Replication starts at specific region called replicon.

DNA unwinding occurs at the replicon which makes a pork like conformation, called replication fork. Helicase and

DNA-replication is semidiscontinuous

DNA replication is bi-directional

Bacterial DNA has a single origin of replication

Eukaryotic DNA have multiple origin of replication

Label first with high radioactive nucleotides, which is followed by weak radioactive nucleotides

Replication of Drosophila (fruit fly) DNA

Origins of DNA replication on mammalian chromosomes origins of DNA replication (every ~150 kb) 5 3 3 5

bidirectional replication

replication bubble fusion of bubbles 5 3 5 3 daughter chromosomes 3 5 3 5

RNA primer direction of leading strand synthesis 3 5 replication fork 5 3

3 5 direction of lagging strand synthesis

Discontinuous synthesis of DNA in the DNA bouble in two irections

5 3 5 3 3 5 3 5 5 3 3 5

replication fork has a leading and a lagging strand

leading strand (synthesized continuously)

replication fork 5 3 5 3 3 5 3 5

replication fork 5 3 3 5

lagging strand (synthesized discontinuously) The leading and lagging strand arrows show the direction of DNA chain elongation in a 5 to 3 direction The small DNA pieces on the lagging strand are called Okazaki fragments (100-1000 bases in length)

Strand separation at the replication fork causes positive supercoiling of the downstream double helix

3 5

5 3

DNA gyrase is a topoisomerase II, which breaks and reseals the DNA to introduce negative supercoils ahead of the fork

3 5

Movement of the replication fork

5 3

5 3

Movement of the replication fork

RNA primer Okazaki fragment RNA primer

nitiation of DNA synthesis at the E. coli origin (ori)

origin DNA sequence 5 3

A A A A

3 5

binding of dnaA proteins

A A

DNA melting induced by the dnaA proteins

dnaA proteins coalesce dnaB and dnaC proteins bind to the single-stranded DNA

A A A

A A

B C

dnaB further unwinds the helix

dnaG (primase) binds...

A A A

B C
A

dnaB further unwinds the helix and displaces dnaA proteins

A A A A A A

...and synthesizes an RNA primer

G
RNA primer

B C

G B C

Primasome dna B (helicase) dna C dna G (primase)

template strand 3 OH RNA primer (~5 nucleotides) 5

DNA polymerase 5 5 RNA primer 3

3 5 newly synthesized DNA

RNA primer 5 3

pol III 5

DNA polymerase III initiates at the primer and elongates DNA up to the next RNA primer 5 3 5

newly synthesized DNA (100-1000 bases) (Okazaki fragment) 5 3

pol I

NA polymerase I inititates at the end of the Okazaki fragment and further elongates the DNA chain while simultaneously moving the RNA primer with its 5 to 3 exonuclease activity

5 3

newly synthesized DNA (Okazaki fragment)

DNA ligase seals the gap by catalyzing the formation f a 3, 5-phosphodiester bond in an ATP-dependent reaction 5 3

What are needed to make lagging strand in E. Coli? One RNA polymerase Two DNA polymerases (I and III) One DNA ligase

Proteins at the replication fork in E. coli

Rep protein (helicase) pol III 3 5

5 3

G
Single-strand binding protein (SSB)

Primasome

DNA ligase

C B

pol III

DNA gyrase - this is a topoisomerase II, which breaks and reseals double-stranded DNA to introduce negative supercoils ahead of the fork pol I

Common structure of DNA polymerase Enzyme has independent domains

Responsible for positioning template correctly at the active site

Binds DNA as it exits the enzyme

Conserved sequence motif for catalytic active site

Process of DNA replicatiton

1. Helicase separates both strands of the DNA 5. Ligation of Okazaki fragments by ligase (lagging strand only)

2. Singlestranded proteins bind and maintain separated strands 3. Prime with 3OH end (difference between leading & lagging strand)

4. Synthesis of DNA by DNA polymerase

(Figure obtained at Ohio State Biosci website