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
What are chromosomes like in E.coil vs eukaryotic chromosomes
The E.coil chromosome is circular - single origin
Most eukaryotic chromosomes are linear - multiple origins
What is the problem of replicating the ends of linear eukaryotic chromosomes (telomeres)
There is no problem with leading strand at the new 3’ end as DNA polymerase making the leading strands keeps going till it falls off
However the problem with the lagging strand at the new 5’ end because the last RNA primer on the lagging strand end is removed but cannot be replaced
Hence DNA replication shrinks our chromosomes overtime
How is the problem of shrinking chromosomes solved
Solved through telomerase
Telomerase is active in which cells
Germ cells (produce eggs + sperm) and not in most somatic cells (body cells
What are the bacterial enzymes involved in DNA synthesis
- DNA Pol I: helps remove RNA primer and replaces with DNA in chromosome replication, also has a major role in repair of damaged DNA
- DNA Pol II: restarting replication when blocked by damaged DNA, also a role in DNA repair
- DNA Pol III: Chromosome replication
- DNA Pol IV and V: allow replication to bypass some types of DNA damage, also involved in DNA repair
What is the main role of DNA polymerase
Synthesis of DNA
What is the main role of Exonucleases
Degrade nucleic acids from the ends
What is the role of (3’-5’ exonuclease)
Degrades from the 3’ end - involved in proofreading (checks the nucleotide it has just inserted is correct)
What is the role of (5’-3’ exonuclease)
used to degrade the RNA primers at the end of Okazaki fragments
DNA polymerase lll which does most chromosomal replication is much faster than DNA pol1 which just synthesises short stretches of DNA
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 is the role of Helicase
Unwinds the DNA duplex to produce the replication fork (DnaB gene in E.coil)
What is the role of Primosome
(makes RNA primer)
Moves with the lagging strand
Contains products of DnaC and DnaG genes and other in E.coil
What is the role of the single-stranded DNA binding protein (SSB in E.coil)
Keeps strands aparts and helps prevent stem-loop formation
What is the role of DnaB (helicase)
unwinds duplex
what is the role of a subunit (catalytic core)
Synthesise DNA
The leading strand is made continuously by the addition of nucleotides to the 3’ end as it passes through the catalytic core of the enzyme
What is the role of tau subunit
ensures dimerisation of polymerase
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
How would descibe the state of DNA in cells
What implications does it have
Supercoils - making DNA more compact
It can have implications for DNA replication creating difficulties
True or False?
Supercoiling can occur in circular DNA molecules and in linear DNA molecules
True
But only in lineae DNA molcules if they are contrained at the ends
What is the difference between positive and negative supercoiling
Positive: when the right-handed double-helical confromation of DNA is twisted in a right handed fashion - i.e. twisted even tighter - extra twist)
Negative: twisted in a left-handed fashion i.e. looser coiling take out a twist
Both can occur as the DNA is unwound by the helicase to allow DNA replication
What are the roles of Topoisomerase and Gyrase
Restore balance in supercoiling regions during DNA replication
Topoisomerase l: can remove both positive and negative supercoils, main function in DNA replication is to remove negative supercoil (tightens underwound molecule)
DNA gyrase: removes positive supercoils (relaxes overwound molecules)
What implication can occur if the DNA is overwound or underwound
DNA is overwound - positive supercoiling - in front of the replication fork
DNA is relatively unwound - negative supercoiling - behind the replication fork
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
How long does it take E.coil to replicate
E.coil has 4.6 x 10^6 bp of DNA in its single circular chromosome
DNA polymerase lll synthesises DNA at 900 nt per second
Divide the total length of the genome by 900 gives 5111 seconds (n/60) is 85mins
However there are two replication forks so the answer is 42mins
Experiments have shown E.coil can divide at much faster rates between 20 and 35 mins in good conditions
Replication process is already going when it starts
At cell division the chromosome is already partly replicated. The newly formed cell inherits a chromosome
