What does it mean bidirectional and semiconservative replication?
semiconservative - each parental strand ends up in one of the daughter moleculeds
bidirectional - progress in 2 directions from the origin of replication
Describe the process of initiation of replication.
- Replication is initiated at oriC origin of replication
- Initiator protein DnaA melts the DNA at the origin of replication
- DNA helicase DnaB is recruited to the melted DNA and starts unwinding process with the use of ATP
- In Eukaryotes ORC functions in a similar way to DnaA, DnaB and DnaC
- Initiation of DNA synthesis requires a template and a primer
In what direction the new strand is synthesised in replication? What is the consequence of this?
DNA is synthesised in 5’-3’ direction. As a result the replication on the leading strand is continuous, while on the lagging strand it is episodal.
Describe the DNA synthesis on the lagging strand.
- Okazaki fragments are synthesised by elongation from primers.
- There are nicks between the 3’ ends of okazaki fragments and the preceeding RNA primers
- Nick translation - DNA polymerase I extends the Okazaki fragments (5’-3’ polymerisation) and removes the RNA primer (5’-3’ exonuclease)
- DNA ligase binds after polymerase dissociation and seals the nick between the fragments
What is replisome? What are the functions of Beta-Clamps, Primerosome and SSB tetramer?
Replisome is the molecular machinery for DNA replication.
- Primosome – contains Primase which synthetises the RNA primase necessary for DNA replication
- β-clamps keep the polymerase complex loaded on DNA
- SSB tetramer – protect the single stranded DNA from damage and improper DNA formation
How many polymerases have E.coli? Which one has the highest processivity?
E. coli has >5 DNA polymerases - Pol I-V
DNA Pol III is the subunit of Pol III holoenzyme with the highest processivity
How many polymerases do Eukaryotes typically have?
> 15
What are the important features of Polymerases?
o Multisubunit enzymes
o Require templates
o Have proofreading activity (3’-5’ exonuclease)
o Replicative polymerases remain bound to the replication fork
o Coordinate synthesis of leading and lagging strands
What is the 3D shape of DNA polymerases?
right hand-shaped
