lecture dna repli transc transl060312(2)

8/2/2019 Lecture DNA Repli Transc Transl060312(2)

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DNA-replication is semi-

discontinuousReplication starts atspecific regioncalled replicon.

DNA unwindingoccurs at thereplicon whichmakes a pork likeconformation,called replication

fork. Helicase and


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DNA replication is bi-directional

Bacterial DNA has a single

origin of replication


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Replication of Drosophila (fruit fly) DNA

Label first with high radioactive nucleotides,

which is followed by weak radioactive nucleotides

Eukaryotic DNA have multiple origin of replication


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origins of DNA replication (every ~150 kb)

replication bubble

daughter chromosomes

fusion of bubbles

bidirectional replication

Origins of DNA replication on mammalian chromosomes

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RNA primer

5

3

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direction of leading strand synthesis

direction of lagging strand synthesis

replication fork


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iscontinuous synthesis of DNA in the DNA bouble in twirections

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5 3

3 5

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5 3

3 5

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leading strand (synthesized continuously)

lagging strand (synthesized discontinuously)

replication fork has a leading and a lagging strand

The leading and lagging strand arrows show the directionof 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)

replication fork replication fork


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5

3

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Strand separation at the replication fork causes positivesupercoiling of the downstream double helix

NA gyrase is a topoisomerase II, which

breaks and reseals the DNA to introduce negativesupercoils ahead of the fork


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5

3 5

3

Movement of the replication fork


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Movement of the replication fork

RNA primer

Okazaki fragment

RNA primer

5


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

5

3

3

5

origin DNA sequence

binding of dnaA proteins

A A A

dnaA proteins coalesce

DNA melting induced

by the dnaA proteinsA

A

A

AA

A

A

A

A

AA

A B C

dnaB and dnaC proteins bind

to the single-stranded DNA

dnaB further unwinds the helix


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AA

A

AA

A B C

dnaB further unwinds the helixand displaces dnaA proteins

G

dnaG (primase) binds...

A

A

A

A A

AB C

G

...and synthesizes an RNA primer

RNA primer


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B C

G

5 3template strand

RNA primer(~5 nucleotides)

Primasomedna B (helicase)dna Cdna G (primase)

OH3 5


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3

5 3

RNA primer

newly synthesized DNA

5

5

DNA polymerase


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RNA primer

5

DNA polymerase III initiates at the primer andelongates DNA up to the next RNA primer

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5

3

5

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

5

3

A polymerase I inititates at the end of the Okazaki fragmentnd further elongates the DNA chain while simultaneouslymoving the RNA primer with its 5 to 3 exonuclease activity

pol III

pol I


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newly synthesized DNA(Okazaki fragment)

5

3

5

3

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

hat are needed to make lagging strand in E. Coli?ne RNA polymerase

wo DNA polymerases (I and III)ne DNA ligase


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3

5

Proteins at the replication fork in E. coli

Rep protein (helicase)

Single-strandbinding protein

(SSB)

BCG

Primasome

pol I

pol III

pol III

DNA ligase

NA gyrase - this is a topoisomerase II, whichreaks and reseals double-stranded DNA to introduceegative supercoils ahead of the fork


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Common structure of DNA polymerase

Enzyme has independentdomains

Conserved sequence motif for catalytic active site

Responsible forpositioningtemplatecorrectly at theactive site

Binds DNA as it exitsthe enzyme


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Process of DNA replicatiton

1. Helicaseseparates both

strands of theDNA

2. Single-stranded proteinsbind andmaintain

separatedstrands

3. Prime with 3-OH end(differencebetween leading& lagging strand)

4. Synthesis ofDNA by DNApolymerase

5. Ligation ofOkazaki

fragments byligase (laggingstrand only)

(Figure obtained at Ohio State Biosci websit

lecture dna repli transc transl060312(2)

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