Lecture 12: DNA Replication Flashcards
what way does the parental template strand run?
it runs 3’-5’
Eukaryotic DNA replication
Multiple large linear
chromosomes
23 pairs in
humans
Multiple origins of
replication (ori)
Bidirectional
what is needed to make a DNA copy?
Progressive addition of new nucleotides (A, C, T or G)
A starting point for nucleotide addition
Unwinding of the helical double-stranded DNA (to give two parental templates)
Release of tension generated by unwinding the DNA helix
Prevention of unwound double-stranded helical DNA, i.e. single-stranded DNA, from reforming and to protect it
Joining of ends of newly synthesised fragments together (lagging as well as leading strands)
Leading Strand
Continuously synthesised in it’s 5’-3’
Lagging strand
discontinuously synthesised in it’s 5’-3’ direction as Okazaki Fragments
What is Primase?
Enzyme (a type of RNA polymerase) that makes an RNA primer = starting point for DNA polymerisation
What is DNA Polymerase III?
Needs an OH group onto which the phosphate group of the incoming nucleotide can be attached
Only makes DNA
in the 5’ → 3’direction
Enzyme that synthesises a new DNA strand by adding nucleotides complementary to
the parental template strands
Cannot bind to single stranded DNA and start copying it
What pulls the two dna strands apart?
the Helicase unzips the DNA
what does the topoisomerase do?
goes ahead of the replication fork and cuts it to release the tension it will then glue them back together after the replication has proceeded
what does the single stranded DNA binding protein do?
they keep the two split strands apart as the C-G and A-T want to go back together really bad.
their other function is that they prevent the single strands of dna from being degraded.
what does primase do?
has an internal 3 prime hydroxyl group, they add a short stand of RNA nucleotides to the 3’ prime strand.
what Dna pol III do?
it utalises the 3prime hydroxyl group from the primase and will add a dna strand all along the strand
what does the primase do to the 3’ prime 5’ prime stand
it goes along and makes a bunch of strands for the DNA pol III to add dna strands inbetween
what does DNA pol I do?
recognises the RNA DNA hybrid and goes along the strand and removes the RNA component
What is ligase’s role in DNA replication?
used the 3 prime hydroxyl group and the 3 prime phosphate group to form a phosphodiester bond between the gaps
What is needed to make a DNA copy revised:
DNA polymerase III
Primase Enzyme makes RNA Primer
Helicase
Topoisomerase nick and rejoins DNA strands
Single-stranded DNA binding protein
DNA polymerase I (RNase H activity removes RNA and DNA polymerase adds the DNA nucleotides)
DNA ligase
When can DNA errors be repaired?
- During replications (using an EXOnuclease)
2. After replication (Using an ENDonuclease)
DNA replication shows high accuracy:
DNA pol III has a replication error rate of 1 in 10^8 base pairs replicated
DNA pol III has a proofreading mechanism
chekcs the newly inserted nucleotide bases against the template
any incorrect bases are removed by a 3’ to 5’ EXOnuclease acitivity of DNA Pol III
What can cause DNA damage or errors?
Incorrectly inserted bases are not corrected by DNA pol III
Radiation damage (e.g. UV)
Chemical modifications of bases (natural & chemical causes)
These types of incorrect or damaged nucleotide bases are removed by an ENDOnuclease
What is the polymerase chain reaction?
In vitro method of making multiple DNA copies so that there is enough DNA material to work with
Only ‘targeted’ DNA region will be copied
Rapid exponential increase of DNA molecules
Method utilises cycles of heating and cooling
What is an exonuclease and endonuclease and what is the difference between them?
Exonucleases are enzymes that catalyze the removal of nucleotides in either the 5-prime to 3-prime or the 3-prime to 5-prime direction from the ends of single-stranded and/or double-stranded DNA. Removal of nucleotides is achieved by cleavage of phosphodiester bonds via hydrolysis.
Endonuclease is an enzyme that breaks down a nucleotide chain into two or more shorter chains by cleaving the internal covalent bonds linking nucleotides — compare exonuclease.
