Lecture 5- DNA replication Flashcards
Which direction does DNA synthesis occur in?
Occurs on a 5’ to 3’ prime direction mediated by the formation of a phosphodiester bonds.
What happens in the chemical reaction for DNA replication?
- The 3’ hydroxyl at the end of the growing DNA strand makes a nucleophile attack on the phosphate of the incoming dNTP
- This forms a diester bond
- Therefore the nucleotide is incorporated and the pyrophosphate is released
Describe DNA synthesis via a replication fork
- DNA helicase separates the double stranded DNA
- This enables a primer to form on the leading strand with DNA synthesis occurring in 5’ to 3’ direction in this DNA replication fork
- On the lagging strand, DNA synthesis still occurs 5’ to 3’ but in the opposite direction to the leading strand
- The antiparallel orientation of parental strands and unidirectional orientation of new DNA synthesis means that both new strands cannot be synthesised continuously
- To facilitate the lagging strand, Okazaki fragments produced by the lagging strand
- To keep up with the leading strand, the lagging strand continuously synthesised short DNA fragments
What type and which direction is the synthesis of the leading and lagging strand?
Both occur 5’ to 3’
Leading strand synthesis is continuous, lagging strand synthesis is discontinuous
What is the job of DNA polymerase?
Use free -OH groups to add nucleotides to the 3’ ends of existing DNA strands
How is a RNA primer made?
The short RNA primer is synthesised using template and NTPs by DNA primase
Outline the steps on synthesising DNA chains
- DNA primase makes RNA primer
- Primase dissociates from the DNA template leaving a primer-template junction
- DNA polymerase extends the RNA primer from the primer-template junction
- The RNA primer are removed by ribonuclease H
- The gap that is left is filled by DNA polymerase and the nick is sealed by DNA ligase
What does DNA helicase do?
Using ATP to separate parental DNA strands by breaking H bonds
Give 2 diseases causes by mutation in genes encoding DNA helicase?
- Werner Syndrome
2. Bloom Syndrome
What increases the processivity of DNA polymerases?
The association with a sliding clamp
What is processivity?
An enzymes ability to catalyse consecutive reactions without releasing its substrate
Explain the function of proceessive DNA polymerases?
- Add multiple nucleotides increasing the rate of DNA synthesis
- DNA polymerase’s ability to slide along the DNA template increased processivity
What is the role of the clamp loader?
The clamp loader used ATP hydrolysis to load the sliding clamp onto the DNA near the primer-template junction
What is the role of the sliding clamp?
- The sliding clamp is locked onto the DNA which causes the displacement of primase and triggers the recruitment of DNA polymerase.
- DNA is then reeled through the replisome and polymerase synthesised DNA continuously on the leading strand
- On the lagging strand, DNA polymerases and synthesises DNA in bursts
What do SSBs do?
Expose single stranded DNA in the replication fork, making it available for templating synthesis of the new DNA strand and stopping the formation of DNA hairpins
How do DNA hairpins form?
The leading and lagging stands can anneal back on itself due to complementary DNA, creating hairpins
Without SSBs, DNA hairpins can form. What is the problem with this?
Hairpins impede the progress of DNA polymerase and compromise its processivity.
What is the role of DNA topoisomerases?
Prevent DNA from becoming tangled during DNA replication and enhancing the processivity of DNA polymerase
How does DNA topoisomerases work?
Relieve tension by transiently nicking and resealing the parental double stranded DNA helix. This prevents DNA from becoming tangled which keeps the replication fork open for maximal processivity of DNA polymerases
Where does replication begin in E.coli?
Replication begins at a genetically defined replicator site called oriC
How is DNA replication initiated?
- An initiator protein binds to oriC and promotes the melting of the double stranded DNA
- This provides access for DNA replication machinery collectively referred to as the replisome
What are the 2 phases (biphasic) of DNA replication initiation in eukaryotes?
- Replicator selection occurs in G1 phases- formation of pre-replication complex
- Origin activation occurs in S phase- unwinding of DNA and recruitment of DNA polymerase
Ensures that each origin is used and each chromosome is only replicated once per cell cycle.
Describe eukaryotic replicator selection leading to the formation of the pre-replication complex
- Origin recognition complex bind to the replicator sequence (eg ARS in yeast)
- Helicase loading proteins Cdc6 and Cdt1 are recruited and bind to the ORC
- ATP hydrolysis opens the two hexametric helicase MCM2-7 rings, allowing them to wrap around the DNA in a head-head orientation
- This concludes the formation of the pre-replication complex
What do high levels of Cdk activate and prevent during S-phase
Activates existing pre-RC complexes but prevents the formation of new pre-RC complexes
