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Bis2A 8.1 DNA Replication
∗
The BIS2A Team
This work is produced by OpenStax-CNX and licensed under the
†
Creative Commons Attribution License 4.0
Abstract
This module will discuss the structure and replication of DNA.
Section Summary
The currently accepted model of the double-helix structure of DNA was proposed by Watson and Crick.
Some of the salient features are that the two strands that make up the double helix are complementary and
anti-parallel in nature.
Deoxyribose sugars and phosphates form the backbone of the structure, and the
nitrogenous bases are stacked inside. A purine always pairs with a pyrimidine; A pairs with T, and G pairs
with C. During cell division, each daughter cell receives a copy of the DNA by a process known as DNA
replication. DNA replicates by a semi-conservative method in which each of the two parental DNA strands
act as a template for new DNA to be synthesized. After replication, each DNA has one parental or old
strand, and one daughter or new strand.
Design challenge
DNA is the storage molecule for hereditary information in the cell.
mate goal - a copy of the DNA must be created.
If the cell is to replicate - its ulti-
So one clear problem statement/question is "how can
the cell eectively copy its DNA?" Some subquestions of relevance might be: What are the chemical and
physical properties that enable DNA to be copied?
With what delity must the DNA be copied?
speed must it be copied at?
As you go through the reading and lecture materials
The list could go on.
What
try to be constantly aware of these and other questions associated with this process. Use the questions as
guideposts for organizing your thoughts and try to nd matches between the "facts" that you think you
might be expected to know and the driving questions.
1 Nucleotide Structure Review
The building blocks of DNA are nucleotides. The important components of the nucleotide are a nitrogenous
base, deoxyribose (5-carbon sugar), and a phosphate group (review module 3.4). The nucleotide is named
according to its nitrogenous base. The nitrogenous base can be a purine such as adenine (A) and guanine
(G), or a pyrimidine such as cytosine (C) and thymine (T).
∗ Version
1.1: Feb 9, 2016 6:50 pm -0600
† http://creativecommons.org/licenses/by/4.0/
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Figure 1:
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Each nucleotide is made up of a sugar, a phosphate group, and a nitrogenous base. The sugar
is deoxyribose in DNA and ribose in RNA. The purines have a double ring structure with a six-membered
ring fused to a ve-membered ring.
Pyrimidines are smaller in size; they have a single six-membered
ring structure. The carbon atoms of the ve-carbon sugar are numbered 1', 2', 3', 4', and 5' (1' is read
as one prime). The phosphate residue is attached to the hydroxyl group of the 5' carbon of one sugar
of one nucleotide and the hydroxyl group of the 3' carbon of the sugar of the next nucleotide, thereby
forming a 5'-3' phosphodiester bond.
2 Discovery of the Double Helix
In the 1950s, Francis Crick and James Watson worked together to determine the structure of DNA at the
University of Cambridge, England. Other scientists like Linus Pauling and Maurice Wilkins were also actively
exploring this eld. Pauling had discovered the secondary structure of proteins using X-ray crystallography.
In Wilkins' lab, researcher Rosalind Franklin was using X-ray diraction methods to understand the structure
of DNA. Watson and Crick were able to piece together the puzzle of the DNA molecule on the basis of
Franklin's data because Crick had also studied X-ray diraction. In 1962, James Watson, Francis Crick, and
Maurice Wilkins were awarded the Nobel Prize in Medicine. Unfortunately, by then Franklin had died, and
Nobel prizes are not awarded posthumously.
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Figure 2:
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DNA has (a) a double helix structure and (b) phosphodiester bonds.
The (c) major and
minor grooves are binding sites for DNA binding proteins during processes such as transcription (the
copying of RNA from DNA) and replication.
Watson and Crick proposed that DNA is made up of two strands that are twisted around each other to
form a right-handed helix. The two strands are
anti-parallel in nature;
that is, the 3' end of one strand
faces the 5' end of the other strand. The sugar and phosphate of the nucleotides form the backbone of the
structure, whereas the nitrogenous bases are stacked inside. The diameter of the DNA double helix is 2 nm,
and it is uniform throughout. Only the pairing between a purine and pyrimidine can explain the uniform
diameter. The twisting of the two strands around each other results in the formation of uniformly spaced
major and minor grooves.
Figure 3:
DNA has (a) a double helix structure and (b) phosphodiester bonds.
The (c) major and
minor grooves are binding sites for DNA binding proteins during processes such as transcription (the
copying of RNA from DNA) and replication.
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Additionally, Watson and Crick postulated about the mode of DNA replication based solely on the
structure. Initially there were three possibilities, conservative, semi-conservative and dispersive models of
replication.
