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Chapter 30

DNA ReplicationPages 984-998

All rights reserved. Requests for permission to make copies of any part of the work

should be mailed to: Permissions Department, Harcourt Brace & Company, 6277Sea Harbor Drive, Orlando, Florida 32887-6777

Learning objectives: Understand what ismeant by the following, and how we knowthese statements to be true

DNA replication is:Semi-conservativeBidirectional

Semi-discontinuous

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The Dawn of Molecular Biology

April 25, 1953 Watson and Crick: "It has not escaped our

notice that the specific (base) pairing we havepostulated immediately suggests a possiblecopying mechanism for the genetic material."

The mechanism: Strand separation, followed bycopying of each strand.

Each separated strand acts as a template for thesynthesis of a new complementary strand.

This is referred to as semi-conservative model

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Parental Strands

Strandduplication

1/2 old1/2 new

Strand separation

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Models for DNA replication

1) Semiconservative model:

Daughter DNA molecules contain one parentalstrand and one newly-replicated strand

2) Conservative model:

Parent strands transfer information to anintermediate (?), then the intermediate gets copied.

The parent helix is conserved, the daughterhelix is completely new

3) Dispersive model:Parent helix is broken into fragments, dispersed,copied then assembled into two new helices.

New and old DNA are completely dispersed

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Testing Models for DNA replicationMatthew Meselson and Franklin Stahl (1958)

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Matthew Meselson and Franklin Stahl more recently

Faculty member at HarvardMechanisms of Molecular Evolution

Faculty Chair for CBW Studies

Faculty member at U. of OregonMeiotic Recombination

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Testing Models for DNA replicationMeselson and Stahl (1958)

Density labeling experiment on E. coli (bacterial) DNA

Bacterial culture

15

NH4Cl(Sole N source)

Grow for several generations

Bacterial culturewith dense DNA

This is the starting material for the experiment

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Meselson and Stahl (continued)

Harvest cells and resuspend in media with14NH4Clas the sole N source

Bacterial culture

with dense DNA

Grow for 1generation

Harvest some cells

1st generation

Grow for anothergeneration

Harvest some cells

2nd generation

Grow for anothergeneration

etc

For each generation isolate the DNA and spin through a density (CsCl) gradient).Detect DNA in the gradient (eg by UV absorption)

Monitor how many DNA bands there are after each generation

Bacterial culture

0 generation

14NH4Cl

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Anticipated Results for each Possible Model

Gray = Heavy

orange = light

3 light, 1 heavyi.e. two bands

2 light, 2 intermediatei.e. two bands

4 intermediatei.e. one band

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Meselson and Stahl Original Data

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DNA Replication

Is semiconservative Matthew Meselson and Franklin Stahl showed

that DNA replication results in new DNA duplex

molecules in which one strand is from the parentduplex and the other is completely new

Since DNA replication is semiconservative,therefore the helix must be unwound.

Unwinding generates torsional stress (supercoils)which must be removed by topoisomerases(Chapter 12)

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Real World Biochemistry

Ciprofloxacin is a synthetic bactericidalantibiotic that inhibits bacterial DNAsynthesis, so that bacteria rapidly die.The target is the enzyme DNA gyrase

(topoisomerase II), which is responsiblefor the supercoiling and uncoiling of theDNA. Uncoiling of the DNA is theinitiative step for replication,transcription and repair of the DNA.Thus, prolonged inhibition will

eventually lead to the death of thebacteria.

CIPRO

http://infections.bayer.com/treatment/ciprofloxacin_ciprobay_en.html

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DNA Replication

Since DNA replication is semiconservative,therefore the helix must be unwound.

John Cairns (1963) showed that initial unwindingis localized to a region of the bacterial circulargenome, called an origin or ori for short.

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E. colichromosome

Localizedunwinding

origin

OR

DNA replication

unidirectionalbidirectional

Replication forks

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John Cairns

Grow cells for several generationsSmall amounts of 3H thymidineare incorporated into new DNA

Grow for briefperiod of time

Add a highconcentration

of 3H- thymidine

in media with lowconcentration of

3H- thymidine

Bacterialculture

*T

*T

*T

*T

Dense label at the replication forkwhere new DNA is being made

*T

*T*T *T

*T*T

*T*T

*T*T*T

*T*T

*T*T

*T

*T*T *T *T

*T*T*T

*T

*T*T*T

All DNA is lightlylabeled with radioactivity

*T*T *T

Cairns then isolated the chromosomes by lysing the cells very very gently

and placed them on an electron micrograph (EM) grid which he exposed toX-ray film for two months.

