DNA replication Flashcards
How is DNA ‘semi-conservative’?
Each parent strand is used as a template
Contains information which dictates the order of the nucleotides in the newly synthesised strand
Each daughter strand contains one strand from the parent strand
Which direction does DNA synthesis occur in?
How is this different to the movement of RNA pol II?
5’ –> 3’
RNA pol II moves along DNA in the 3’ –> 5’ direction
Which bonds are present in DNA?
Phosphodiester bonds
How does DNA replication occur?
Sequential polymerisation of of new nucleotide at the 3’ end:
Nucleophillic attack by the 3’ -OH on the sugar on the incoming DEOXYRIBONUCLEOSIDE TRIOSPHOSPHATE
Why is DNA synthesis effectively an ‘irreversible reaction’?
It is coupled to the breakdown of PPi (pyrophosphate) to 2Pi (2 x inorganic phosphate)
The breakdown of the 2 high energy phosphate bond provides the free energy for DNA synthesis
Why is DNA synthesis a high exothermic reaction?
Production of 2 inorganic phosphates from pyrophospahte as each deoxyribonucleoside phosphate is added to the growing DNA chain
Breaking a phosphate bond releases high energy required for the DNA synthesis
Why are molecules of DNA added as deoxyribonucleoside TRIphosphates?
COUPLED REACTION:
To able the 2 of phosphate molecules to be released as PRYOPHOSPHATE
And then, to allow pyrophosphate to be broken down into 2 individual phosphate molecules
Breakage of TWO high energy bonds
What enzyme break pyrophosphate into 2 inorganic phosphate molecules?
Pyrophosphatase
What is the ‘equation’ for DNA synthesis?
dNTP + (dNMP)n –> (dNMP)n+1 + 2Pi
Deoxyribonucleoside TRIphosphate + Deoxyribonucleoside MONOphosphate –> …
How is DNA synthesis initiated?
By creating a replication fork where DNA strands are separated
By the extension of an RNA primer
Which enzyme separates the paired DNA strands?
DNA helicase
How does DNA synthesis occur on the ‘lagging strand’?
What does this mean?
In the OPPOSITE direction to the replication fork
Still in the 5’ –> 3’ direction (DNA is anti-parallel)
Means that the lagging strand cannot be synthesised continuously
What are Okazaki fragments?
Short fragments formed on the lagging strand of DNA synthesis, due to the antiparallel and unidirectional orientation of DNA
What enzyme synthesises the RNA primer on DNA?
DNA primase (a type of RNA polymerase)
How long is the DNA primer?
20 nucleotides in length
How does DNA primase bind to DNA?
WITHOUT the need for a primer
What does DNA polymerase do?
On the lagging strand?
Extends the RNA primer laid down by DNA primase
On the lagging strand:
- Extends the primer to the 5’ end of the previous Okazaki fragment
- THEN, extends the 3’ end of the UPSTREAM Okazaki fragments after ribonuclease H has removed the RNA template
What does ribonuclease H do?
Removes the RNA primer between the junction of Okazaki fragments
Creating a gap
What is ribonuclease H specific for?
The RNA component of the RNA-DNA hybrid molecule
What does DNA ligase do?
Seals the nick between 2 Okazaki fragments in a 2 step reaction to create one CONTINUOUS strand
Where does DNA ligase get the energy from to ligate Okazaki fragments together?
ATP hydrolysis
From the breakdown of pyrophosphate by pyrophosphatase
What is the first step of catalysation by DNA ligase ‘sealing the nick’?
- Covalent attachment of AMP from ATP to the 5’ end of the nick, forming ADP (adenosine diphosphate)
Uses 1 molecule of ATP
What is the second step of DNA ligase ‘sealing the nick’?
- NUCLEOPHILLIC attack (by the 3’ hydroxyl) on the phosphate bond between the 2 phosphates within the ADP
- Releases AMP
- Creates a new phosphodiester bond in the backbone of DNA
Why is ‘sealing the nick’ energetically favourable and irreversible?
COUPLED REACTION of conversion of PPi to 2Pi
What does DNA helicase use to separate parental strands of DNA at the replication fork?
How is it used?
ATP
Used to give DNA helicase its forwards momentum to unwind DNa
How does DNA helicase move on DNA?
SPINS around and wraps itself around ONE of the strands parental DNA
What is Werner syndrome?
What is is caused by?
Premature ageing
Caused by:
- Autosomal recessive mutations in the WRN gene encoding RecQ helicase (a DNA helicase)
- Causes inefficient replication of the genome during GROWTH and REGENERATION of adult tissues
What is bloom syndrome?
What is is caused by?
Cancer
Caused by:
- Loss-of-function mutation in RecQ-family DNA helicase which maintains GENOME INTEGRITY (in DNA repair)
What is the processivity of DNA polymerase enhanced by?
Association with the SLIDING CLAMP
Describe processivity
Once the first nucleotide has been polymerised, there is a HIGH PROBABILITY that the next and next will be
How does the sliding clamp work?
