Name 3 things that lead to the High fidelity of DNA replication (in bacteria)
1. Induced fit (specificity pocket) only allowing Watson-Crick base pairing
2. Pol exonuclease activity (both 3'->5' and 5'->3')
3. DNA repair enzymes (NER, BER) Mismatch repair, etc.
You and the incredibly cool and studly Dr. Dennis Fineberg are studying a new disease. Upon further investigation, you notice that DNA replication is taking far longer than it should. Via electron microscopy, you realize that there are very few replication bubbles present on the Replicating DNA. You follow your incredible biologist intuition and test the DNA along with the replisome proteins and find no errors. The origin looks good. What is likely the cause of the ailment?
The pre-replication (G1) proteins ORC, Cdt1, and Cdc6 proteins are not functioning properly, only partially allowing the Helicase to attach replication to start.
Likely due to under-expression or mutants that decrease binding affinity
There are 4 main types of DNA repair we have covered(split into two categories). In broad strokes Explain how each works and what they are used to fix.
Types of repair: Alkylated nucleotides, Pyrimidine dimers, NER (Nucleotide excision repair (E.coli)), BER (base excision repair).
Bonus points: What are the categories?
Direct reversal of damage
Alkylated nucleotides: An alkyl transferase removes the alkyl group (via cysteine residue)
Pyrimidine dimers: An enzyme called Photolyase, using FADH- as a cofactor, splits the connected pyrimidine bases
Excision repair
NER: Thymine dimers or bulky groups to a base that lead to uneven DNA (helix distortion)
1) Uvra searches the DNA, finds the Lesion, recruits Uvrb, then leaves
2) Uvrc binds and cleaves 7bp upstream, while Uvrb cleaves 3-4 bp downstream
3) Uvrd helicase unwinds the strand releaing the 11-12 bp, then Pol 1 fills the gap and DNA ligase seals it
BER: Miss matched base
1) Glycosylase removes the base
2) AP endonuclease cleaves the backbone
3) Pol 1 adds new bases, and Ligase seals
Describe the process of how Helicase attaches in Humans and E.coli
Note: there are 2 separate but similar methods
E.coli
1. DnaA binds to DnaA boxes, 6 of these proteins interact to form a hexamer with DNA wrapped around its exterior. This formation favors seperation of strands at A=T sites forming ssDNA
2. DnaC facilitates the binding of hexameric DnaB to the ssDNA
3. DnaB creates space for the primase to attach
Humans
1. Orc 1-6 recognize the OriC site and aggregate around it forming the ORC (origin replication complex)
2. The ORC recruits proteins Cdc6 and Cdt1, 2 of each load onto both ends of the ORC (the Cd proteins are often called licensing factors)
3. The ORC, Cdc6, and Cdt1 recruit the MCM complex (hexamer of Mcm 2-7)
4. Protein kinases phosphorylate ORC, MCM (to make it active), and Cdc6 (inactivates Cdc6 and causes it to move to the cytoplasm)
Note on Cdc6: as far as I can tell its a licensing factor that if not removed will lead to over-replication. If it remained in the nucleus, it could cause the reassociation of the MCM and produce more DNA (bad obviously)
-Note about the Note: Cdc6 is phosphorylated by a cyclin-dependent kinase (CDK) as it signals to end replication around the S and G1 phase