DNA Replication - Advanced
Douglas Wilkin, Ph.D.
Say Thanks to the Authors
Click http://www.ck12.org/saythanks
(No sign in required)
To access a customizable version of this book, as well as other
interactive content, visit www.ck12.org
CK-12 Foundation is a non-profit organization with a mission to
reduce the cost of textbook materials for the K-12 market both
in the U.S. and worldwide. Using an open-content, web-based
collaborative model termed the FlexBook®, CK-12 intends to
pioneer the generation and distribution of high-quality educational
content that will serve both as core text as well as provide an
adaptive environment for learning, powered through the FlexBook
Platform®.
Copyright © 2015 CK-12 Foundation, www.ck12.org
The names “CK-12” and “CK12” and associated logos and the
terms “FlexBook®” and “FlexBook Platform®” (collectively
“CK-12 Marks”) are trademarks and service marks of CK-12
Foundation and are protected by federal, state, and international
laws.
Any form of reproduction of this book in any format or medium,
in whole or in sections must include the referral attribution link
http://www.ck12.org/saythanks (placed in a visible location) in
addition to the following terms.
Except as otherwise noted, all CK-12 Content (including CK-12
Curriculum Material) is made available to Users in accordance
with the Creative Commons Attribution-Non-Commercial 3.0
Unported (CC BY-NC 3.0) License (http://creativecommons.org/
licenses/by-nc/3.0/), as amended and updated by Creative Commons from time to time (the “CC License”), which is incorporated
herein by this reference.
Complete terms can be found at http://www.ck12.org/terms.
Printed: February 3, 2015
AUTHOR
Douglas Wilkin, Ph.D.
www.ck12.org
C HAPTER
Chapter 1. DNA Replication - Advanced
1
DNA Replication Advanced
• Describe how DNA is replicated.
• Explain the importance of the fact that during DNA replication, each strand serves as a template to make a
complementary DNA strand.
What do you need to copy?
Obviously, you need something to copy. The same is true for DNA. To copy or replicate DNA, you use both strands
as templates.
DNA Replication
DNA replication is the process in which a cell’s entire DNA is copied, or replicated. The identification of the
structure of DNA suggested that each strand of the double helix would serve as a template for synthesis of a new
strand. DNA replication process occurs during the Synthesis (S) phase of the eukaryotic cell cycle. As each DNA
strand has the same genetic information, both strands of the double helix can serve as templates for the reproduction
of a complementary new strand. The two resulting double helices, which each contain one "old" strand and one
"new" strand of DNA, are identical to the initial double helix. DNA replication is said to be semi-conservative
1
www.ck12.org
because of this process of replication, where the resulting double helix is composed of both an old strand and a new
strand.
For an animation of DNA replication, see http://www.hhmi.org/biointeractive/media/DNAi_replication_vo1-sm.mov
.
FIGURE 1.1
DNA replication occurs when the DNA
strands “unzip”, and the original strands
of DNA serve as a template for new nucleotides to join and form a new strand.
The replication fork is obvious at the point
of separation of the double helix.
The semi-conservative mechanism of replication was one of three models originally proposed for DNA replication:
1. Semiconservative replication would produce two copies that each contained one of the original strands and
one new strand.
2. Conservative replication would leave the two original template DNA strands together in a double helix, with
the new DNA composed entirely of two new strands.
3. Dispersive replication would produce two copies of the DNA, both containing a mixture of old and new
material.
Of these three models, the semi-conservative model seemed most reasonable since it would allow each new daughter
strand to remain associated with its template strand. The semiconservative model was confirmed by the MeselsonStahl experiment.
2
www.ck12.org
Chapter 1. DNA Replication - Advanced
FIGURE 1.2
DNA replication is a semi-conservative
process. Half of the parent DNA molecule
is conserved in each of the two daughter
DNA molecules.
The Meselson-Stahl Experiment
In 1958, Matthew Meselson and Franklin Stahl identified evidence that supported the hypothesis that DNA replication was semiconservative. The experiment took advantage of the fact that nitrogen is a major constituent of DNA,
and that two forms of nitrogen are available: 14 N and a heavier isotope, 15 N. The DNA of cells grown in 15 N medium
have a higher density than cells grown in normal 14 N medium. The experiment was performed as follows:
1. E. coli were grown for several generations in a medium with 15 N.
2. After that, E. coli cells with only 15 N in their DNA were transferred to a 14 N medium and were allowed to
divide.
3. After one round of replication, DNA was isolated and was compared to pure 14 N DNA and 15 N DNA. After
one round of DNA replication, the resulting DNA was found to have close to the intermediate density between
14 N labelled DNA and 15 N labelled DNA. Since conservative replication would result in equal amounts of
DNA of the 15 N and 14 N densities, but no DNA of an intermediate density, the conservative replication model
was excluded. However, this result was still consistent with both semiconservative and dispersive replication.
Semiconservative replication would result in double-stranded DNA having an intermediate density, with one
strand of 15 N DNA and one of 14 N DNA. Dispersive replication would result in double-stranded DNA also of
an intermediate density, with both strands having mixtures of 15 N and 14 N DNA.
