Lesson 18: DNA Replication 2 Flashcards
DNA polymerase requires what
an origin of replication
DNA Polymerases:
initiation and mechanism of action
where does replication occur at
replication forks
what direction can replication from the origin move in
unidirectionally or bidirectionally
what direction does DNA synthesis occur in
unidirectional from 5’ to 3’
—> synthesis only occurs in 1 direction - meaning nucleotides can be only added to the 3’ end
reaction mechanism for DNA synthesis
(1) each incoming dNTP is positioned by base pairing with the appropriate template nucleotide
(2) a phosphodiester bond is created by nucleophilic attack of the primer-strand 3’ hydroxyl group of the alpha-phosphate on the incoming dNTP
(3) after phosphodiester bond formation then next nucleotide, dATP, is fitted into the template
why is Mg2+ required
metal-ion-catalysis
– neutralizing negative charges
leading strand
continuous
lagging strand
discontinuous
okazaki fragemets
short fragments on lagging strand
what direction does the leading strand go in
– same direction of the replication fork
excision and end of ookazaki fragments
- RNA primers need to be removed
- okazaki fragments need to be ligated
—— repliacted DNA shouldn’t contain RNA at the end of the process
3 traits of DNA polymerase (DNA pol I)
a – 5’ –>3’ synthesis activity
b – 2 exonuclease activities
c – “slow”, low processivity ( # of nucleotides that can be added before DNA pol I dissociates from DNA)
exonuclease activity
- synthesis is 5’–>3’ direction therefore proofreading is 3’–>5’
5’–>3’: RNA primer removal (nick translation) –> removes RNA then fills gap with DNA
cellular function of exonuclease activity and function of DNA Pol I
remove RNA primer and fill in with dNMP using dNTPs as substrates
DNA pol III
- only proofreading exonuclease activity - fast and highly processive
- where a bulk of DNA synthesis comes form
components of the replisome: how many poll III enzymes per replication fork
- 2 poll III enzymes
components of the replisome: gyrase
(topoisomerase) unwinds the superhelical tension ahead of the fork as replication proceeds
components of the replisome: helicase (DnaB protein)
locally “melts” dsDNA —> ssDNA so Pol III can access information in template strands
components of the replisome: primosome
enzymatic component of the replisome that synthesizes short RNA primers de novo (from nothing)
components of the replisome: DNA ligase
catalyzes the formation of phosphodiester bond (seals in nicks)
both leading and lagging strands are copied by how many replisomes
1
single-stand binding protein (SSB)
coats the unwound DNA to prevent it from reannealing
what directions does the replisome encounter the DNA strands
in both 5’–>3’ and 3’ –> 5’ direction
how many errors occur in bacteria for DNA replication
1 mistake every 1000 bp –> rate of 1X10^-3
sources of fidelity:
a: dNTP concentrations: cell tightly controls [dNTPs], too high = increased mutation rate
b: 2 step polymerase reaction: bind correct dNTP via AT: GC base pairing –> ensure proper geometry at active site; then incorporatre dNMP
correct base pairing leads to
purine: pyrimidine orientation with a consistent spacial geometry
incorrect base pairing leads to
misaligned geometry shown functional groups outside the “ correct region”
proofreading by Pol I and Pol III
- both DNA pol I and pol III possess 5’–>3’ synthesis activity with an intrinsic error rate
– therfore, both require a 3’ –> 5’ exonuclease or proofreading function
– the catalytic site for dNMP incorporation is separate from the proofreading sire
