DNA Recplicaton Flashcards
Directions of DNA synthesis:
- DNA (or RNA) is synthesised in the 5’ —-> 3’ direction (remember the 3rd carbon OH group)
- thus parents template strands run the 3’ ——> direction
Eukaryotic DNA replication
- Hydrogen bonds are broken at AT rich regions and synthesised from that single point in opposite directions
- multiple larger linear chromosomes, 23 pairs in humans
- multiple origins of replication (ori)
- bi directional
What is needed to make a DNA copy?
- progressive addition of new nucleotides (A,C,T,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 is…
Continuously synthesised in its 5’ —-> 3’ direction
Lagging strand:
Discontinuously synthesised in it’s 5’ —-> 3’ direction as Okazaki fragments
AT rich area
- helicase pulls strands apart - recognising rich regions
- as each is strand is synthesised - helicase continues to pull apart
Primase
- an enzyme (type of RNA polymerase) that makes an RNA primer= starting point of DNA polymerisation
DNA polymerase 111
- an enzyme that synthesises a new DNA strand by adding nucleotides complementary to parental template strands
- cannot bind to a single stranded DNA and start copying it - needs an OH group onto which the phosphate group of the incoming nucleotide can be attached
- 5’ to 3’ direction
DNA pol 1 function
Removes RNA primers and fills the gap with DNA nucleotides
DNA Ligase
- Joins the newly synthesised Okazaki fragments together (creates phosphodiester binds), once the RNA primers have been removed and replaced by DNA nucleotides
- Joined together the newly synthesised fragments form the multiple replication bubblers, including the leading strands.
DNA pol 1 carries out 2 activities
- RNase Activity:
- RNase H is an endonuclease enzyme that recognises RNA:DNA hybrids and degreases the RNA part - DNA Polymerase Activity:
- synthesises DNA by adding nucleotides ( complementary to the parental DNA template of the lagging strand)
Topoisomerase
- cuts strands apart to release tension then glues them back together
Single stranded binding proteins
- prevents strands from joining back together once separated
- Protects single strands of DNA from enzymes
- DNA pol 111 moves along and knocks off said proteins
Responsible for progressive addition of new nucleotides (A,C,T,G)
DNA polymerase 111
What enzyme is responsible for making the starting point for nucleotide addition
- primase enzyme makes RNA primer
What unwinds the helical double stranded DNA to give two parental strands
Helicase
Prevention of unwound double- stranded helical DNA, i.e. single-stranded DNA, from reforming and to protect it from degradation
Single-stranded DNA binding protein
Removes RNA primer and fills gap with DNA nucleotides
DNA polymerase 1 (RNase H activity removes RNA and DNA polymerase adds the DNA nucleotides)
What is responsible for joining newly synthesised fragments together (lagging as well as leading strands, within and between replication bubbles)
DNA ligase
When can DNA errors be repaired?
- During replication (using an EXOnuclease) - will move from end to end ( usually 3-5 sometimes 5-3)
- After replication (using an ENDOnuclease) - can move within, doesn’t need an end - can remove from end
Replication error rare of DNA pol 111
1 in 10^8 - 10^10 base-pairs replicated
(DNA pol 111 has a proofreading mechanism which checks the newly inserted nucleotide bases against the template)
How are incorrect bases corrected during DNA synthesis
- through the 3’ to 5’ activity of DNA pol 111
-Damaged/ incorrect DNA
I
V
- incorrect base is removed by the 3’ to 5’ exonuclease activity of DNA pol 111
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V
- DNA synthesis conitinues
What can cause DNA errors or damage after DNA replication?
- incorrectly inserted bases are not corrected by DNA pol 111
- radiation damage (U.V)
- chemical modifications of base (natural and Chemical causes)
These types of incorrect or damaged nucleotide bases are removed by endonuclease
How damaged nucleotide bases are removed by removing an endonuclease
- Damaged / incorrect DNA
- damage, including some surrounding (flaking) regions, is removed by endonuclease
- A DNA polymerase makes new DNA
- DNA ligaments joins new DNA to existing DNA
What happens if the DNA is not corrected?
- DNA error becomes part of DNA template thus permemant DNA change (mutation)
DNA helix (where one of the strands carry’s an error) separated for the replication process what’re each DNA strand will be used as a template
The DNA strand being synthesised using incorrect DNA template will carry a permanent error. All future DNA molecules arising form this ‘mutated’ DNA will carry the incorrect DNA base.
What happens in the polymerase chain reaction (PCR) (test tube)
- in vitro method of making multiple DNA copies do 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
PCR can be used in:
- medical applications
- forensic applications
- infectious disease detection and identification
- molecular biology research and applications
How PCR works:
- Denaturation: is increased, denaturing hydrogen bonds and separating DNA strands
- Annealing: temp is lowered to allow primers to base pair to complementary DNA template
- Extension: polymerase extends primer to form nascent DNA strand
THIS PROCESS REPEATES UP TO 35 TIMES
1st cycle - 2^2
2nd - 3^2
3rd - 4^2
Functions of PCR components unused ‘in vitro’ DNA replication
DNA template:
- DNA molecule to which complementary nucleotides can be matched to make identical copies via DNA synthesis
Primers:
- provides a free ‘OH group, the chemical group that is essential to initiate DNA synthesis
- defines the region of the DNA molecule to be replicated
DNA polymerase:
- enzyme which addds nucleotides, (complementary to the DNA template), and joins them together forming a phosphodiester bond
dNTPs:
- free nucleotides (equal amount of each) - the building b locked used by the DNA polymerase
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