4. The cells were then allowed to progress through another round of DNA replication in the 14 N medium. DNA
from cells after two replications had been completed was found to consist of equal amounts of DNA with
both an intermediate density and a lower density. This was inconsistent with the dispersive replication model,
which would have resulted in a single density, lower than the intermediate density of the one-replication cells,
but still higher than cells grown only in 14 N DNA medium, as the original 15 N DNA would have been split
evenly among all DNA strands. The result was consistent with the semiconservative replication hypothesis.
See A half DNA ladder is a template for copying the whole at http://www.dnaftb.org/20/animation.html to see
Meselson and Stahl talk about their work.
Helicase and Polymerase
DNA replication begins as an enzyme, DNA helicase, breaks the hydrogen bonds holding the two strands together
and forms a replication fork. The resulting structure has two branching strands of DNA backbone with exposed
bases. These exposed bases allow the DNA to be “read” by another enzyme, DNA polymerase, which then builds
the complementary DNA strand. As DNA helicase continues to open the double helix, the replication fork grows.
3
www.ck12.org
5’ → 3
The two new strands of DNA are “built” in opposite directions, through either a leading strand or a lagging strand.
The leading strand is the DNA strand that DNA polymerase constructs in the 5’ → 3’ direction. This strand of DNA
is made in a continuous manner, moving as the replication fork grows. The lagging strand is the DNA strand at
the opposite side of the replication fork from the leading strand. It goes in the opposite direction, from 3’ to 5’.
DNA polymerase cannot build a strand in the 3’ → 5’ direction. Thus, this "lagging” strand is synthesized in short
segments known as Okazaki fragments. On the lagging strand, an enzyme known as primase builds a short RNA
primer. DNA polymerase is then able to use the free 3’-OH group on the RNA primer to make DNA in the 5’ → 3’
direction. The RNA fragments are then degraded and new DNA nucleotides are added to fill the gaps where the RNA
was present. Another enzyme, DNA ligase, is then able to attach (ligate) the DNA nucleotides together, completing
the synthesis of the lagging strand ( Figure 1.3).
FIGURE 1.3
DNA replication. The two DNA strands
are opened by helicase.
The strands
are held open by a single strand of binding proteins, preventing premature reannealing.
Topoisomerase solves the
problem caused by tension generated by
winding/unwinding of DNA. This enzyme
wraps around DNA and makes a cut
permitting the helix to spin and relax.
Once DNA is relaxed, topoisomerase reconnects broken strands. DNA primase
synthesizes a short RNA primer which
initiates the Okazaki fragment. Okazaki
fragments are attached by DNA ligase.
Many replication forks develop along a chromosome. This process continues until the replication forks meet, and the
all of the DNA in a chromosome has been copied. Each new strand that has formed is complementary to the strand
used as the template. Each resulting DNA molecule is identical to the original DNA molecule. During prophase of
mitosis or prophase I of meiosis, these molecules of DNA condense into a chromosome made of two identical "sister"
chromatids. This process ensures that cells that result from cell division have identical sets of genetic material, and
that the DNA is an exact copy of the parent cell’s DNA.
Vocabulary
• DNA helicase: The enzyme that breaks the hydrogen bonds holding the two DNA strands together during
DNA replication.
• DNA ligase: Enzyme that can attach together (ligates) strands of DNA with double strand breaks.
• DNA polymerase: The enzyme that builds a new DNA strand during DNA replication.
• DNA replication: The process of copying DNA prior to cell division (eukaryotes) or reproduction (prokaryotes).
4
www.ck12.org
Chapter 1. DNA Replication - Advanced
• lagging strand: The DNA strand at the opposite side of the replication fork from the leading strand.
• leading strand: The DNA strand that DNA polymerase constructs in the 5’ → 3’ direction.
• Okazaki fragments: Short fragments of DNA that comprise the lagging strand.
• primase: An enzyme that builds a short RNA primer on the lagging strand during DNA replication.
• replication fork: Site where DNA helices unwinds the DNA, allowing DNA replication to proceed.
• semi-conservative: DNA replication process where half of the parent DNA molecule is conserved in each of
the two daughter DNA molecules.
Summary
• DNA replication is the semi-conservative process by which a cell’s entire DNA is copied, or replicated.
• During DNA replication, the two new strands of DNA are “built” in opposite directions, starting at replication
forks.
Explore More
Use this resource to answer the questions that follow.
• http://www.hippocampus.org/Biology → Non-Majors Biology → Search: Replication
1.
2.
3.
4.
5.
Why must DNA be replicated?
When does replication occur?
Describe the first step of replication.
Why is each strand of DNA able to serve as a template for replication?
Explain the meaning of semi-conservative replication.
Review
1. How is DNA replicated?
2. What are the roles of the following enzymes?
a.
b.
c.
d.
DNA polymerase.
DNA helicase.
DNA ligase.
primase.
3. Why is DNA replication called a "semi-conservative" process?
4. Outline the Meselson-Stahl Experiment.
References
1. Zachary Wilson. CK-12 Foundation . CC BY-NC 3.0
2. Laura Guerin. CK-12 Foundation . CC BY-NC 3.0
3. Mariana Ruiz Villarreal (User:LadyofHats/Wikimedia Commons). http://commons.wikimedia.org/wiki/Fi
le:DNA_replication_en.svg . Public Domain
5