Fidelity of DNA polymerase
Shape selectivity:
DNA polymerase's conformational change for determination of fidelity for each nucleotide
Induced fit:
Structure determines function
Matched nucleotide
Fidelity of DNA polymerase
Proofreading activity:
DNA polymerase's enzymatic activity for determination of fidelity for DNA polymerization
3’ to 5’ exonuclease
Exonuclease vs Endonuclease
Excision vs Incision
Fidelity of DNA polymerase
DNA replication with a proofreading polymerase:
DNA polymerase's enzymatic activity for determination of fidelity
https://www.youtube.com/watch?v=6O0qD6KCOVE
DNA Polymerase
DNA polymerase synthesizes DNA only in the 5’ to 3’ direction:
adding a dNTP to the 3’ hydroxyl group of a growing chain.
Why is DNA replication performed in the 5’ to 3’ direction?
Proofreading activity for fidelity
DNA polymerization requires deoxynucleoside 5’-triphosphates
DNA polymerization requires
deoxynucleoside 3’-triphosphates
DNA Polymerase
DNA polymerase requires a primer to begin DNA synthesis
[ NO de novo DNA synthesis ]
[ Primed DNA synthesis ]
Why does DNA polymerase require the primer for replication?
Stepwise proofreading activity for
fidelity
This end will be from already right or proofreaded nucleotide?
DNA Polymerase
DNA polymerase requires a 1] Primer to begin 2] 5’to 3’ DNA synthesis
Replication fork
The replication forks represent the regions of
active DNA synthesis [replication] by DNA polymerse
HOWEVER;
1] DNA polymerase synthesizes DNA in 5’ to 3 direction
2] Double-helical DNA run in opposite direction
Semi-continuous DNA replication
DNA Strand in continuous synthesis: Leading strand
DNA strand in discontinuous synthesis : Lagging strand [Okazaki fragments]
Elongation of double strands of DNA at the replication fork
1] 5’ to 3’ direction
2] Same time [NO same location] in opposite direction
Synthesis of
leading & lagging strands of DNA
1] The leading strand is made continuously & in one piece
2] The lagging strand is made small chunks, Okazaki fragments
in order to follow the 5’ to 3’ direction
3] Okazaki fragments are then joined together by DNA ligase [Spot welder]
4] DNA replication is semiconservative
Figure 6.3
How is the synthesis of Okazaki fragments initiated?
1] DNA polymerase requires a primer
2] DNA polymerase cannot initiate synthesis de novo
1] Primase synthesizes primer
2] Primase synthesizes RNA fragments
[RNA priming]
[RNA-DNA hybrid]
Continuous synthesis of
lagging strands of DNA
Discontinuousness of lagging strands of DNA
Q1] Lagging strand is synthesized in small pieces, Okazaki fragment
A1: Okazaki fragments are joined together by DNA ligase
Q2] Newly synthesized Okazaki fragment contain an RNA-DNA joint
A2: RNA primers must be removed and replaced with DNA
How is RNA primer removed and replaced with DNA?
1] RNA primer is removed by 5’ to 3’ exonuclease
2] DNA gap is filled by DNA polymerase
3] DNA fragments are joined by DNA ligase
Figure 6.5
Prokaryote
Eukaryote
DNA pol I
Rnase H
DNA pol I
DNA pol d
DNA ligase
DNA ligase
Different polymerases in
procaryotic and eukaryotic cells
Figure 6.6
Prokaryote
Eukaryote
DNA pol III
DNA pol e
Primase
Primase
+ DNA pol a
DNA Pol III
DNA Pol d
Polymerase accessory proteins
DNA polymerase must maintain the stable association with the DNA template
1] Sliding-clamp proteins (PCNA) : loading of the DNA polymerase at primer-template junction
2] Clamp-loading proteins (RFC) : loading of the sliding-clamp proteins at primer-template junction
Helicase and
Single-stranded DNA-binding proteins
The parental DNA has to be unwounded and the single-stranded regions has to be stabilized
For serving as template for new DNA synthesis
1] Helicase: unwinding of the two strands of parental DNA ahead of the replication fork
2] Single-stranded DNA-binding proteins (SSB): stabilization of extended single-stranded state
DNA polymerase holoenzyme
The DNA polymerase holoenzyme consists of 2 copies of the polymerase core enzyme
linked to a central structure: Coordinated & simultaneous replication