Later, Meselson and Stahl performed a famous experiment to show that DNA replication is
semi-conservative (gure 4), where each strand is used as a template for the creation of the new strand. To
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learn more about this experiment watch The Meselson-Stahl Experiment
Figure 4:
.
DNA has an anti-parallel double helix structure, the nucleotide bases are hydrogen bonded
together and each strand complements the other. DNA is replicated in a semi-conservative manner, each
strand is used as the template for the newly made strand.
3 DNA Replication
When a cell divides, it is important that each daughter cell receives an identical copy of the DNA. This is
accomplished by the process of DNA replication. In Eukaryotic cells the replication of DNA occurs during
the synthesis phase, or S phase, of the cell cycle, before the cell enters mitosis or meiosis, a subject we
will discuss later in the quarter. In bacteria and archaea, DNA replication is regulated by the cell's energy
demands and biomass. Regardless of how the process is regulated, the synthesis of DNA is a similar process,
though the proteins and machinery used by each varies.
1 https://www.youtube.com/watch?v=JcUQ_TZCG0w
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3.1 Initiation of Replication
Where along the DNA does the replication machinery start DNA replication?
There are specic nucleotide sequences called origins of replication along the length of the DNA at which
replication begins. At these sites, an enzyme called helicase unwinds and opens up the DNA helix, separating
the two strands from one another. As the DNA opens up, Y-shaped structures called replication forks
are formed. Two replication forks are formed at the origin of replication, and these get extended in both
directions as replication proceeds. There are multiple origins of replication on the eukaryotic chromosome,
such that replication can occur simultaneously from several places in the genome. In the bacterium, E. coli,
there is only one origin of replication located on the DNA.
note: Why would dierent organisms have dierent numbers of replication origins? What could
the benet be to having more than one? Is there a drawback to having more than one?
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Figure 5:
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At the origin of replication, a replication bubble forms. The replication bubble is composed
of two replication forks, each traveling in opposite directions along the DNA.
Source: https://www.mun.ca/biology/scarr/IG1_10_Fig16.html
3.2 Elongation of Replication
During elongation, an enzyme called
DNA polymerase polymerizes the new strand of DNA nucleotides.
The main synthetic enzyme is called DNA polymerase III. DNA polymerase, and any polymerase, will 'read'
the template strand from 3' to 5'. Due to the antiparallel nature of the DNA double helix, DNA polymerase
will polymerize the new strand from 5' to 3'. (Recall that the 3' and 5' terminology refers to the functional
groups bound to the 3' and 5' carbons in the ribose ring of the nucleotides.) In eect, DNA polymerase adds
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the phosphate group (5') from the incoming nucleotide to the existing hydroxyl group (3') of the previously
added nucleotide.
Figure 6:
DNA polymerase catalyzes the addition of the 5' phosphate group from an incoming nucleotide
to the 3' hydroxyl group of the previous nucleotide. This process creates a phosphodiester bond between
the nucleotides while hydrolyzing the phosphoanhydride bond in the nucleotide.
Source: http://bio1151.nicerweb.com/Locked/media/ch16/elong.html
note: Create an energy story for the addition of a nucleotide onto a polymer as shown in gure 6
above.
DNA polymerase can only add new nucleotides to a 3' hydroxyl group. Which means that DNA polymerase
cannot initiate creation of the newly synthesized strand, it needs to have a 3' hydroxyl group already present
in order to start replication.
An enzyme called
primase
supplies an RNA
primer,
which provides this
starting point. This RNA primer is removed later, and the nucleotides are replaced with DNA nucleotides.
Leading and Lagging Strand
As the replication fork elongates, one strand is synthesized continuously. This strand is called the
strand.
leading
In the leading strand, DNA polymerase polymerizes the DNA in the same direction the replication
fork is traveling. As helicase continues to unwind the DNA in one direction, DNA polymerase synthesizing
the leading strand follows close behind helicase.
Because DNA polymerase can only synthesize DNA in a 5' to 3' direction, the polymerization of the other
strand needs to occur in the opposite direction that helicase is traveling. This strand is called the
strand and has to be polymerized in short segments called Okazaki fragments.
lagging
The Okazaki fragments
each require a primer made of RNA to start the synthesis.
As synthesis proceeds, a dierent DNA polymerase (DNA polymerase I) removes the RNA primer, and
replaces it with DNA nucleotides. The gaps in the sugar phosphate backbone between the fragments are
sealed by an enzyme called
DNA ligase.
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Figure 7:
(A) The lagging strand is created in multiple segments. (B) A replication fork showing the
leading and lagging strand. (C) A replication bubble showing the leading and lagging strands.
