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