L2

Question 1

dna polymerase

Answer 1

● Holds a loose nucleotide (with 3 PO4)
● The 3’ strand of the DNA reacts with the PO4
● DNA polymerase needs to be attached to a DNA or RNA first

Question 2

rna polymerase

Answer 2

does not need a primer

Question 3

replication process

Answer 3

initiation, elongation, termination
● Proteins attach to the DNA, and put it under torsion and de methylate it
● Helicase gets added, which separates the DNA strands
● DNA polymerase is added, and it works from 5’ to 3’, where 3’ gets longer,
in the normal strand
● In the lagging strand, which goes 3’ to 5’, which means it needs to be
synthesized in the opposite direction, for this, primers are put down, which
allow short replication in segments, which are called Okazaki fragments, at
the end of each, the dna polymerase has to move back and do it again.
● DNA polymerase 3 stops at the primer of the next Okazaki fragment
● DNA polymerase 1 degrades the primer, which leaves the two fragments
barely disconnected; they are then fused into a single line by DNA ligase

Question 4

dna ligase reaction

Answer 4

1. DNA ligase grabs a phosphate bound to ribose and Adenine (also called
   AMP)
2. In the zone where the DNA is nicked, there is a loose phosphate on one
   side, and a loose OH on the other. The AMP connects to the loose
   phosphate.
3. The enzyme connects the previously loose phosphate to the loose OH,
   sealing the nick in the DNA and spending. The Ligase and the AMP
   dissociate from each other and the DNA afterwards

Question 5

dna polymerase 1

Answer 5

● Slowest and shortest (20 nucleotides per second)
● Can add between 3 and 200 nucleotides
● Can remove primers
- in e coli also removes okazaki fragments

Question 6

dna polymerase type 2

Answer 6

● Fast-ish, 40 nucleotides per second
● Can add 150 nucleotides before dying

Question 7

dna polymerase tup 3

Answer 7

● Between 250 and 1000 nucleotides per second
● Can add up to 500k nucleotides

Question 8

dna polymerase

Answer 8

needs a primer with 3’OH end to add nucleotide to growing chain

Question 9

rna primer removal and okazaki fragment ligation

Answer 9

• DNA polymerase III stops at the primer of
  the next Okazaki fragment.
• DNA polymerase I uses its 5’ → 3’
  exonuclease domain to degrade the RNA
  primer and its 5’ → 3’ DNA synthesis
  activity to replace the RNA primer with
  dNTPs.
• This leaves one 3’OH – 5’PO4 break.
• E.coli DNA ligase catalyses the covalent
  closure of this gap at the cost of one NAD+
  molecule.
• Phage T4 DNA ligase uses one ATP for the
  same ligation reaction.

Question 10

beta clamp

Answer 10

polymerase strongly bound to template via ring, prevents dissociation of the polymerase from the template dna enabling synthesis of dna strand that are long

Question 11

dna plolymerase choises

Answer 11

1. keep elongating
2. reverse and use 3’to 5’ exonuclease activity to break down nascent rna (when faulty base pair in binding site which proof reads)
3. fall of template (inhibited by beta clamps)