Define DNA replication
It is a semiconservative replication
Each strand of the double helix is used as a template strand for the synthesis of 2 new strands, resulting DNA will have 1 parental strand and 1 new strand
Leading strand replication - continuous (5)
- Helicase - enzyme that finds AT rich region and unwinds the DNA in order for DNA polymerase to get into the middle of DNA. It moves along the DNA helix, forming a replication bubble
- Topoisomerase - enzyme that cuts the tightly wind DNA helix to release tension and glues the strands back together to allow helicase to continue (moves in from of helicase)
- ssbp (single stranded DNA binding proteins)
- Protein that stabilises the unwound template strands until it is used as a template
- Protein that protects the separated single strand DNA from enzyme attack
- Prevents separated DNA strands from joining back together before replication has finished - Primase (type of RNA polymerase) - enzyme that synthesises an RNA primer at the 5’ end - the starting point for DNA polymerisation
- DNA polymerase (DNA Pol III) - enzyme that uses the parental DNA as a template, synthesises new DNA strand by adding complimentary nucleotides to an RNA primer or a pre-existing DNA strand. As DNA polymerase III moves along the DNA strand, it kicks off the ssbp proteins
Lagging strand replication - discontinuous (5)
- Helicase, Topoisomerase and ssbp do their jobs
- Enzyme primase begins synthesis of RNA primer at the 5’ end of each okazaki fragment with OH group at 3’ end
- DNA pol III synthesises each okazaki fragment by attaching to the 3’ OH group and adding nucleotides complementary to the parental template strands
- DNA polymerase I - 2 activities
- RNase activity: endonuclease enzyme that recognises DNA-RNA hybrids and degrades the RNA part by removing the RNA primer
- DNA polymerase activity: fill the gap and synthesises DNA by adding nucleotides to the 3’end of each okazaki fragment, complimentary to the parental DNA template of the lagging strand
After the last addition, the backbone is left with a free 3’ end - DNA ligase joins newly synthesised okazaki fragments together by creating phosphdiester bonds
Where does the origin of replication occur and why
AT rich region because only 2 hydrogen bonds present so easier to pull apart as it is held together more weakly
Direction of leading strand replication vs. lagging strand replication
Leading strand: 5’ - 3’
Lagging strand: 3’ - 5’
why can primase make RNA primers from scratch
Because it contains an internal 3’ OH group
Why can’t DNA polyerase bind directly onto ssDNA and start replication without an initial RNA or DNA primer
The primer provides an 3’OH group to which the phosphate group of the incoming nucleotide can be attached
Prokaryotic replication vs. eukaryotic replication (4)
Prokaryotic replication - occurs in the cytoplasm - single circular chromosome - single origin of replication (ori) - rapid Eukaryotic replication - occurs in the nucleus - Multiple linear chromosome - multiple origin of replication (ori) - slower than prokaryotic replication
when is the X-shaped chromosome formed
After DNA replication, just before mitosis
What is nuclease
Enzyme that removes nucleotides
Repair of DNA errors - during replication (3)
- DNA Pol III has proofreading mechanism, it checks the newly inserted nucleotide bases against the template
- Incorrect bases are moved by the 3’ - 5’ EXOnuclease activity of DNA pol III
- DNA synthesis continues
Only nucleotides at the ends can be removed
Repair of DNA errors - after replication (3)
- Damaged/incorrect DNA removed by enzyme ENDO nuclease (removes nucleotides from within a sequence)
- DNA polymerase makes new DNA
- DNA ligase joins new DNA to existing DNA
DNA damage/error causes (3)
- Incorrectly inserted bases are not corrected by DNA pol III
- Radiation damage eg. UV –> pyrimidine dimers
- chemical modifications of bases
Importance of correcting DNA errors
If a DNA error is not corrected , it becomes part of the DNA template causing a permanent DNA change - DNA damage/mutation
‘in vitro’ DNA replication by the Polymerase Chain Reaction (PCR) - (4)
- DNA replication carried out in test tube to make multiple copies of DNA
- only ‘targeted’ DNA region will be copied
- Rapid exponential increase of DNA molecules
- Method utilises cycles of heating and cooling
PCR components (6)
- DNA template (chromosomal/genomic)
- Primers (short stretch of DNA nucleotides, usually 20)
- Heat stable DNA polymerase (special enzyme that will polymerase at high temperature)
- dNTPs (deoxynucleotide triphosphates)
- Buffer solution (maintain the pH that the DNA polymerase requires to work)
- Divalent cations (Mg2+)/co-factors (ions essential for DNA polymerase to work)
what are dNTPs
Free nucleotides with equal amount of A,C,G,T that act as the building blocks used by DNA polymerase in PCR
Process of PCR
- Denaturing (98°C) - Temperature increased to separate DNA strands by breaking H-bonds
- Annealing (48°C - 72°C) - Temperature decreased to allow primers anneal/form H-bonds to their target DNA following the base paring rules
- Extension (68°C - 72°C) - (Taq) Polymerase (works optimally at this temperature) starts polymerisation by extending primer (adding complimentary nucleotides to the 3’ end) to form nascent DNA strand
Above steps are repeated 25-30 times resulting in exponential increase of DNA molecules
Up to how many times is PCR repeated
35 times
Direction of DNA synthesis
5’ - 3’
Parental template DNA strand direction
Run from 3’ - 5’
Product of 1 complete cycle of PCR
2 double stranded copies of the target DNA
most common polymerase used in PCR
Taq polymerase (derived from hot spring bacteria)
