Lecture 5 - DNA Replication Flashcards
DNA double helix consists of:
sugar-phosphate backbone with paired bases hydrogen bonded together
DNA double helix can assemble from its component strands:
- unzipped by breaking hydrogen bonds between the bases
- this allows new strands to be synthesised by matching up bases to the old strands
- this process of DNA replication allows DNA to be copied when cells divide
how is DNA synthesis catalysed?
DNA synthesis is catalysed by enzymes known DNA polymerases and they occur in the 5’-3’ direction
DNA replication is semi-conservative:
because a newly made DNA molecule therefore has one daughter strand and one parent strand
what are the three possible principles of DNA replication:
(a) semi-conservative → accepted model
(b) dispersive
(c) conservative
meselson-stahl experiment:
bacterium were grown in a light medium and a heavy medium and their centrifuged DNA showed two separate band locations - however when these bands were transferred to their heavier/lighter counterpart their newly centrifuged values were in between - indicating semi-conservative/dispersive replication
how does the new strand form in DNA synthesis?
The 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 paired nucleotide
RNA primes DNA synthesis:
because DNA polymerases cannot initiate a new DNA chain on their own, each new DNA strand must be initiated by a synthesis of a short RNA (primer) that is extended by the DNA polymerase
the RNA primer part of the chain is then removed
why always 5’-3’?
DNA synthesis always goes in a 5’-3’ direction
its likely always in this direction because proofreading is chemically difficult if synthesis occurred in the 3’-5’ direction
higher eukaryotic DNA polymerases:
eukaryotic cells express a range of DNA polymerases with slightly different characteristics
some are focused of bulk DNA replication at the replication fork whilst others are focused on the bypass of damaged DNA / repair pathways
rate of encorporation of incorrect dNTPs=
10,000 slower
rate of encorporation of rNTPs=
1000 fold slower
what prevents DNA polymerase from rNTP binding?
“discriminator” amino acids
how does the DNA polymerase interact with the RNA primer?
DNA polymerase grips the primer template junction like a hand
what is the final error rate during DNA replication?
the final error rate is 1 mistake per every 1010 nucleotides added
what do DNA polymerases used in DNA replication have next to the active site?
next to the active site DNA polymerases have an exonuclease site
what is the purpose of exonuclease activity?
incorrectly incorporated bases can be caught by the exonuclease activity, allowing the DNA polymerase to have another go at incorporating the correct base
because DNA polymerase can only synthesise 20-100 base pairs before releasing the template, how does it stay in place and not fall off?
•Sliding clamp encircles newly synthesised dsDNA and holds DNA pol
• DNA pol prevented from diffusing away from primer: template junction
the replisome:
DNA replication requires a mix of DNA unwinding, DNA polymerase and DNA stabilization factors to come together in a highly processive molecular machine – the replisome
“trombone slide” model of replication:
- the leading strand can be synthesised continuously in a 5’-3’ direction
- the lagging strand is looped out so that it passed through the polymerase active site in a 3’-5’ direction, allowing synthesis to occur in the 5’-3’ direction
how are RNA primer fragments removed from the DNA molecule?
RNA fragments are removed via the RNAase enzyme for DNA polymerase to fill the gap for DNA ligaments to make the final seal
enzymes for DNA replication:
•Primase
•DNA polymerases (2)
•Clamps
•Single-stranded DNA-binding protein, to keep strands apart
•DNA ligase
•Helicase, to unwind DNA
•Topoisomerase, to relax DNA
what is different about the origins of replication in eukaryotes and prokaryotes?
bacteria have one single origin per chromosome whilst eukaryotic chromosomes have many origins to compensate for larger genomes and slower speed
how do RNA primers differ in eukaryotes and prokaryotes?
RNA primers are longer in eukaryotes and shorter in prokaryotes
how do okazaki fragments differ in prokaryotes and eukaryotes?
okazaki fragments are longer in prokaryotes than they are in eukaryotes
what is the function of PCR (polymerase chain reaction)?
‘amplifies’ (i.e. selectively makes lots of) specific DNA sequences
what does PCR require?
PCR requires: DNA polymerase, oligonucleotide ‘primers’
each cycle of PCR requires 3 steps:
(1) separating DNA strands (95C)
(2) ‘annealing’ of primers. (40-60C)
(3) DNA synthesis (70C)
because the polymerase chain reaction has stages that exceed to 95C what is crucial that we do?
use thermal protected enzymes such taq DNA polymerase which comes from thermophilic organisms used to these temperatures
PCR summary:
• Development of the PCR has revolutionised
molecular biology and has widespread applications
• Tiny amounts of DNA can be amplified in a few hours
• PCR works because DNA polymerases require primers
where does a replisome assemble?
a replisome assembles at each replication fork with a complex set of enzymes
orientation of DNA replication:
DNA replication is bi-directional
