Lecture 2- duplication Flashcards
Origin of replication in E.coli
oriC
This is 245 base pairs, 5 repeats of a 9bp sequence that is a binding site for the initiator protein DnaA
Name 2 DNA bending proteins which separate the strands
IHF and Fis
DNA unwinding element (DUE)
AT rich sequence adjacent to where DnaA assembles. This is where DNA is pulled apart for replication.
When DUE melts (base pairs pulled apart) DnaA becomes compact to force the strands apart
DUE is where the strand opens to form the initial bubble
DnaB - function and problem
Opens the replication form
Ring shaped protein - difficult to load onto a long molecule of DNA that is also circualr
How is DnaB loaded onto DNA?
DnaC-ATP ring binds the DnaB ring and opens it for loading
DnaC-ATP will direct DnaB to the origin opened by DnaA
Binding of the DnaBC complex to DnaA promotes assembly of two DnaB rings, one for each replication fork
ATP hydrolysis releases DnaC leaving DnaB bound to the DNA
SSB
Single stranded DNA binding protein
Accessory pigment
Function of SSB
Stops unwound parent strands from re-annealing
Prevents intramolecular strand annealing- separated single strand that may have complementary sections may anneal to form a hair pin shape, this results in deletions
Protects DNA strands from degradation or damage
Acts as an assembly point for multiple proteins involved in DNA replication
Can block access of proteins involved in DNA replication
DNA gyrase - topoisomerase II
As DnaB unwinds parental strands ahead of the replication fork the unwound parental DNA can coil up (supercoiling) and result in a clock to fork progression
DNA gyrase alleviates supercoiling by cutting the two strands of DNA and passing a strand through the break before resealing the cut strands.
What do all DNA polymerases have in common?
A proof reading ability so they can check errors when nucleotides are inserted.
Fidelity
The faithful copying of DNA and how accurate it is
DNA polymerase II and its proofreading subunit
DNA polymerase II has an epsilon proofreading subunit with exonuclease activity.
Without this an incorrect nucleotide would be inserted every one in 7 million base pairs.
Function of DNA polymerase I
Removes RNA primer and replaces with DNA
single subunit and has 5’ to 3’ exonuclease activity
Function of DNA polymerase II
Fills in gaps following repair of DNA damage
DNA polymerase III
Chromosome replication
Initiates DNA synthesis by extending an RNA primer
The core polymerase has… (3 points)
DNA polymerase III
A separate epsilon subunit that specifies the 3’ - 5’ exonuclease (for proof reading)
two alpha subunits
Two tau subunits that attach the alpha polymerase subunits and hold them together
DNA polymerase III
Gamma complex
Gamma complex/clamp loader Binds tau (T) and loads the beta sliding clamp onto the parental strands
DNA polymerase III
Beta clamp
Ring (homodimer) that encircles the parental strand on each daughter template to ensure the polymerase does not fall off during DNA synthesis (enhances processivity)
What allows opening of the beta clamp ring?
gamma complex bound to ATP while ATP hydrolysis allows clamp release and ring closure
Priming different strands
The leading strand is primed one at the replication origin (twice as there are two bidirectional forks).
On the lagging strand DnaG primase syntheises an RNA primer for every Okazaki fragment as synthesis is directed on a parental loop and synthesised discontinuously.
Beta clamps and RNA primers
A new B clamp is laoded on each RNA primer by the clamp loader (gamma complex).
As new okazaki fragments are formed the clamp loader (gamme) opens a new clamp (Beta) and the helicase (DnaB) recruits primase (DnaG) to the replication fork to initiate the next fragment.
What happens after the synthesis of the RNA primer?
The clamp loader displaces primase (DnaG) and loads the clamp onto the new primer.
How long does it take E.coli to replicate?
DNA polymerase III works at 900 nucleotides per second. It should take 42 minutes for the chromosomal DNA to be replicated but it is less than this as the next round of replication is initiated before the firsst round has been completed
Summarise all the key component features of bacterial DNA replication (12)
Single origin of replication DnaA opens up DNA initiation coordinated with cell cycle DnaC loads DnaB DnaB unwinds DNA at the fork SSB keeps unwound strands apart DnaG makes RNA primer gamma complex loads the Beta clamp B clamp keeps polymerase on DNA DNA polymerase is Pol III RNA primer removed by Pol I DNA ligase seals the nick