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Evidence points to bidirectional replication

Label at both replication forks

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Features of DNA Replication

DNA replication is semiconservative

Each strand of both replication forks is

being copied.

DNA replication is bidirectional

Bidirectional replication involves tworeplication forks, which move in oppositedirections

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Arthur Kornberg (1957)

Protein extracts from E. coli+

Template DNAIs new DNA synthesized??

- dNTPs (substrates) all 4 at once- Mg2+ (cofactor)- ATP (energy source)- free 3OH end (primer)

In vitro assay for DNA synthesis

Used the assay to purify a DNA polymerizing enzyme

DNA polymerase I

Currently a faculty member atStanford School of Medicine

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3

Kornberg also used the in vitroassay to characterizethe DNA polymerizing activity

- dNTPs are ONLY added to the 3 end of newlyreplicating DNA

-therefore DNA synthesis occurs only in the

5 to 3 direction

3

3

53

5

535

535

535 3

Parental template strandNew progeny strand

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THIS LEADS TO A CONCEPTUAL PROBLEM

Consider one replication fork:

5

3

5

3

Direction of

unwinding

Continuous replication

5

3Primer

5

3

53

Discontinuous replication

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Evidence for the Semi-Discontinuous replicationmodel was provided by the Okazakis (1968)

Reiji Okazaki was born near Hiroshima, Japan, in 1930.

He was a teenager there at the time of the explosion of the first of twonuclear bombs that the US dropped at the end of World War II.

His scientific career was cut short by his untimely death from cancerin 1975 at the age of 44, perhaps related to his exposure to the falloutof that blast.

Tuneko Okazaki, until recently, was a professor at

The University of Nagoya where she was the firstwoman at that institution to be named a professor.

Currently she is on the faculty of Medicine in Fujita,and does research on centromeres.

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Evidence for Semi-Discontinuous Replication(pulse-chase experiment)

Bacteria arereplicating

Bacterialculture

Add 3H Thymidine

For a SHORT time(i.e. seconds)

Flood with non-radioactive T

Allow replicationTo continue

Harvest the bacteriaat different timesafter the chase

Isolate their DNASeparate the strands(using alkali conditions)Run on a sizing gradient

smallest

largest

Radioactivity will onlybe in the DNA that wasmade during the pulse

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smallest

largest

Results of pulse-chase experiment

Pulse

5

3

5

3

Direction of

unwinding

3

5

Primer

5

3

5

3

***

Chase

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Continuous synthesis

Discontinuous synthesis

DNA replication is semi-discontinuous

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Features of DNA Replication

DNA replication is semiconservative

Each strand of template DNA is being copied.

DNA replication is bidirectional

Bidirectional replication involves two replicationforks, which move in opposite directions

DNA replication is semidiscontinuous

The leading strand copies continuously

The lagging strand copies in segments(Okazaki fragments) which must be joined

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The Enzymologyof DNA Replication

In 1957, Arthur Kornberg and colleaguesdemonstrated the existence of a DNA

polymerase - DNA polymerase I Pol I needs all four deoxynucleotides, a template

and a primer - a ss-DNA (with a free 3'-OH) thatpairs with the template to form a short double-stranded region

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DNA Polymerase I has THREE

different enzymatic activities:

a 5 to 3 DNA polymerizing activity

a 3 to 5 exonuclease activitya 5 to 3 exonuclease activity

DNA Pol Ifrom E. coli is 928 aa (109 kD) monomer

- a single polypeptide that packs a punch!

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The protein is folded into discretedomains

Hans Klenow used proteases (subtilisin ortrypsin) to cleave between residues 323 and

324, separating 5'-exonuclease (on the smallfragment) and the other two activities (on thelarge fragment, the so-called "Klenow fragment)

Tom Steitz has determined the structure of theKlenow fragment

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The 5 to 3 DNA polymerizing activity

Subsequenthydrolysis of

PPi drives the

reaction forward

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DNA Polymerase I

Replication occurs 5' to 3'

Nucleotides are added at the 3'-end of thestrand

Pol I catalyzes about 20 cycles of polymerizationbefore the new strand dissociates from template

20 cycles constitutes moderate "processivity"

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More on Pol I

Why the exonuclease activity?

The 3'-5' exonuclease activity serves aproofreading function

It removes incorrectly matched bases, so

that the polymerase can try again

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For Next Class:

We will finish up DNA replicationChapter 30

Sections 30.3 30.4, 30.5, 30.6

We will NOT cover section 30.7