2 additional proteins assemble onto the DNA PRIOR to the recruitment of DNA polymerase at the PRIMER-TEMPLATE JUNCTION
1) Sliding clamp - FIXES DNA pol to the primer-template junction
2) Clamp loader - Attaches the sliding clamp to the primer-template junction
Prevents DNA polymerase from FALLING off the DNA template
How is DNA polymerase attached to the primer-template junction?
1) Clamp loader with ATP takes the clamp and fixes it around the primer-template junction
2) Once the ring of the clamp is complete:
- ATP –> ADP + Pi
- Clamp loader dissociates and DNA polymerase is recruited
What do single-stranded binding proteins do?
Keep single-stranded DNA in the replication fork UNBOUND
Make the DNA nucelotides readily available to be read by DNA polymerase
What would happen is SSBs were not present?
2 parental strands (between the point of DNA helicase and DNA polymerase) would rebind to eachother, once DNA helicase gets far enough ahead
How do SSBs attach to DNA?
Rapid, cooperative way
Decorate the parental strands between DNA helicase and DNA polymerase
What do DNA topoisomerases do?
What does this allow?
Prevent DNA becoming tangled during DNA replication:
- Nick and reseal the backbone of DNA
- To allow the strands to rotate around eachother and release the stress on the DNA strands
Why are DNA topoisomerases needed?
Unwinding of DNA at the replication fork by DNA helicase induces SUPERCOILING into the DNA helix, ahead of DNA helicase
Prevents progression of the enzymes
What do Type I topoisomerases do?
Nick and reseal ONE of the 2 DNA strands
What do Type II topoisomerases do?
Nick and reseal BOTH of the 2 DNA strands
Which type of topoisomerase uses ATP?
Type II (nick and reseal BOTH)
What is highly conserved between E.coli and human replication forks?
KEY components:
RNA primers DNA polymerase SSBPs Sliding clamp Ribonuclease H DNA ligase DNA topoisomerases I and II
Where is DNA replication initiated?
At the replication origin
What does the replicator origin in DNA do?
Recruit INITIATOR proteins
Which direct the initiation of DNA replication
Describe the initiation of DNA replication in eukaryotes
Biphasic:
1) Replicator Selection: formation of a pre-replicative complex
2) Origin Activation: unwinding of DNA and recruitment of DNA polymerase
When does the replicator selection phase of DNA replication initiation occur?
In G1
When does the origin activation phase of DNA replication initiation occur?
S
What ensures that each replication origin is only used ONCE per cell cycle?
Temporal seperation of the 2 stages:
1) Replicator selection
2) Origin activation
Describe the replicator selection phase of DNA replication initiation
Formation of a Pre-Replicative Complex (pre-RC):
1) Origin Recognition Complex (ORC) binds to replicator sequence in the replicator origin
2) ORC recruits helicase-loading proteins Cdc6 and Cdt1 (which bind to the ORC)
3) Helicase Mcm2-7 binds to the complete formation of the pre-RC
- Forming a STABLE complex at a SELECTED replicator origin
How many molecules of helicase Mcm2-7 are needed in DNA replication and why?
2
As there are 2 replication forks which move away from eachother
Describe the origin activation phase of DNA replication initiation
High levels of cyclin-dependant kinase (Cdk) activity:
- BIND to and ACTIVATE the existing Pre-RC
- PREVENT new Pre-RC from forming
Why can the selected origins not be activated in G1?
There is no Cdks present to activate the Pre-RC
How do Cdk levels ensure that chromosomes are replicated exactly ONCE per cell cycle?
- Levels of Cdks remain HIGH in G2 - preventing the formation of any new pre-RC
- Levels drop at the end of M phase - allowing new pre-RC to form at the stat of G1
Describe the ‘End Replication Problem’
- Created by the need for RNA primers for DNA replication
- RNA primer removed by ribonuclease H
- All the gaps which are created by the removal of the primer can be closed by DNA polymerase and DNA ligase
- APART FROM ONE: at the far end, as there is no DNA sequence before it for DNA polymerase to elongate
- If nothing done about this: progressive shortening of this DNA strand, which could result in the loss of important genes
What prevents the loss of DNA sequence at the 5’ end of the lagging strand?
TTAGGG repeats added to the 3’ end of the template strand by telomerase
What is the structure of telomerase?
- Ribonucleoprotein: made of many proteins and ONE intrinsic RNA
- Intrinsic RNA has a sequence of 9 nucleotides long: AAUCCCAAU
- Which has 6 nucleotides complimentary to TTAGGG
What is the ‘telomerase shuffle’?
- The synthesis of the telomere repeat sequences, which is done as a step-wise process
- Joins to the DNA sequence with the 6 complimentary nucleotides and then shuffles forwards 6 nucleotides so that the back end of the RNA is anneled to the front end of the newly synthesised DNA
