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Lecture Topic 10: DNA Replication - Part 1. Mode of Replication to DNA Polymerase III
Welcome to Lecture Topic 10!

Please read and study the discussion below taken from Chapter 11 of our reference book. Students will answer the
short Quiz all at the same time on Oct 26, 2023.

Introduction

Following Watson and Crick’s proposal for the structure of DNA, scientists focused their attention on how this
molecule is replicated. Replication is an essential function of the genetic material and must be executed precisely if
genetic continuity between cells is to be maintained following cell division. It is an enormous, complex task. Consider
for a moment that more than 3 × 109 (3 billion) base pairs exist within the human genome. To duplicate faithfully the
DNA of just one of these chromosomes requires a mechanism of extreme precision. Even an error rate of only 106
(one in a million) will still create 3000 errors (obviously an excessive number) during each replication cycle of the
genome. Although it is not error free, and much of evolution would not have occurred if it were, an extremely accurate
system of DNA replication has evolved in all organisms.

As Watson and Crick wrote at the end of their classic 1953 paper that announced the double helical model of DNA,
“It has not escaped our notice that the specific pairing (A-T and C-G) we have postulated immediately suggests a
copying mechanism for the genetic material.” Called semiconservative replication, this mode of DNA duplication
was soon to receive strong support from numerous studies of viruses, bacteria, and eukaryotes. Once the general
mode of replication was clarified, research to determine the precise details of DNA synthesis intensified. What has
since been discovered is that numerous enzymes and other proteins are needed to copy a DNA helix. Because of the
complexity of the chemical events during synthesis, this subject remains an extremely active area of research.

In this topic, we will discuss the general mode of replication, as well as the specific details of DNA synthesis.
The research leading to such knowledge is another link in our understanding of life processes at the molecular level.

DNA Is Reproduced by Semiconservative Replication

Watson and Crick recognized that, because of the arrangement and nature of the nitrogenous bases, each strand of
a DNA double helix could serve as a template for the synthesis of its complement (Figure 8.1). They proposed that, if
the helix were unwound, each nucleotide along the two parent strands would have an affinity for its complementary
nucleotide.

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Figure 8.1 Generalized model of semiconservative replication of DNA. New synthesis is shown in blue.

As we learned earlier in the previous topic, the complementarity is due to the potential hydrogen bonds that can be
formed. If thymidylic acid (T) were present, it would “attract” adenylic acid (A); if guanidylic acid (G) were present, it
would attract cytidylic acid (C); likewise, A would attract T, and C would attract G. If these nucleotides were then
covalently linked into polynucleotide chains along both templates, the result would be the production of two identical
double strands of DNA. Each replicated DNA molecule would consist of one “old” and one “new” strand, hence the
reason for the name semiconservative replication.

Two other theoretical modes of replication are possible that also rely on the parental strands as a template (Figure
8.2). In conservative replication, complementary polynucleotide chains are synthesized as described earlier.
Following synthesis, however, the two newly created strands then come together and the parental strands
reassociate. The original helix is thus “conserved.”

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Figure 8.2 Results of one round of replication of DNA for each of the three possible modes by which replication
could be accomplished.

In the second alternative mode, called dispersive replication, the parental strands are dispersed into two new double
helices following replication. Hence, each strand consists of both old and new DNA. This mode would involve
cleavage of the parental strands during replication. It is the most complex of the three possibilities and is
therefore considered to be least likely to occur. It could not, however, be ruled out as an experimental model. Figure
8.2 shows the theoretical results of a single round of replication by each of the three different modes.

The Meselson–Stahl and Taylor–Woods–Hughes Experiments

In 1958, Matthew Meselson and Franklin Stahl published the results of an experiment providing strong evidence
that semiconservative replication is the mode used by bacterial cells (E. coli) to produce new DNA molecules. They
also ruled out both the conservative and dispersive modes of DNA replication in bacteria.

In 1957, the year before the work of Meselson and Stahl was published, J. Herbert Taylor, Philip Woods, and
Walter Hughes presented evidence that semiconservative replication also occurs in eukaryotic organisms. They
experimented with root tips of the broad bean Vicia faba, which are an excellent source of dividing cells.

