092013 Replication 3 Post

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Structure and Mechanismof DNA Replication


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Okazakis experiments:Labeling the DNA in cells with a mutant ligase


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Text

Objectives:

1. Analyze data from and propose experiments to probe the

function of the components of the replication machinery.2. Predict the effect on replication when a topoisomerase is

mutated or deleted.

3. Describe the basic elements for replication initiation and

predict the consequence of mutations in one of thecomponents in replication initiation.

4. Explain the end replication problem and describe therequirements for replicating DNA ends.

Goal: To understand how the genetic material isduplicated and assess how duplication is achieved with

extraordinary accuracy.


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Combining the chemistry and the polymerase


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Structure determination using X-ray crystallography

Courtesy D. Jeruzalmi


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DNA polymerase

fingers

- template - incoming base - quality control

palm

- chemistry - qc

thumb - product duplex

3-5 exonuclease - proofreading


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primer

template


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primer

template


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fingers - bind incoming dNTP - conformational change - sense base-pair geometry

open


P

P

P

3'-OH

ion B

primer template5'

5'

3'

O-helix ofDNA polymerase(open)

ARG

Lys

Tyr

C

G

ion A

incomingdNTP

++

++

Catalytic site

closed

P

P

P

40

3'-OH

5'

5' 3'

O-helix(closed)

rotation of O-helix

ARGLys

Tyr

C G ++

++


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open

P

P

P

3'-OH

ion B

primer template5'

5'

3'


ARG

Lys

Tyr

C

G

ion A

incomingdNTP

++

++

Catalytic site

closed

P

P

P

40

3'-OH

5'

5' 3'

O-helix(closed)

rotation of O-helix

ARGLys

Tyr

C G ++

++



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open

P

P

P

3'-OH

ion B

primer template5'

5'

3'


ARG

Lys

Tyr

C

G

ion A

incomingdNTP

++

++

Catalytic site

closed

P

P

P

40

3'-OH

5'

5' 3'

O-helix(closed)

rotation of O-helix

ARGLys

Tyr

C G ++

++

Tyr



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fingers

palmthumbexonuclease

primertemplate


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incomingdNTP

templatebase


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space forincoming

dNTP

templatebase


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DNA replication is very complex.

There are multiple polymerases.


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Replication changes the supercoiling state of theDNA and therefore requires topoisomerases.


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Peter et al. (1998) Cell, 94.

Replication generates positive supercoils ahead of thereplication fork


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Peter et al. (1998) Cell, 94.

Replication generates positive supercoils ahead of thereplication fork


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Two special cases in replication:

Initiation

Termination


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Origins of replication

Replication of chromosomes commences at specificsites called origins.

Initiator proteins bind to specific DNA elements toactivate replication.

Replication proceeds bidirectionallyfrom origins withtwo replication forks that move away from each other.

Replication of circular chromosomes yields interlockeddaughter chromosomes and replication of linearchromosomes yields tangled chromosomes.


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Initiator proteinsbind to specific DNA elements to activate replication.

Origin


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Divergent replication forks extendout from the origin


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topoisomerase II


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It takes ~40 minutes to replicate the E. colichromosome.In the lab, E. colican divide every 20 min.

How is this possible?

Multi-fork Replication

!"#$%&'()*$+ #$%&'()*$+

,"($

#$%&'()*$+

*-'($

#$%&'()*'".

(/#,0,1,0$1

1$.#$.)*$+


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Initiation is very tightly controlled so that each origin isactivated once and only once per cell cycle.

Eukaryotic chromosome contain multiple origins


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The lagging strand cannot be

completely replicated.

Template for lagging DNA strand

Template for leading DNA strand

!"

#"

template DNA new DNA RNA primer

Replicating chromosome ends


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Genes lost!

Essential genes here

- cells die!

Information loss during replication


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Telomere repeat

- cells die!

Delaying information loss during replication


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Telomerase!

Telomere repeat


Solving the information loss problem


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Telomerase!

Elizabeth H. Blackburn Carol W. Greider Jack W. Szostak

The Nobel Prize in Physiology or Medicine 2009


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The end replication problem

+ last Okazaki

fragment

repair Okazaki

fragments

+ incompletely

replicated DNA

3'5'

3'5'

3'5'

5'3'

5'3'

5'5'3'

3'5'

5'3'

3'5'

5'3'

3'


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+

+

replicate again

chromosome

is shorter

+

3'5'

5'3'

3'5'

5'3'

3'

5'

5'

3'

3'5'

5'3'


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Telomerase solves the end replication problem


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From MBOG Interactive Animations, Chapter 9 "Action of Telomeres"


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The End

of DNA replication

Special thanks to

Prof. David Jeruzalmi

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092013 Replication 3 Post