DNA Replication Flashcards
What does dna pol do
what types are there for e. Coli
It is an enzyme that catalyzes the addition of deoxyribonecleotide to a dna strand
Pol 1
Pol 111
In E. coli what does dna pol 1 do
DNA pol 111
Pol 1: removed the rna primer during dna replication, its a 5-3 and 3-5 exonuclease
Pol 111: a replication polaymerase , 3-5 exonuclease
What happens when an incoming dntp binds to dna pol
A conformational change of the enzyme,
The dntp goes into the tight pocket
How is a new dntp added to the growing 5-3 dna strand in replication
The incoming dntp has 3 phosphates ppp
The p that stay and bind to the 3’ oh of the growing strand is the alpha
beta and gamma phosphates leave as ppi
PPi gets hydrolyzed by pyrophosphatase and turns into 2pi
This makes energy for next dntp to be added
Slide 10
Equation
What type of catalysis does dna pol do
Catalysis by approximation and orientation because it orient the dNTPs to the growing strand
What is in the dna pol active site and why
Mg2+ cofactors
Is makes the 3’ oxygen of the previous base a better nucleophile
And it stabilizes the negative charge on the phosphates of the incoming dntp
What does replication require
DNTPs (datp,dctp,d GTP,dttp) and mg2+
Primer to start synthesis
What direction if dna synthesis and where is it assembled
How does dna pol correct mistakes
5’ to 3’
Read from the template strand
Used 3-5 exonuclease activity to correct mistakes (proofread),
does this by the mismatch first causing a stall in replication, then exonuclease uses hydrolysis to remove incorrect bases
What separates the dna to two seepage stands to be replicated
Helicase (ring like hexamers)
A single strand passers through the hole of the helicase and bind to two of its subunits. The helicase does a confirmatioal change by hydrolyzing atp
It now acts as a wedge to split the two strands apart
What is step 1 of E. coli dna replication
What does it contain
The origin of replication (OriC) is the start site of replication
Contains
DnaA which binds to start the prepriming complex
On DNAA is
SSB (single stranded binding protien) which stops the split dna duplex from
reannelaing
DNAB which is a helicase the uses atp to unwind the dna
What is step 2 of E. coli replication
Primase which is an rna polymerase add an rna primer in the 5-3’ detection to the temple strand
Around 10nucletides long
What is step 3 of E. coli replication
The leading and lagging strand are made
The lagging strand is made in Okazaki fragments on the bottom 3-5 template
Leading is one is made continuous on the 5-3 template
Does the dna pol 111 make the leading and lagging strand at the same time
What keeps the dna pol on the dna
Yes it makes them both at the same time at the replication fork
The sliding clamp (beta 2)
What is the dna oolaymerase 111 holoenzyme
It’s the full complex that helps in replication
Is has the a clamp loader and two dna polymerase cores
interacts with. DNAb (helicase)
Why are there Two opolymerase cores on the dna poll 111 holoenzyme
What is the trombone model
One makes the leading stand and one makes the lagging strand
It makes the lagging strand by forming it into a loop and making the 3’ end face the top and 5’ bottom.
It then makes the fragments, make around 1000 nucleotides, then increase the size of the loop
Add a rna primer to the new loop and makes more fragments
Pol 1 fills the fragment gaps and removes rna primer by 5-3 exonuclease activity
Ligase seals the fragments
This is the trombone model
How does ligase seal the Okazaki fragment
It joins the 3’ oh and the 5’ phosphate of two backbone fragments by hydrolyzing atp
What is the steps of ligase brining together the back bone
Intial charging
Transfer:
Phosphodiester bond formation
What happens in the intitial charging step of ligase
A catalytic lysine in the ligase attacks the aplha phosphate of atp
This makes lysyl amp (ligase+amp) and releases pyrophosphate
What happens in the transfer step of ligase
The adenylate ligase (lysyl amp) transfers amp to the 5’ phosphate of the nicked backbone
This makes amp+dna
What happens in the bond formation step of ligase
The 3’ oh of the mucked back bone attack the 5’ dna amp
This makes the phosphodiester bond and released amp
