L2 Flashcards
dna polymerase
● 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
rna polymerase
does not need a primer
replication process
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
dna ligase reaction
- DNA ligase grabs a phosphate bound to ribose and Adenine (also called
AMP) - 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. - 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
dna polymerase 1
● Slowest and shortest (20 nucleotides per second)
● Can add between 3 and 200 nucleotides
● Can remove primers
- in e coli also removes okazaki fragments
dna polymerase type 2
● Fast-ish, 40 nucleotides per second
● Can add 150 nucleotides before dying
dna polymerase tup 3
● Between 250 and 1000 nucleotides per second
● Can add up to 500k nucleotides
dna polymerase
needs a primer with 3’OH end to add nucleotide to growing chain
rna primer removal and okazaki fragment ligation
- 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.
beta clamp
polymerase strongly bound to template via ring, prevents dissociation of the polymerase from the template dna enabling synthesis of dna strand that are long
dna plolymerase choises
- keep elongating
- reverse and use 3’to 5’ exonuclease activity to break down nascent rna (when faulty base pair in binding site which proof reads)
- fall of template (inhibited by beta clamps)