Together, these two experiments soon led to the general acceptance of the semiconservative mode of
replication. Later studies with other organisms reached the same conclusion and also strongly supported Watson
and Crick’s proposal for the double-helix model of DNA.

Origins, Forks, and Units of Replication

To enhance our understanding of semiconservative replication, let’s briefly consider a number of relevant issues.
The first concerns the origin of replication. Where along the chromosome is DNA replication initiated? Is there only
a single origin, or does DNA synthesis begin at more than one point? Is any given point of origin random, or is it
located at a specific region along the chromosome? Second, once replication begins, does it proceed in a single
direction or in both directions away from the origin? In other words, is replication unidirectional or bidirectional?

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To address these issues, we need to introduce two terms. First, at each point along the chromosome
where replication is occurring, the strands of the helix are unwound, creating what is called a replication fork. Such a
fork will initially appear at the point of origin of synthesis and then move along the DNA duplex as
replication proceeds. If replication is bidirectional, two such forks will be present, migrating in opposite directions
away from the origin. The second term refers to the length of DNA that is replicated following one initiation event at a
single origin. This is a unit referred to as the replicon.

The evidence is clear regarding the origin and direction of replication. John Cairns tracked replication in E. coli,
using radioactive precursors of DNA synthesis and autoradiography. He was able to demonstrate that in E. coli there
is only a single region, called oriC, where replication is initiated. The presence of only a single origin is characteristic
of bacteria, which have only one circular chromosome. Since DNA synthesis in bacteria originates at a single point,
the entire chromosome constitutes one replicon. In E. coli, the replicon consists of the entire genome of 4.6 Mb (4.6
million base pairs).

Figure 8.3 illustrates Cairns’s interpretation of DNA replication in E. coli. This interpretation does not answer the
question of unidirectional versus bidirectional synthesis. However, other results, derived from studies
of bacteriophage lambda, demonstrated that replication is bidirectional, moving away from oriC in both
directions. Figure 8.3 therefore interprets Cairns’s work with that understanding. Bidirectional replication creates two
replication forks that migrate farther and farther apart as replication proceeds. These forks eventually merge,
as semiconservative replication of the entire chromosome is completed, at a termination region, called ter.

Figure 8.3 Bidirectional replication of the E. coli chromosome. The thin black arrows identify the
advancing replication forks.

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DNA Synthesis in Bacteria Involves Five Polymerases, as Well as Other Enzymes

To say that replication is semiconservative and bidirectional describes the overall pattern of DNA duplication and the
association of finished strands with one another once synthesis is completed. However, it says little about the more
complex issue of how the actual synthesis of long complementary polynucleotide chains occurs on a DNA
template. Like most questions in molecular biology, this one was first studied using microorganisms. Research on
DNA synthesis began about the same time as the Meselson–Stahl work, and the topic is still an active area of
investigation. What is most apparent in this research is the tremendous complexity of the biological synthesis of
DNA.

DNA Polymerase I

Studies of the enzymology of DNA replication were first reported by Arthur Kornberg and colleagues in 1957.
They isolated an enzyme from E. coli that was able to direct DNA synthesis in a cell-free (in vitro) system. The
enzyme is called DNA polymerase I, because it was the first of several similar enzymes to be isolated.

Kornberg determined that there were two major requirements for in vitro DNA synthesis under the direction of DNA
polymerase I: (1) all four deoxyribonucleoside triphosphates (dNTPs) and (2) template DNA. If any one of the four
deoxyribonucleoside triphosphates was omitted from the reaction, no measurable synthesis occurred. If
derivatives of these precursor molecules other than the nucleoside triphosphate were used (nucleotides or
nucleoside diphosphates), synthesis also did not occur. If no template DNA was added, synthesis of DNA occurred
but was reduced greatly.

Most of the synthesis directed by Kornberg’s enzyme appeared to be exactly the type required for
semiconservative replication. The reaction is summarized in Figure 8.4, which depicts the addition of a single
nucleotide. The enzyme has since been shown to consist of a single polypeptide containing 928 amino acids.

Figure 8.4 The chemical reaction catalyzed by DNA polymerase I. During each step, a single nucleotide is added to
the growing complement of the DNA template, using a nucleoside triphosphate as the substrate. The release of
inorganic pyrophosphate drives the reaction energetically.

