Chapter 30

DNA Replication Pages 984-998

Learning objectives: Understand what is meant by the following, and how we know these statements to be true DNA replication is: Semi-conservative Bidirectional 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 have postulated immediately suggests a possible copying mechanism for the genetic material." The mechanism: Strand separation, followed by copying of each strand. Each separated strand acts as a template for the synthesis of a new complementary strand. This is referred to as semi-conservative model

Parental Strands

Strand separation

Strand duplication

1/2 old 1/2 new

Models for DNA replication

1) Semiconservative model: Daughter DNA molecules contain one parental strand and one newly-replicated strand 2) Conservative model: Parent strands transfer information to an intermediate (?), then the intermediate gets copied. The parent helix is conserved, the daughter helix 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

Testing Models for DNA replication

Matthew Meselson and Franklin Stahl (1958)

Matthew Meselson and Franklin Stahl more recently

Faculty member at Harvard Mechanisms of Molecular Evolution Faculty Chair for CBW Studies

Faculty member at U. of Oregon Meiotic Recombination

Testing Models for DNA replication

Meselson and Stahl (1958) Density labeling experiment on E. coli (bacterial) DNA

Bacterial culture

Grow for several generations

15NH Cl 4

(Sole N source)

Bacterial culture with dense DNA

This is the starting material for the experiment

Meselson and Stahl (continued)

Harvest cells and resuspend in media with

14NH Cl 4

as the sole N source

14NH Cl 4

Bacterial culture with dense DNA

Bacterial culture 0 generation

Grow for 1 generation

Grow for another generation

Grow for another generation

etc

Harvest some cells 2nd generation

Harvest some cells 1st generation

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

Anticipated Results for each Possible Model

Gray = Heavy

orange = light

3 light, 1 heavy i.e. two bands

2 light, 2 intermediate i.e. two bands

4 intermediate i.e. one band

Meselson and Stahl Original Data

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 parent duplex 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)

Real World Biochemistry

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

Ciprofloxacin is a synthetic bactericidal antibiotic that inhibits bacterial DNA synthesis, so that bacteria rapidly die. The target is the enzyme DNA gyrase (topoisomerase II), which is responsible for the supercoiling and uncoiling of the DNA. Uncoiling of the DNA is the initiative step for replication, transcription and repair of the DNA. Thus, prolonged inhibition will eventually lead to the death of the bacteria.

CIPRO

DNA Replication
Since DNA replication is semiconservative, therefore the helix must be unwound. John Cairns (1963) showed that initial unwinding is localized to a region of the bacterial circular genome, called an origin or ori for short.

Replication forks

E. coli chromosome

Localized unwinding

origin

DNA replication

bidirectional

OR

unidirectional

John Cairns
Bacterial culture
*T *T *T *T

in media with low concentration of 3H- thymidine

Grow cells for several generations Small amounts of 3H thymidine are incorporated into new DNA

*T *T

*T

All DNA is lightly labeled with radioactivity Add a high concentration of 3H- thymidine

Grow for brief period of time

*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

Dense label at the replication fork where new DNA is being made Cairns then isolated the chromosomes by lysing the cells very very gently and placed them on an electron micrograph (EM) grid which he exposed to X-ray film for two months.

Evidence points to bidirectional replication

Label at both replication forks

Features of DNA Replication

DNA replication is semiconservative
Each strand of both replication forks is being copied.

DNA replication is bidirectional

Bidirectional replication involves two replication forks, which move in opposite directions

Arthur Kornberg (1957)

Protein extracts from E. coli + Template DNA Is new DNA synthesized??

Currently a faculty member at Stanford School of Medicine

- 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

Kornberg also used the in vitro assay to characterize the DNA polymerizing activity

- dNTPs are ONLY added to the 3 end of newly replicating DNA

5 3 5 3 5 3 5 3 3 New progeny strand 3 5 Parental template strand 5 3

5 3

-therefore DNA synthesis occurs only in the 5 to 3 direction

THIS LEADS TO A CONCEPTUAL PROBLEM

Consider one replication fork:
5 Primer

Continuous replication

5
3
Direction of unwinding
5 3

Discontinuous replication
5 3

Evidence for the Semi-Discontinuous replication model 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 two nuclear bombs that the US dropped at the end of World War II.
His scientific career was cut short by his untimely death from cancer in 1975 at the age of 44, perhaps related to his exposure to the fallout of that blast.

Tuneko Okazaki, until recently, was a professor at The University of Nagoya where she was the first woman at that institution to be named a professor. Currently she is on the faculty of Medicine in Fujita, and does research on centromeres.

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

Bacterial culture

Add 3H Thymidine Flood with non-radioactive T Harvest the bacteria at different times For a SHORT time Allow replication after the chase (i.e. seconds) To continue

Bacteria are replicating smallest Isolate their DNA Separate the strands (using alkali conditions) Run on a sizing gradient largest Radioactivity will only be in the DNA that was made during the pulse

Results of pulse-chase experiment

Pulse Chase
5 Primer

smallest

Direction of unwinding
5 3 5 3
largest

*** 5

DNA replication is semi-discontinuous

Continuous synthesis

Discontinuous synthesis

Features of DNA Replication

DNA replication is semiconservative
Each strand of template DNA is being copied.

DNA replication is bidirectional

Bidirectional replication involves two replication forks, 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

The Enzymology
of DNA Replication In 1957, Arthur Kornberg and colleagues demonstrated 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) that pairs with the template to form a short doublestranded region

DNA Pol I from E. coli is 928 aa (109 kD) monomer - a single polypeptide that packs a punch!

DNA Polymerase I has THREE different enzymatic activities:

a 5 to 3 DNA polymerizing activity a 3 to 5 exonuclease activity a 5 to 3 exonuclease activity

The protein is folded into discrete domains

Hans Klenow used proteases (subtilisin or trypsin) to cleave between residues 323 and 324, separating 5'-exonuclease (on the small fragment) and the other two activities (on the large fragment, the so-called "Klenow fragment) Tom Steitz has determined the structure of the Klenow fragment

The 5 to 3 DNA polymerizing activity

Subsequent hydrolysis of PPi drives the reaction forward

DNA Polymerase I
Replication occurs 5' to 3'
Nucleotides are added at the 3'-end of the strand Pol I catalyzes about 20 cycles of polymerization before the new strand dissociates from template 20 cycles constitutes moderate "processivity"

More on Pol I
Why the exonuclease activity? The 3'-5' exonuclease activity serves a proofreading function It removes incorrectly matched bases, so that the polymerase can try again

For Next Class: We will finish up DNA replication Chapter 30 Sections 30.3 30.4, 30.5, 30.6 We will NOT cover section 30.7