1.5 - DNA replication Flashcards
direction of DNA synthesis
5’-3’
3 possible principles of DNA replication
- semiconservative
- dispersive
- conservative
how is a primer located
ends with OH group
dNTP
deoxynucleotide triphosphate
- fundamental building block of DNA
components of deoxynucleotide triphosphate (3)
- nitrogenous base
- deoxyribose sugar
- triphosphate group
triphosphate group in dNTP
3 phosphates attached to 5’ carbon of deoxyribose sugar, provides energy for DNA polymerisation
DNA synthesis (2)
- dNTP base pairs with the template strand through H-bonding
- DNA polymerase catalyses formation of a new phospho-diester bond between the 3’OH of the preceding nucleotide in the chain with the new base pair nucleotide
rate of incorporation of incorrect dNTP
10,000-fold slower
rate of incorporation of rNTP
1000-fold slower
“discriminator” amino acids
in DNA polymerase, prevent rNTP binding via hydroxyl group on discriminator amino acid creating clash with rNTP (preventing binding)
rNTP
primary substrate for RNA synthesis
how are RNA and DNA polymerases different
unlike RNA polymerase, DNA polymerase cannot initiate a new DNA chain
what is each new DNA chain initiated by
synthesis of a short RNA chain (RNA primer catalysed by a primase) extended by DNA polymerase
RNA primer
synthesised short RNA chain that initiates new DNA chain, catalyses by primase and extended by DNA polymerase
what happens to RNA primer when new DNA chain is initiated
RNA primer part of chain removed
different characteristics of DNA polymerases in eukaryotic cells (2)
- some focused on bulk DNA replication at replication fork
- some focused on bypass of damaged DNA or DNA repair pathways
how many bp does DNA polymerase synthesise before releasing template?
20-100bp
how is DNA polymerase prevented from falling off?
sliding clamp encircles newly synthesised dsDNA and holds DNA polymerase (preventing diffusion away from primer: template junction)
post replication error rate
1 mistake per 10^10 nt added
DNA polymerase insertion error rate
1 incorrect dNTP per 10^5 nt added
(base pair accuracy alone not sufficient to account for accuracy - 1 - 10^10)
how is DNA proofread? (2)
- DNA polymerases that read for DNA replication have an exonuclease site next to active site
- incorrectly incorporated bases can be caught by exonuclease activity (allowing DNA polymerase another go at incorporating correct base)
why is DNA synthesis always 5’-3’?
proofreading is chemically difficult if synthesis occurred in 3’-5’ direction (proofreading critical for high fidelity of action by DNA polymerases)
replication fork
only one of 2 strands can be continuously synthesised (because of 5’-3’ direction)
okazaki fragments
short DNA nucleotide sequences synthesised discontinuously on lagging strand, later joined by DNA ligase to form continuous DNA strand
replisome
mix of DNA unwinding, DNA polymerase and DNA stabilisation factors that come together in highly processive molecular machinery for DNA replication
trombone slide model of replication (3)
- leading strand can be synthesised continuously in a 5’-3’ direction
- lagging strand looped out so that it passes through polymerase active site in a 3’-5’ direction, allowing synthesis to occur in 5’-3’ direction
- one asymmetric DNA polymerase dimer synthesises both strands
how are RNA fragments removed and gaps resealed in newly synthesised DNA (3)
- RNA fragments removed by RNase enzyme
- DNA polymerase fills the gap
- DNA ligase makes final seal
tropoisomerase
relaxes DNA allowing DNA helicase to unwind
bidirectional replication (3)
- replication starts at distinct sequences (origins)
- replication machinery initially assembled on DNA
- 2 replication machineries set off in opposite directions
origins of replication in eukaryotes (specifically humans) compared to prokaryotes
- humans - 10s of thousands
- prokaryotes - 1 per chromosome
why do eukaryotic chromosomes have many more origins of replication?
to compensate for larger genome and slower speed
RNA primers in eukaryotes compared to prokaryotes
longer in eukaryotes
okazaki fragments in eukaryotes compared to prokaryotes
longer in prokaryotes
PCR cycle stages and temp they occur at (3)
- separating DNA strands (95*C)
- annealing primers (40-60*C)
- DNA synthesis (ca. 70*C)
how does DNA polymerase survive at 95*C in PCR?
come from thermophilic organism
