fidelity of dna polymerase -...
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Shape selectivity: DNA polymerase's conformational change for determination of fidelity for each nucleotide
Fidelity of DNA polymerase
Induced fit: Structure determines function
Matched nucleotide
Proofreading activity: DNA polymerase's enzymatic activity for determination of fidelity for DNA polymerization
Fidelity of DNA polymerase
3’ to 5’ exonuclease
Exonuclease vs Endonuclease
Excision vs Incision
DNA replication with a proofreading polymerase: DNA polymerase's enzymatic activity for determination of fidelity
Fidelity of DNA polymerase
https://www.youtube.com/watch?v=6O0qD6KCOVE
DNA polymerase synthesizes DNA only in the 5’ to 3’ direction: adding a dNTP to the 3’ hydroxyl group of a growing chain.
DNA Polymerase
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 requires a primer to begin DNA synthesis
DNA Polymerase
[ 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 requires a 1] Primer to begin 2] 5’to 3’ DNA synthesis
DNA Polymerase
The replication forks represent the regions of active DNA synthesis [replication] by DNA polymerse
Replication fork
HOWEVER; 1] DNA polymerase synthesizes DNA in 5’ to 3 direction 2] Double-helical DNA run in opposite direction
DNA Strand in continuous synthesis: Leading strand DNA strand in discontinuous synthesis : Lagging strand [Okazaki fragments]
Semi-continuous DNA replication
Elongation of double strands of DNA at the replication fork 1] 5’ to 3’ direction 2] Same time [NO same location] in opposite direction
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
Synthesis of leading & lagging strands of DNA
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]
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
Continuous synthesis of lagging strands of DNA
How is RNA primer removed and replaced with DNA?
Figure 6.5
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
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
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