Lecture 12 - DNA replication Flashcards
What direction is DNA (or RNA) always synthesised?
5’ to 3’
What way is the parental template strand run?
3’ to 5’
Where do you find origins of replication and why?
At AT rich regions because A-T strands are easier to pull apart than G-C strands because A-T has two carbons as opposed to three.
What are the steps needed to make a copy of DNA?
Progressive addition of new nucleotides
A starting point for nucleotide addition
Unwinding of the helical double stranded DNA
Release of tension generated by unwinding the DNA helix
Prevention of unwound double stranded helical DNA from reforming and to protect it
Joining ends of the newly synthesised fragments together
Why is replication of DNA considered semi-discontinuous?
The leading strand is continuously synthesised in its 5’ to 3’ direction but the lagging strand is discontinuously synthesised in its 5’ to 3’ direction as Okazaki fragments
What is the function of helicase?
To pull the two DNA strands apart. When this happens the strands get tighter
What is primase and its function?
An enzyme (a type of RNA molecule) that makes an RNA primer = starting point for DNA polymerisation.
What is DNA polymerase lll and it’s function
An enzyme that synthesises a new DNA strand by adding nucleotides complimentary to the parental template strands. It does this in the 5’ to 3’ direction.
How are primase and DNA polymerase lll connected?
Pol lll needs an -OH group onto which the phosphate group of the incoming nucleotide can be attached. Primase has an internal -OH group which the strands can add to. Pol lll will add one nucleotide at a time to the RNA primer.
What is the function of topoisomerase?
To cut the two DNA strand so they can unwind which releases tension. After it sticks the pieces together
What is the function of DNA polymerase l?
To remove RNA primers (RNase H) and fills the gap with DNA nucleotides (DNA polymerase)
How does DNA polymerase l complete its function?
DNA pol l can recognise DNA RNA hybrids and it removes the RNA nucleotides and uses the -OH of the next door fragments and extends to the other fragment (can’t join them but extends)
What is the function of DNA ligase?
To join the newly synthesised Okazaki fragments together (creates phosphodiester bonds), once the RNA primers have been removed and replaced by DNA nucleotides
Also joins the newly synthesised fragments from multiple replication bubbles, including the leading strands.
What two activities does DNA pol l carry out?
- RNase activity: RNase H is an endonuclease enzyme that recognises DNA:RNA hybrids and degrades the RNA cell
- DNA polymerase activity: synthesises DNA by adding nucleotides (complementary to the parental DNA template of the lagging strand)
What is the order of enzymes used to make a DNA copy?
Helicase Primase DNA polymerase lll Topoisomerase Single-stranded DNA binding proteins DNA polymerase l DNA ligase
What is the function of the single-stranded DNA binding protein?
Prevent two parent strands from snapping back together
Single strand DNA will prevent DNA ends from being degraded
When can DNA errors be repaired and with what?
During replication - using an exonuclease
After replication - using an endonuclease
What does exonuclease do?
Cuts DNA from the inside
What does endonuclease do?
Cuts DNA from the outside
How does the cell check for DNA errors and fix them during DNA replication?
DNA pol lll has a proofreading mechanism that checks the newly inserted nucleotide bases against the template as they are being added. If there is an incorrect base, it is removed by a 3’ to 5’ exonuclease activity of DNA polymerase lll.
What is the reason for DNA errors found after DNA replication and how is it fixed?
DNA errors found after DNA replication can be from:
Incorrectly inserted bases are not corrected by DNA pol lll
Radiation damage (e.g. UV)
Chemical modifications of bases (natural and chemical causes)
These types or errors are removed by an endonuclease
Why is it important to correct DNA errors?
If it’s not corrected the DNA error can become part of the DNA template which means a permanent DNA change (DNA damage/mutation)
What is in vitro DNA synthesis?
DNA replication in the test tube
What is a polymerase chain reaction (PCR)?
an in vitro method of making multiple DNA copies so that there is enough DNA material to work with
What happens during PCR?
Only ‘targeted’ DNA region will be copied
Rapid exponential increase of DNA molecules
Method utilises cycles of heating and cooling
What is PCR used for?
Medical applications
Forensic applications
Infectious disease detection and identification
Molecular biology research applications
What is In vivo DNA synthesis?
DNA synthesis in cells
What is the function of DNA template in ‘in vitro’ DNA replication?
DNA molecule to which complementary nucleotides can be matched to make identical copies via DNA synthesis
What is the function of primers in ‘in vitro’ DNA replication?
Provide a free ‘3 OH group, the chemical group that is essential to initiate DNA synthesis
Defines the region of the DNA molecule to be replicated
What is the function of DNA polymerase in ‘in vitro’ DNA replication?
Enzyme which add nucleotides, (complementary to the DNA template), and joins them together forming a phosphodiester bond.
What is the function of dNTPs in ‘in vitro’ DNA replication?
Free nucleotides (equal amount of A, G, C and G) - the building blocks used by the DNA polymerase
Compare primers In vivo vs In vitro
In vivo: RNA primer, synthesised by primase, removed and replaced by DNA nucleotides
In vitro: DNA primer, added completed, part of newly-synthesised DNA
Compare DNA strands In vivo vs In vitro
In vivo: one continuous strand (leading), and discontinuous strand (lagging)
In vitro: two continuous strands
Compare start In vivo vs In vitro
In vivo: at ‘origin of replication’ sites (multiple ori sites in eukaryotes)
In vitro: two sites where DNA primers are designed to bind
Compare synthesis In vivo vs In vitro
In vivo: 5’ to 3’ direction all DNA (whole genome) synthesised
In vitro: 5’ to 3’ direction. Region between the primers synthesised.
Compare temperature In vivo vs In vitro
In vivo: one constant temperature -37C
In vitro: cycling of different temperatures (45-98C)
Compare proteins and enzymes In vivo vs In vitro
In vivo: Several enzymes and proteins (DNA helicase, topoisomerase, single-stranded DNA binding proteins, primase, DNA pol lll, DNA pol l, DNA ligase)
In vitro: one enzyme - heat stable DNA polymerase adding nucleotides (all other steps are regulated by temperatures)
