Lecture 4 - DNA replication Flashcards
What is DNA dependent DNA synthesis?
The process where new DNA strands are synthesised at the replication form
Needs a primer to initiate synthesis
What direction does DNA dependent synthesis occur in?
5’-3’
Which enzymes carries out DNA dependent DNA synthesis?
DNA-dependent DNA polymerase
What is exonuclease activity?
When DNA polymerases degrade DNA
What are the two types of exonuclease activity?
3’-5’
5’-3’
What is 3’-5’ exonuclease activity?
Polymerase removes nucleotides its just inserted
This is proofreading and lets errors be corrected
DNA polymerase III has 3’-5’ activity
What is 5’-3’ exonuclease activity?
The polymerase can remove DNA that already attached to the template
DNA polymerase I has 5’-3’ activity
Which polymerases are involved in prokaryotes?
DNA polymerase I
DNA polymerase III
Which polymerases are involved in eukaryotic replication?
DNA polymerase alpha
DNA polymerase delta
What occurs at the replication fork in humans?
- Helicase breaks the base pairs
- Single strand binding proteins protect the bare single strands
- DNA topoisomerase unwinds the strands
- Primase or DNA polymerase alpha makes primers on the leading and lagging strand
- Two copies of DNA polymerase delta synthesises DNA
- FEN1 and DNA ligase removes primers and joins the Okazaki fragments
- The proliferating cell nuclear antigen sliding clam holds DNA polymerase delta tightly onto DNA
What happens during the replication of E.coli (bacteria)?
- DnaA proteins bind close to the origin of replication
- The DNA becomes wound around these proteins
- This forces base pairs to break at the origin of replication and it opens
- DnaB ptoteins attach to the origin to prepare the replication forks which forms the prepriming complex
- DnaB breaks more base pairs and the replication forks move away from the origin
- The primosome is formed by attachment of two primase enzymes
- These make the RNA primers that initiate replication of the leading strands
What occurs at the replication fork in E.coli (bacteria)?
- DnaB or Helicase breaks the base pairs
- Single strand binding proteins protect the bare single strands
- DNA topoisomerase unwinds the strands
- Primase makes primes on the lagging and leading strand
- Two copies of DNA polymerase III synthesise DNA
- DNA polymerase I and DNA ligase remove primers and join Okazaki fragments
- The gamma complex attaches and detaches polymerase III from the lagging strand
- The beta complex holds polymerase III onto the template which allows it to slide
What are the differences between human and E.coli genomes?
Human: Comprises 3200Mb DNA
Ecoli: Comprises 4.64Mb DNA
Human: Genome is split into 24 linear DNA molecules
Ecoli: Contained in circular DNA molecule
What are single strand binding proteins? (SSBS)
Proteins that protect the separated DNA strands
What is the leading strand?
Strand of DNA that can be copied by continuous DNA synthesis
A primer is needed to copy it
As replication fork moves its extended by DNA synthesis
What is the lagging strand?
Must be copied from a primer at the replication fork
Must be made in sections called Okazaki fragments
What are Okazaki fragments?
Sections that make up the lagging strand
They must be joined together and RNA primers must be removes to complete synthesis of lagging strand
What are primers like in prokaryotes?
Made of RNA
Made by the primase enzyme
4-15 nucleotides long
When the primers made DNA polymerase III makes the new strand
What are primers like in eukaryotes?
Made of RNA
Primer is extended by DNA polymerase alpha which adds 20 nucleotides
DNA polymerase delta makes the rest of the new strand
How does joining work in prokaryotes?
Polymerase I and III
III is on lagging strand and hits the primer and stops as doesn’t have 5’-3’
Switches to I which removes the primer and enters DNA
Small nick linked with DNA ligase
How does joining work in eukaryotes?
Uses alpha and delta
Delta and helicase push aside the primer which makes a flap
FEN1 cuts the flap away which makes a missing phosphodiester bond
Polymerase continues until it reaches the missing bond
DNA ligase links two fragments together
DNA polymerase I is:
Bacterial
3’-5’ and 5’-3’
DNA repair and replication
DNA polymerase III is:
Bacteria
3’-5’
Replicating enzyme
DNA polymerase a is:
Eukaryotic
Neither 3-5 or 5-3
Priming
DNA polymerase delta is:
Eukaryotic
3-5
Replicating enzyme