Sources: https://www.mun.ca/biology/scarr/iGen3_03-08.html
https://www.mun.ca/biology/scarr/iGen3_03-09.html
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3.2.1 Replication Design Challenge: Proofreading
When the cell begins the task of replicating the DNA, it does so in response to environmental signals that
tell the cell it is time to divide. The goal in DNA replication is to produce two identical copies of the DNA
template. This is a daunting task when you consider that there are 3,000,000,000 base pairs in the human
genome. As DNA polymerase synthesizes the new strand there are two features of replication that come into
play: the fact that replication needs to be accurate, and the conicting desire to be fast.
note: Why would DNA replication need to be fast? Consider the environment the DNA is in, and
compare that to the structure of DNA while being replicated.
There is an apparent trade o that nature has assessed between a polymerase that is fast versus a polymerase
that is accurate. DNA polymerase (unlike RNA polymerase) proofreads its work. When a wrong nucleotide
is added to the growing polymer, the misshaped double helix will cause DNA polymerase to stall, the newly
made strand will be ejected from the polymerizing site on the polymerase and will enter into the exonuclease
site. In this site, DNA polymerase is able to cleave o the last several nucleotides that were added to the
polymer. This proofreading capability comes with some trade-os: It requires time and energy.
note: What are the pros and cons for DNA polymerases' proofreading capabilities?
3.3 Termination of Replication
Telomeres and Telomerase
The ends of linear eukaryotic chromosomes pose a specic problem for DNA replication.
Because DNA
polymerase can add nucleotides in only one direction, the leading strand allows for continuous synthesis
until the end of the chromosome is reached; however, on the lagging strand there is no place for a primer to
be made for the DNA fragment to be copied at the end of the chromosome. This chromosomal end remains
unpaired, and over time these ends get progressively shorter as cells continue to divide. The ends of the linear
chromosomes are known as
telomeres,
which have repetitive sequences that do not code for a particular
gene. As a consequence, it is telomeres that are shortened with each round of DNA replication instead of
genes. For example, in humans, a six base-pair sequence, TTAGGG, is repeated 100 to 1000 times. The
discovery of the enzyme
telomerase helped in the understanding of how chromosome ends are maintained.
Telomerase is an enzyme composed of protein and RNA. Telomerase attaches to the end of the chromosome
by complementary base pairing between the RNA component of telomerase and the DNA template. Once
the lagging strand template is suciently elongated by telomerase, primase will create a primer followed by
DNA polymerase which can now add nucleotides that are complementary to the ends of the chromosomes.
Thus, the ends of the chromosomes are replicated.
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Figure 8:
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The ends of linear chromosomes are maintained by the action of the telomerase enzyme.
Telomerase is not active in adult somatic cells. Adult somatic cells that undergo cell division continue
to have their telomeres shortened. This essentially means that telomere shortening is associated with aging.
In 2010, scientists found that telomerase can reverse some age-related conditions in mice, and this may have
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potential in regenerative medicine. Telomerase-decient mice were used in these studies; these mice have
tissue atrophy, stem-cell depletion, organ system failure, and impaired tissue injury responses. Telomerase
reactivation in these mice caused extension of telomeres, reduced DNA damage, reversed neurodegeneration,
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and improved functioning of the testes, spleen, and intestines. Thus, telomere reactivation may have potential
for treating age-related diseases in humans.
4 DNA Replication in Bacteria
DNA replication has been extremely well-studied in bacteria, primarily because of the small size of the
genome and large number of variants available.
Escherichia coli has 4.6 million base pairs in a single circular
chromosome, and all of it gets replicated in approximately 42 minutes, starting from a single origin of
replication and proceeding around the chromosome in both directions. This means that approximately 1000
nucleotides are added per second. The process is much more rapid than in eukaryotes. Table 1 summarizes
the dierences between bacterial and eukaryotic replications.
Dierences between Prokaryotic and Eukaryotic Replications
Property
Prokaryotes
Eukaryotes
Origin of replication
Single
Multiple
Rate of replication
1000 nucleotides/s
50 to 100 nucleotides/s
Chromosome structure
circular
linear
Telomerase
Not present
Present
Table 1
:
3
Click through a tutorial
on DNA replication.
Exercise 1
(Solution on p. 12.)
You isolate a cell strain in which the joining together of Okazaki fragments is impaired and suspect
that a mutation has occurred in an enzyme found at the replication fork. Which enzyme is most
likely to be mutated?
3 http://openstaxcollege.org/l/DNA_replicatio2
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Solutions to Exercises in this Module
to Exercise (p. 11)
Ligase, as this enzyme joins together Okazaki fragments.
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