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BCHM

218
(Molecular Biology)
Lecture 10:

DNA Replica9on
Dr. John Allingham
Bo8erell Hall Rm. 652
Department of Biomedical and Molecular Sciences
allinghj@queensu.ca
February 24, 2015

Please read MBPP, pp. 32-36, 364-372



Allingham Lecture 10- BCHM218

Review of SecLon 1 and 2


We have covered the fundamental principles that
determine the informaLon-storing and funcLonal
capabiliLes of geneLc informaLon molecules:

The structures of protein and DNA building


blocks, as well as genomes and gene products.
The mechanisms by which they are organized.
Their chemical properLes and reacLvity.
Methods with which to isolate, manipulate, and
study them.
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Preamble to SecLon 3 and 4


We will now use these principles to understand
the molecular basis by which genomes are copied
and edited, and geneLc informaLon is regulated:

Basis for DNA replicaLon delity.


Mechanisms of geneLc recombinaLon.
Processes involved in decoding DNA-based
messages into molecular devices that enable
life.
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The EukaryoLc Cell Cycle

(mitosis)

(quiescent)

(four copies
of each
chromosome)

(each chromosome
is duplicated)

(two copies
of each
chromosome)

Some consideraLons:
Every Lme a cell divides, it replicates all of its DNA;
a process that takes place in a ma8er of hours.
Eukaryote genomes are large - nearly 3 billion
bases in the human genome!
Considering that the human body houses about 15
million, million cells, each one harboring a (near)
perfect copy of the genome (two copies in fact), it
is important that mistakes are minimized.

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Some consideraLons:
A large number of factors are associated with the
process of DNA replicaLon to ensure rapid and
faithful reproducLon of the geneLc material.

Beginnings of ReplicaLon Enzymology


How i s the DNA polymer made?

Kornberg et al. followed the fate of


radioacLve [14C]thymidine (ensured that
whatever radioacLve polymer made was
DNA, not RNA)
Puried DNA polymerase I (Pol I).
Demonstrated that Pol I received
instrucLons on what nucleoLdes to
incorporate from an exisLng template
(parental DNA strands).
Marked the beginnings of biotechnology.
Arthur Kornberg

Pg. 403

Some Biochemical Principles


Enzymes and other components involved in replicaLon can be
idenLed, puried, and have their funcLons analyzed biochemically.

Some Biochemical Principles


Biochemical studies ogen involve an assay for the acLvity
under invesLgaLon, which is usually a measurement of the
product of the reacLon being catalyzed by the enzyme.

Materials and Methods


Incorpora9on Assay:
A simple way to observe the
acLvity of a DNA polymerase is
to measure the incorporaLon of
a radioacLvely or uorescently
labeled dNTP into the high
molecular weight polymer DNA.

Materials and Methods


Detec9on of DNA polymer:
This can involve precipitaLon of
the polymer with a strong acid,
such as trichloroaceLc acid
(unincorporated nucleoLdes or
nucleosides do not precipitate).
Analyze radioacLvity by Geiger
counter, scinLllaLon counLng,
or electrophoresis and
autoradiography.

[32P] dTTP

incorporation

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Detec9on of DNA polymer:


This can alternaLvely involve
use of a posiLvely charged lter
(in low salt) to which negaLvely
charged polynucleoLde
backbone will bind, but
unincorporated nucleoLdes will
pass through.
Analyze uorescence signal by
uorescence detector.

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Materials and Methods


IdenLcaLon and isolaLon of the DNA polymerizing acLvity from
other proteins can be done by chromatography.
Each fracLon from a chromatographic column is assayed for
protein (e.g., the absorbance at 280 nm, gray line) with the ability
to catalyze the incorporaLon of [32P] dTTP into DNA (black line).

RadioacLvity

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Results
PuricaLon of Polymerase by Kornberg et al. involved a mulLstep
process in which protein concentraLon and specic enzyme acLvity
(DNA polymerizaLon) were monitored.

(Lehman, I. R., Bessman, M. J., Simms, E. S., and Kornberg, A. (1958) J. Biol. Chem. 233, 163170)

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Results

Requirements of the PolymerizaLon ReacLon were deduced by


Kornberg et al. by showing that the maximal incorporaLon of
labeled deoxyribonucleoLdes into DNA (monitored by amount of
precipitated DNA with dTP* incorporated) was dependent on the
presence of polymerized DNA, Mg++, and the deoxynucleoside
triphosphates of thymine, cytosine, guanine, and adenine.

(Bessman, M. J., Lehman, I. R., Simms, E. S., and Kornberg, A. (1958) J. Biol. Chem. 233, 171177)

Conclusions:
DNA Polymerase ReacLon Components

Requirements:
All DNA polymerases require a template strand.

All DNA polymerases require a primer strand.
Deoxyribonucleoside 5-triphosphates (dNTPs)
Polymerase enzyme + associated cofactors

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These two classics were declined by the JBC when submitted in


the fall of 1957. Among the critical comments were:

It is very doubtful that the authors are entitled to speak of the

enzymatic synthesis of DNA; Polymerase is a poor name
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More consideraLons:

Is DNA replicaLon a new invenLon?

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DNA replica9on apparently evolved


twice.

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Koonin and MarLn:


From detailed gene sequence comparisons


Eugene Koonin found that bacteria and
archaea broadly share the same mechanisms
(and enzymes) of protein synthesis (i.e. how
DNA is read into RNA, and how that RNA is
then translated into proteins).
This is not the case for the enzymes needed
for DNA replicaLon. Most have nothing at all
in common between bacteria and archaea.
Nucleic Acids Res. 1999 September 1; 27(17): 33893401

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DNA polymerases

Viral

Bacterial

Human

Viral

Allingham Lecture 7- MBIO218


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Proteins and mechanisms of Okazaki


fragment synthesis

Why Are There So Many Diverse Replication Machineries?


Journal of Molecular Biology, Volume 425, Issue 23, 2013, 4714 - 4726

Koonin and MarLn:


There are, however, many important


funcLonal parallels among all known cellular
systems (bacteria, archaea, and eukaryotes)
of DNA replicaLon.
These common features can be roughly
summarized as follows:

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(1) DNA replicaLon is semi-conservaLve

Each daughter chromosome contains one parental


strand and one newly synthesized strand.
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(2) ReplicaLon is iniLated at specic sites

Origins of replicaLon are used in conjuncLon with


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an origin recogniLon system.

(3) ReplicaLon is typically bidirecLonal

ReplicaLon fork moves away from the origin of


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replicaLon in both direcLons.

(4) ReplicaLon is conLnuous on the leading


strand and disconLnuous on the lagging strand

Both daughter strands cannot be replicated in the


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same direcLon that the replicaLon fork moves.

(5) RNA primers are needed to start DNA


replicaLon

Primer strands must be complementary to the


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template and contain a free 3-OH group.

(6) nucleases, polymerases and ligases


replace the RNA primers with DNA and seal
the remaining nick

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KEY CONVENTION

The direcLon of synthesis by DNA polymerases refers to the


direcLon in which each new nucleoLde is chemically linked to
the growing daughter strand. All DNA polymerases funcLon in
the 53 direcLon, linking the -5-phosphate of a new dNTP
to the 3 posiLon of the nucleoLde residue at the end (i.e., the
3 end) of the chain.

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