DNA Replication Flashcards
Requirements for synthesis
- Deoxynucleoside triphosphates (dNTPs) - DNA - rNTPs - RNA
2. Primer - template junction
Types of polymerase
- DNA-directed polymerase
-replication of DNA
-requires a 3’ end - DNA-directed RNA polymerase
-transcription of DNA
-primer formation during DNA replication (does not require 3’
end)
Compare RNA polymerase and DNA polymerase in ability when starting replication
- RNA polymerase can start new RNA chains de novo
- DNA polymerase just needs a 3’ end ( can use one on RNA or DNA)
In what direction does synthesis occur
Occurs by extension of 3’ end - replication is 5’-3’ but moves along the template 3’-5’
Sn2 reaction
- dNTP pairs with its base pair (A-T, C-G) by hydrogen bonding
- 3’OH attacks αphosphate on the dNTP.
- phosphate β and γ stay together (pyrophosphate) but separates from dNTP
- the strong phosphodiester bond is not formed under this reaction (not enough energy)
Creation of the phosphodiester bond
- Hydrolysis of pyrophosphates yields the required energy
- the phosphodiester bond forms
- extremely favorable form - hard to break down
DNA polymerase ensures that the correct nucleotide is incorporated into the growing strand of DNA by __________.
A. detecting the identity of the incoming nucleotide
B. specifically binding only the correct nucleotide in the active site
C. selecting the correct nucleotide in a exonuclease active site in the DNA polymerase
D. monitoring the ability of the incoming nucleotide to form either an A:T or G:C base pair
D. monitoring the ability of the incoming nucleotide to form either an A:T or G:C base pair
How does DNA polymerase know it has the correct base?
No base pairing if incorrect base
Why are rNTPs not incorporated?
rNTP cannot bond because is pushed to far that it cannot bind it’s phosphate group to the 3’OH
Hand model of DNA polymerase - palm function
Catalytic site
Hand model of DNA polymerase - fingers
Bend template and interact with dNTPs
Hand model of DNA polymerase - thumb
Holds polymerase and DNA together
What are the important amino acids found on the “finger” of polymerase
- Tyr
- Arg
- Lys
Function of Tyr amino acid on finger of polymerase
Interacts with base - pushes base so hydrogen bonding can occur
Function of Arg/Lys amino acid on finger of polymerase
Interacts with phosphate group - puts in place with right orientation
Catalysis in polymerase
- Happens in palm
- A preps primer
- B preps dNTP
- B stabilizes pyrophosphate
Function of metal ions in catalysis that occurs in palm
Two metal ions present
A. Interacts with 3’-OH - reduces association between O-H
B. interacts with triphosphate group of dNTP - neutralizes negative charge
Mispaired base pair
- mismatch disrupts palm-DNA interaction
- creates higher affinity between exonuclease and ss 3’ DNA compared to polymerase site.
- end of prom moves to exonuclease active site
- mismatch removed - affinity changes
- primer moves back to polymerase active site
Compare replication between leading and lagging strands
- Leading strand -> continuous replication
2. Lagging strand -> segmented (Okazaki fragments)
Unwinding of DNA
- Helicase unwinds DNA
- Ring-shaped = high degree of processivity
- Pulls DNA through center of ring
- Requires ATP hydrolysis for energy
- Usually bonds to lagging strand
Problem of separating DNA strands
- Supercoiling occurs ahead of replication fork
2. If not relieved replication stops
Solution to the problem of unwinding DNA
- Topoisomerase relieves the supercoiling of DNA
- Cuts DNA ahead of fork, changes the flips the direction of the coil (positive to negative supercoil).
- Reseals DNA
How does the cell prevent annealing?
Single stranded DNA-binding proteins prevent annealing
Also protects against possible mutations (DNA is at its most vulnerable form).
Basic needs of DNA polymerase
- Template DNA
- Primer
- dNTPs
- Metal ions
DNA polymerase can initiate DNA replication
False - needs a primer (requires primase)
Function of the sliding clamp
Quickens polymerase reattaching to DNA for replication increasing processivity
- Polymerase detaches every 20-100base pairs
- Sliding clamps keeps the polymerase from diffusing away
Prokaryote polymerase
DNA pol I-V
- DNA pol I
- DNA pol III
Eukaryote DNA polymerase
Have DNA pol (Greek letters)
- DNA pol switching
- DNA pol α
- DNA pol δ and ε
Prokaryotic DNA polymerase III holoenzyme
- 3 pol III
- primary enzyme involved in chromosome replication
- polymerase activity
- exonuclease activity
- 2 sliding clamps
- 1 clamp loader
- Highly processive
- Proof reading function
Prokaryotic DNA pol I
- Removes RNA-DNA linkage left behind by RNase H (5’ exonuclease)
- Fills in gaps left behind by RNase H
- Not highly processive
- Has no clamp
Removing a primer
- RNase removes almost all RNA strands on DNA
- Cleaves only RNucleotide
- The H of RNase H = hybrid
Binding backbone
DNA polymerase cannot bind backbone
Ligase binds backbone
Eukaryotic DNA polymerases switch
Have multiple subunits
- DNA pol α - primase
- involved in initiating new DNA strands
- creates RNA primer and then start DNA copy
- DNA pol ε - synthesizes leading strand
- DNA pol δ - synthesizes lagging strand
