Exam 2-2 Flashcards
What are the 3 fundamental rules of replication?
- Replication is semi-conservative.
- Replication begins at an origin and process bidirectionally.
- Synthesis of a new DNA strand occurs in the 5’ -> 3’ direction and is semidiscontinuous.
Fundamental Rule 1.
What does it mean that replication is semi-conservative?
It means that:
- The strands separate.
- New complementary strands synthesize using each separate strand as a template.
Meaning that after replication. The DNA has one old-strand and one new-strand.
Fundamental Rule 2.
What does it mean that replication is bidirectional?
That DNA is synthesized in two directions. (i.e. Leading and lagging strand)
Fundamental Rule 3.
Why is the new strand synthesized in the 5’->3’ direction and said to be semi-discontinuous?
DNA replication goes in the 5’ to 3’ direction because DNA polymerase acts on the 3’-OH of the existing strand for adding free nucleotides
It is said to be semi-discontinuous due to the leading and lagging strand)
What is DNA polymerase?
Is an enzyme that produces DNA molecules by assembling nucleotides and pairing them onto existing DNA strands.
What are the requirements for DNA polymerase?
- Template -> Basis for heredity
- dNTP (not ATP, nDP, nMP) ->
Building blocks - Mg2+ -> promotes reaction
- Primer -> needed to start the reaction
DNA Polymerase has 3 roles, what are they?
- 5’ -> 3’ DNA polymerase activity
- 3’ -> 5’ Exonuclease activity
- 5’ -> 3’ Exonuclease activity
What amino acid is present in the active site of DNA polymerase? What is its role?
You will always have 2 aspartate groups within the enzyme.
They aspartate groups stabilize and coordinate the Mg2+ groups
What is role of Mg2+ in DNA polymerase?
The Mg2+ stabilizes the charges of the phosphate groups of the dNTP.
It helps to orientate the bases correctly into the active site.
1 of the Mg2+ ions interacts with the 3’ oxygen atom of the sugar-phosphate backbone making it more nucleophilic.
How does the addition of the new dNTP occur?
The oxygen from the lastly synthesized nucleotide will attack the phosphate of the new nucleotide to produce a phosphodiester bond.
What three amino acids help to ensure that the right substrate is entering the active site?
Lys, Arg, and Tyr.
Lys and Arg will interact with the phosphates and Tyr will interact with the base to help the nucleotide come into the correct position by “closing the hand” ensuring there is a good fit.
DNA polymerase has only one active site for synthesis, how is able to distinguish the correct base for pairing?
A lot of it has to do with GEOMETRY. (i.e. A, D, and H properties -> acceptor, donator, h-bonding)
If the geometry is correct the alpha-phosphate of the incoming dNTP will be in close proximity to the 3’-Hydroxyl group for a nucleophilic attack.
Anything distorting the active site will inhibit how close the two factors need to be for a nucleophilic attack, resulting in no DNA replication.
rNTPs and dNTPs are extremely similar in structure, how does DNA polymerase distinguish between the two specifically?
Within the active site, there is a hydrophobic pocket. rNTPs has a 2’ hydroxyl group that would not allow for binding.
The hydroxyl group would also cause geometrical distortion, inhibiting how close the phosphate is to the 3’ Hydroxyl group needed for a nucleophilic attack.
In general what is 3’ -> 5’ exonuclease activity?
It is “Proofreading.”
It is a way for DNA Polyermase 1 to check on its errors and reduce the chances of a mutation.
Explain how the 3’ -> 5’ exonuclease activity works.
Occasionally, DNA Polymerase I will insert the wrong nucleotide onto the strand.
When there is a mismatch, DNAP1 will function very slowly or even come to a stop. The incorrect nucleotide causes the active site to become distorted.
This will initiate the proofreading function of DNAP1.
There is an active site on DNAP1, called the “Exonuclease Active Site” that cleaves the phosphodiester bond of the incorrect base.
DNAP1 will then add random bases into the active site until it get the correct base. After 5 tries, if the correct base is not inserted, DNAP1 will move on, leaving behind the incorrect base.
What is 5’ - 3’ Exonuclease Activity?
It is Nick translation.
It is responsible for DNA repair and primer removal during replication.
It is going to cleave nucleotides in front of it, opposite of 3’->’ exonuclease, which cleaves behind it.
DNA Polymerase is good and all, but what is the name enzyme synthesizing DNA in our body?
Polymerase III
When comparing DNA Polyermase I and III what does DNAP1 have that DNAP3 doesn’t have?
DNAP III does NOT have 5’ -> 3’ exonuclease activity.
What advantages does DNAP III have over DNAP I?
It is extremely fast at replication.
What is the structure of DNA Polymerase consist of?
- It has many subunits.
- Clamp loader
- Helicase
- 2 Core proteins
How were the factors of DNA replication discovered?
Via “temperature sensitive” mutations.
What is the role of DNA gyrase in prokaryotic DNA synthesis?
It relieves strain during replication, preventing supercoiling.
DNA replication is controlled at what step in synthesis?
At the initiation step.
What is DNA-A?
The protein that activates initiation of DNA replication.
What is OriC?
The area where DNA replication will start.
What is DNA-B?
The helicase that opens the replication fork, separating the two DNA strands.
What is DNA-C?
The helicase loader.
What is the general initiation scheme of DNA replication in E.Coli?
DNA-A recognizes OriC, and promotes the loading of DNA-B (helicase) by ultizing DNA-C (helicase loader)
DNA-A and DNA-C are coupled with what to help their role?
They are coupled with ATP.
DNA-A -> to attach to the OriC
DNA-C -> to attach DNA-B to the strand
Explain how initiation of the DNA strand in prokaryotes begins from start to end. (terms you can use (OriC, supercoiling, DUE region)
OriC has specific active sites that allow DNA-A to bind to the DNA strand.
These active sites have regions that allow for curvature when DNA-A is bound (similarly to histones).
Once DNA-A is bound to OriC, it will induce positive supercoiling, resulting in helical strain.
This strain will cause denaturation of the DUE region (which is rich in A-T), opening up the strand, creating single stranded DNA.
Once the single-stranded DNA has been available, DNA-B (helicase) will bind to the strand with the help of DNA-C-ATP.
How is DNA replication in prokaryotes regulated?
In short, SeqA binds to a newly synthesized hemimethylated DNA strand, moving the OriC to the cell membrane This will inhibit any DNA replication by preventing DNA-A from binding to the DNA molecule.
In long, once replication has completed, you end up with hemimethylated DNA strands.
Since Dam methylase doesn’t doesn’t associated itself with DNA right away, SeqA binds to the strand, inhibiting DNA-A-ATP from binding to the DNA molecule.
SeqA carries the OriC to the membrane of the cell for a period of time until it disassociates.
Once it disassociates, DNA is available for methylation via DAM methylase and DNA-A can bind again to the OriC.
THE IMPORTANcE OF HEMIMETHYLATED STRANDS!
Firstly, OriC becomes inaccessible to DnaA, and secondly, DnaA binds better to fully methylated DNA than hemi-methylated DNA.
What is SeqA and DAM methylase?
SeqA is a protein that binds to the OriC fragment GATC, and moves the hemi methylated strand to the exterior of the cell (cell membrane)
Dam methylase is an enzyme that adds methyl groups to the base adenosine.
Problem: Where and how to start replication?
What is the solution? is ATP involved?
DNA-ATP binds to origins with the help of helicase and SSB (single-stranded binding proteins)
ATP is involved. (via DNA-A-ATP and DNA-C-ATP)
