DNA replication Flashcards
DNA replication is vital for what
passing on genetic information from cells to cells as cells divide and from generation to generation as organisms reprooduce
Which individuals suggested a mechanism of how DNA could be replicated when they proposed the double helical structure of DNA
Watson and Crick
Which is the semi-conservative method of replication
Double-stranded DNA is unwound to produce 2 single strands (parent strand) and each parent strand is used to produce a daughter strand
Each new molecule has one original parent strand and one new daughter strand
In each resulting molecule one strand is conserved from the parent
What was the experiment Meselson and Stahl used to prove semi-conservative replication
Growing bacteria in heavy nitrogen and then light nitrogen and seeing the bands which are formed by the DNA when it was centrifuged
How does the structure of DNA affect replication
The problem when replicating DNA is the two strands are antiparallel
DNA can only be extended at the 3’ prime end
Effecting the process of replication
DNA replication can only be in which direction
Occur left to right
How does DNA polymerase add nucleotides to DNA
Adds nucleotides to the 3’ end of a chain
Scientists extracted E.coil proteins and looked for polymerase activity
What was needed for this experiment
1) dNTP’s (dGTP, dATp, dCTP, dTTP) triphosphate groups
2) A template (parent strand to copy)
3) A primer (another strand paired with a template to give a 3’ end)
What occurs when an incoming nucleotide with a 5’-triphosphate
A diphosphate is released when a nucleotide is added to the chain
Explain the processes of a nucleotide being added to a DNA strand
- The 3 prime sides are opposite to another
- Hence on the strand being used as a template, a nucleotide is added to the top stand
- DNA polyermase bring in a nucleotide triphospahte with a base complementary to the template DNA strand and 2 of the phosphate groups are being released
- Repeated for the next base and so on
DNA replication is described as what
Discontinuous
What is the ‘leading strand’ and what is the ‘lagging stand’ in terms of DNA replication
Leading strand - made continuously - primer extended by DNA polymerase from left to right (primer orientated 5’ to 3’ and extended from 3’ end)
Lagging strand - made discontinuously - can’t be made in same direction as DNA being unwound. Need to wait until DNA unwound before adding primer with lagging strand being made in a series of frangments
How does replication ever get started if you need a 3’ end to prime DNA synthesis
DNA is primed by RNA
RNA synthesis doesn’t need a primer
RNA primers are removed by what
DNA polymerase 1
Ribonucleases (Rnase)
How are the nicks in DNA backbone between the Okazaki fragments closed
DNA ligase is used
The Semi-conservative model of replication predicts that replicating DNA would have a what
replication fork
a replication fork is the point at which the double stranded DNA is unwound by the helicase enzyme
When replication initiates within the DNA strand
DNA replication could be
When replication initiates it doesn’t initiate at one end of a linear piece of DNA but initiates somewhere within the double-stranded region
DNA replication could therefore be bidirectional or unidirectional
What is used in the detection of fork movement
Radioisotopes
What would be the method behind working out the direction of fork movement
1) Grow E.coil in medial without radioisotope
2) Grown briefly in low levels of radioisotope (3H thymidine labels new DNA)
3) Grown in higher level of radioisotope
4) Isolate DNA em grid. Detect label via photographic emulsion
If the fork only moves in one direction we would only have a thin band on one side
What else can be used to demonstrate the bidirectional nature of DNA replication
An Autoradiograph
When new DNA strands are being synthesised, how does replication occur across both the strand being replicated
How is the problem of shrinking chromosomes solved
Solved through telomerase
What are the main molecules required for DNA synthesis
- DNA polymerase lll (in E.coil)
- Clamp - fits around DNA - templaye runs through the middle of the clamp
- Clamp loader
- Catalytic core
- Tau (dimerisation) - holds the two halfs of enzyme together
(bar the clamp loader) the whole thing is symmetrical as it synthesises both strands at the same time
What happens when sufficient template DNA for lagging strand synthesis has unwound (1-2kb of DNA)
Primase synthesis a RNA primer for lagging strand synthesis
The template strand is then pulled through the catalytic core allowing the addition of nucleotides to the 3’ end of the primer
Because the DNA polymerase lll is symmetrical the lagging strand template forms a loop so pulled through in the same direction
What are the different forms of DNA polymerase in eukaryotes
a synthesises the inital 20-30 nucleotides at the 3’ end of the primer
3’-5’ exconuclease is crital for proof reading
In Eukaryotes: the lagging strand is make how
as a series of Okazaki fragments by DNA polymerase delta
What is the differences in the ways RNA primers are removed in mammals and E.coil
In E.coil RNA primers are removed by DNA polymerase 1 which degrades from the 5’ end and replaced with DNA
In mammals the RNA primers are removed by an endonuclease FEN1
When an Okazaki fragment is synthesised and reaches the 5’ end of the next fragment, it displaces the RNA primer used to initiate that fragment and ‘flaps’ around
FEN1 is known as a ‘flap endonuclease’ - it cuts of the RNA primer and degrades internally rather than at the ends (as DNA pol 1 does)
DNA ligase then fills the gaps
Bacterial replication forks initiate and terminate where
initiate at the origin and terminate at the terminus
The terminators keep the process in check - causes the replication forks to terminate if either replication fork goes beyond half way point (e.g. if a fork is slower due to DNA damage)
Bidirectional replication forks that should each replicate half the chromosome (stopping at half way point)
How does DnaA initate and aid in replication at the bacterial origin
DnaA binds to 9bp repeats by DnaA wrapping the DNA around itself which then leads to
DNA melts at 13bp repeats (AT rich) i.e. denatures - 2 strands come apart
Allows Helicase, primase and DNA polymerase to start replication
