DNA Replication Flashcards
What direction is DNA replicated in?
5’ to 3’ end
5’ = phosphate group
3’ = OH group
Why is DNA synthesis an irreversible reaction?
It is coupled to a breakdown of PPi by 2Pi
Where is pyrophosphatase found?
It is ubiquitous to all cells
DNA synthesis is exothermic or endothermic?
Exothermic - -7kcal/mole
dNTP + (dNMP)n =
(dNMP)n+1 +2Pi
The energy required for DNA synthesis comes from what?
The breakage of 2 high energy phosphate bonds
Where is DNA synthesis initiated?
At the replication fork
Which enzyme separated paired DNA strands?
DNA Helicase
The leading strand runs in which direction?
The 5’ end to the 3’ end
What does the lagging strand form?
Okazaki fragments
Which enzyme synthesises the primer for DNA Synthesis?
DNA primase - synthesises a short RNA
How long are primers usually?
About 10 nucleotides long
How does DNA polymerase differe from DNA primase?
Unlike DNA polymerase, DNA primase can start a new polynucleotide chain by joining two nucleotide triphosphates together
What four enzymes are required for lagging strand synthesis?
DNA Primase
DNA Polymerase
Ribonuclease H
DNA Ligase
What is the role of ribonuclease H?
Removes the RNA Primer
What are the steps of DNA replication in the lagging strand?
1) New RNA primer synthesis by DNA primase
2) DNA polymerase adds to RNA primer to make a new okazaki fragment
3) DNA polymerase finishes the DNA fragment
4) The old RNA primer erased and replaced by DNA
5) Nick sealing by DNA ligase joins new okazaki fragment to the growing chain
How does DNA ligate newly synthesised okazaki fragments?
DNA ligase seals a broken phosphdiester bond
DNA ligase uses a molecule of ATP to activate the 5’ end at the nick before forming a new bond:
- ATP + 5’p = p-p + 5’-p-AMP
- p-p = 2Pi + free energy
Describe the structure of DNA Helicase
Has a symetrical hexometric structure
6 subunits
What is the rate at which DNA Helicase pulls apart the helix?
1000 nucleotide pairs per second
What are single strand binding proteins? SSBs
Bind tightly and cooperatively to exposed single stranded DNA without covering the bases
They aid DNA helicase by stabilising unwound DNA
They straighten out the lagging strand template to prevent hairpin helices forming
Why are hairpin helices indesirable?
They can impede DNA synthesis by DNA polymerase
What is Werner Syndrome?
Progeria (premature aging)
Mutations are autosomal recessive, occuring in RECQ helicase gene WRN
What causes blood syndrome?
Loss of function mutation in a Recq- family DNA helicases which maintain genome integrity
What is the role of the sliding clamp?
Encircles DNA like a nut on a bolt and helps to move DNA polymerase forward along the DNA
What is the role of DNA topoisomerases?
These prevent DNA from becoming tangled during replication
Unwinding of DNA at the replication fork introduces superhelical tension into the DNA helix
Tension is relaxed by topisomerases which nick and reseal the back bone of the parental helix
What is the specific role of type 1 topoisomerases?
They nick and reseal one of the 2 DNA strands, no ATP required
What is the specific role of type 2 topoisomerases?
They nick and reseal both DNA strands, ATP is required
When does replication selection occur on eukaryotes?
During G1
What occurs in origin activation?
Unwinding of DNA and recruitment of DNA polymerase
Occurs in S phase
How long does DNA replication take in mammals?
Approx 8 hours
Temporal separation of growth and synthesis ensures what?
That each origin is used and that each chromosome is replicated exactly once in each cell cycle
How is the pre-replicative complex formed?
The origin recognition complex binds to a replicator sequence
Helicase loading proteins Cdc6 and Cdt1 bind to the ORC
The Helicase Mcm2-7 binds to complete the formation of pre-RC
What happens if Cdk activity levels are low in S phase?
pre-RC formation is allowed
But pre-RCs are not activated
What happens if Cdk activity levels are high?
New pre-RC formation inhibition
Existing pre-RC activation is allowe
In what phases are Cdk levels low?
G1
In what phases are Cdk levels high?
S, G2 and M phases
What is the problem with RNA primer removal?
It causes progressive shortening of linear DNA sequences
Telomerase extends which end of DNA?
The 3’ end
