Flashcards in Module 2 part one Deck (38):
What are the 4 phases of the cell cycle? Explain briefly what occurs in each
G0 phase; occurs when cells are not dividing, quiescent
1) G1 phase; cells are not dividing, but are growing
2) S phase; entire replication of cell genome
3) G2; second growth
4) M phase; mitosis where the parent becomes 2 daughters
What was Arthur Kornberg's three discoveries?
1) A way to measure the amount of DNA synthesis after lysing bacteria cells
2) Heat stable factors for DNA synthesis
3) DNA polymerase 1 found in the lysate
What were the heat stable factors that Arthur Kornberg found?
Nucleoside triphosphate aka dNTP's
What was Kornberg's "Purification of polymerase" briefly explain the process
A multi-step dilution / purification to isolate and monitor DNA pol Activity
1) lyse bacteria via sonication
2) remove DNA and RNA from lysate
3) precipitate proteins out
4) column chromatography for purification
Explain Kornberg's study of "requirements" of the DNA polymerization rxn
they performed 8 different experiments taking away different one synthesis factor each time. They used dTTP to monitor the effect of the effect on incorporation into DNA
What were the results of Kornberg's Requirements of Polymerization experiment
1) all 4 dNTP's must be present
2) intact DNA template
3) Mg++ cofactor
What occurred when one of the components of replication was removed in Kornberg's experiments?
There was almost a complete loss in enzyme activity. Radio-labelled dTTP incorporation measured this.
What are the six principles of DNA replication?
1) it is semi-conservative
2) replication is initiated at specific sites
3) it is bi-directional
4) it is semi discontinuous
5) requires RNA primers
6) Pol, nuclease, and ligases are needed
True or false, linear eukaryotic chromosomes have many origins of replication
What is the replication fork, what is the replication bubble?
Fork: point where helicase splits the template DNA
Bubble: The Opened DNA being replicated
In which direction is DNA synthesized?
5' to 3' direction. Therefore it runs along the template in the 3' to 5' direction
True or false, circular plasmids replicate bi - directionally
False, E. coli plasmid Col E1 is circular and replicates in one direction from a single origin of replication
Which way does the leading and lagging strands move inside the replication bubble?
The leading strand synthesizes moving toward the replication fork
the lagging strand moves away from the replication fork producing Okazaki fragments
(Okazaki fragments are longer in bacteria than eukaryotes)
Explain the linkage that occurs between incoming dNTP's and already bound dNTP's in terms of 5' and 3'
5'------3'OH alphaP dNTP
the top designates the newly growing 5'-3' strand. the alpha 5' phosphate of the new dNTP links to the 3'O- nucleophile (Mg++ takes of H). this is a covalent linkage
Explain the what RNA primers are? How are they made? where are they located?
RNA primers are synthesized by enzymes called Primases
They are 10-13 base pairs long and must be complementary to DNA template since they are the first 10-13 bound molecules which start the polymerization process. they also MUST HAVE A FREE 3'OH GROUP at the end.
True or false, primers can only be composed of RNA
False, some have been known to be DNA
What is a nick in DNA during replication?
On the lagging strand the RNA primers need to be removed which leaves gaps called nicks which need to be filled in
How does pol 1 fill in the nick in general?
DNA pol 1 degrades the RNA primer in the 5' to 3' direction using its 5'-3' exonuclease. This releases dMNP's. Simultaneous to this, pol 1 fills in the gaps in the same 5'-3' direction
What are the 4 components of pol 1 structure? what do they do
pol 1 resembles a right hand
1) Fingers domain: where new dNTP's enter. this closes when they are to be added
2) Thumb: holds Template in place
3) Palm: active site. attachment site of new dNTP's
4) 3'-5' exonuclease: proof reading function
explain pol 1 open conformation
the free 3'OH group of the latest added base pair is sitting in the active site. the fingers domain has bound a free dNTP
explain pol 1 closed conformation
the fingers domain approaches the palm domain so that the chemical rxn between alpha phosphate and free 3'OH can occur.
how does dna pol 1 incorporate the correct dntp each time?
shape recognition in active site. If G tries to pair with A, the complex wont fit within the closed form properly. This is governed by hydrogen bonding, van der waals forces, and ionic interactions
what are moieties?
elements required specifically for characteristic chemical rxn's
what are the 3 moieties in replication?
Aspartic acid residues: negative at physiological pH, they coordinate Mg++ ions.
M++ one: this is the top ion, it deprotonates the previously added dNTP's 3'OH (now a dMN). the created nucleophile can attack the alpha P of new dNTP
Mg++ two: bottom ion, binds to to the other phosphate groups for stability. also ensures the PPi group leaves rapidly
true or false, the 3'O- group attacks the alpha P group in an "in line attack"
what is pyrophosphate?
the leaving group
What are the two sites of the palm active site? Explain whats in both
Insertion site: contain template strand dNMP
Post insertion site: contains the 3'OH group of the previously added base pair.
How is the pol 1 able to move along the template to further replication?
Translocation whereby the pol 1 dissociates with DNA and glides along it to create a new insertion site. This is able to be done due to positive lysine residues in the palm interacting with the negative phosphate backbone of the template
explain what pol 1 processivity is?
the ability for it to catalyze many reactions without letting go of the substrate (template). The higher the processivity number the longer it holds on
what is processive synthesis
the concept of polymerase not letting go of DNA as it catalyses multiple cycles of rxns
What is distributive synthesis?
The pol completely dissociates from the template and rebinds so that the 3'OH group is now in the post insertion site
what occurs when DNA pol 1 senses a mis-paired base pair.
the pol 1 will fray the double stranded DNA by 4 nucleotides disrupting the hydrogen bonds of 3 correct and 1 incorrect base pair. By doing so, the incorrect dNMP ends up in the 3'-5' exonuclease domain which cleaves off the incorrect dMNP
Explain the 3' - 5' exonuclease active site
Also uses a two metal Mg++ system.
1) One Mg++ deprotonates a random water molecule which creates an OH- nucleophile. OH- attacks the phosphate group. the added nucleophile causes the phosphodiester bond to break
2)the second Mg++ facilitates the departure of the dNMP s
Why are Mg++ used for this?
the magnesium ions lower the pKa of the water molecule (or the 3'OH in polymerization)
which prompts the departure of an H group.
Explain why proof reading is no the reverse of polymerization
In proof reading, the final product is a dNMP not a dNTP
True or false, the 5'-3' exonuclease uses a unique system to facilitate the removal of dMNP's when removing primers
false, it uses a very similar two metal divalent system to the 3'-5' exonuclease
Why is 3'-5' exonuclease called 3'5'?
it digests the dNMP's shortening the strand from the 3' side while the 5' side remains intact. The opposite is true for 5'-3' exonuclease