DNA Replication

Question 1

What does dna pol do

what types are there for e. Coli

Answer 1

It is an enzyme that catalyzes the addition of deoxyribonecleotide to a dna strand

Pol 1

Pol 111

Question 2

In E. coli what does dna pol 1 do

DNA pol 111

Answer 2

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

Question 3

What happens when an incoming dntp binds to dna pol

Answer 3

A conformational change of the enzyme,
The dntp goes into the tight pocket

Question 4

How is a new dntp added to the growing 5-3 dna strand in replication

Answer 4

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

Question 5

Slide 10

Answer 5

Equation

Question 6

What type of catalysis does dna pol do

Answer 6

Catalysis by approximation and orientation because it orient the dNTPs to the growing strand

Question 7

What is in the dna pol active site and why

Answer 7

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

Question 8

What does replication require

Answer 8

DNTPs (datp,dctp,d GTP,dttp) and mg2+

Primer to start synthesis

Question 9

What direction if dna synthesis and where is it assembled

How does dna pol correct mistakes

Answer 9

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

Question 10

What separates the dna to two seepage stands to be replicated

Answer 10

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

Question 11

What is step 1 of E. coli dna replication

What does it contain

Answer 11

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

Question 12

What is step 2 of E. coli replication

Answer 12

Primase which is an rna polymerase add an rna primer in the 5-3’ detection to the temple strand

Around 10nucletides long

Question 13

What is step 3 of E. coli replication

Answer 13

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

Question 14

Does the dna pol 111 make the leading and lagging strand at the same time

What keeps the dna pol on the dna

Answer 14

Yes it makes them both at the same time at the replication fork

The sliding clamp (beta 2)

Question 15

What is the dna oolaymerase 111 holoenzyme

Answer 15

It’s the full complex that helps in replication

Is has the a clamp loader and two dna polymerase cores

interacts with. DNAb (helicase)

Question 16

Why are there Two opolymerase cores on the dna poll 111 holoenzyme

What is the trombone model

Answer 16

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

Question 17

How does ligase seal the Okazaki fragment

Answer 17

It joins the 3’ oh and the 5’ phosphate of two backbone fragments by hydrolyzing atp

Question 18

What is the steps of ligase brining together the back bone

Answer 18

Intial charging

Transfer:

Phosphodiester bond formation

Question 19

What happens in the intitial charging step of ligase

Answer 19

A catalytic lysine in the ligase attacks the aplha phosphate of atp

This makes lysyl amp (ligase+amp) and releases pyrophosphate

Question 20

What happens in the transfer step of ligase

Answer 20

The adenylate ligase (lysyl amp) transfers amp to the 5’ phosphate of the nicked backbone

This makes amp+dna

Question 21

What happens in the bond formation step of ligase

Answer 21

The 3’ oh of the mucked back bone attack the 5’ dna amp

This makes the phosphodiester bond and released amp