The way in which each nucleotide is added to the growing chain is a function of the specificity of DNA polymerase I.
As shown in Figure 8.5, the precursor dNTP contains the three phosphate groups attached to the 5' carbon
of deoxyribose. As the two terminal phosphates are cleaved during synthesis, the remaining phosphate attached to
the 5' carbon is covalently linked to the 3'-OH group of the deoxyribose to which it is added. Thus, chain
elongation occurs in the 5' to 3' direction by the addition of one nucleotide at a time to the growing 3' end. Each step
provides a newly exposed 3'-OH group that can participate in the next addition of a nucleotide as DNA synthesis
proceeds.

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Figure 8.5 Demonstration of 5' to 3' synthesis of DNA.

Having isolated DNA polymerase I and established its catalytic activity, Kornberg next sought to demonstrate the
accuracy, or fidelity, with which the enzyme replicated the DNA template. Because technology for ascertaining
the nucleotide sequences of the template and newly synthesized strand was not yet available in 1957, he initially
had to rely on several indirect methods.

One of Kornberg’s approaches was to compare the nitrogenous base compositions of the DNA template with those
of the recovered DNA product. Table 8.1 shows Kornberg’s base-composition analysis of three DNA templates.
Within experimental error, the base composition of each product agreed with the template DNAs used. These data,
along with other types of comparisons of template and product, suggested that the templates were replicated
faithfully.

Table 8.1 Base Composition of the DNA Template and the Product of Replication in Kornberg’s Early Work

DNA Polymerase II, III, IV, and V

While DNA polymerase I (DNA polymerases are abbreviated as DNA Pol I, II, etc.) clearly directs the synthesis of DNA,
a serious reservation about the enzyme’s true biological role was raised in 1969. Paula DeLucia and John
Cairns discovered a mutant strain of E. coli that was deficient in polymerase I activity. The mutation was designated
polA1. In the absence of the functional enzyme, this mutant strain of E. coli still duplicated its DNA and successfully
reproduced. However, the cells were deficient in their ability to repair DNA. For example, the mutant strain is highly
sensitive to ultraviolet (UV) light and radiation, both of which damage DNA and are mutagenic. Nonmutant bacteria
are able to repair a great deal of UV-induced damage. These observations led to two conclusions:

1. At least one enzyme other than DNA Pol I is responsible for replicating DNA in vivo in E. coli cells.
2. DNA Pol I serves a secondary function in vivo and is now believed to be critical to the maintenance of
fidelity of DNA synthesis.

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To date, four other unique DNA polymerases have been isolated from cells lacking polymerase I activity and
from normal cells that contain DNA Pol I. Table 8.2 contrasts several characteristics of DNA Pol I with DNA Pol II and
III. Although none of the three can initiate DNA synthesis on a template, all three can elongate an existing DNA
strand, called a primer, and all possess 3' to 5' exonuclease activity, which means that they have the potential to
polymerize in one direction and then pause, reverse their direction, and excise nucleotides just added. As we will later
discuss, this activity provides a capacity to proofread newly synthesized DNA and to remove and replace incorrect
nucleotides.

Table 8.2 Properties of Bacterial DNA Polymerases I, II, and III

DNA Pol I also demonstrates 5' to 3' exonuclease activity. This activity allows the enzyme to excise nucleotides,
starting at the end at which synthesis begins and proceeding in the same direction of synthesis. Two final
observations probably explain why Kornberg isolated polymerase I and not polymerase III: polymerase I is present in
greater amounts than is polymerase III, and it is also much more stable.

What then are the roles of the polymerases in vivo? DNA Pol III is the enzyme responsible for the 5' to 3'
polymerization essential to in vivo replication. Its 3' to 5' exonuclease activity also provides a proofreading function
that is activated when it inserts an incorrect nucleotide. When this occurs, synthesis stalls and the polymerase
“reverses course,” excising the incorrect nucleotide. Then, it proceeds back in the 5' to 3' direction, synthesizing the
complement of the template strand. DNA Pol I is believed to be responsible for removing the primer, as well as for
the synthesis that fills gaps produced after this removal. Its exonuclease activities also allow for its participation in
DNA repair. DNA Pol II, as well as DNA Pol IV and V, are involved in various aspects of repair of DNA that has been
damaged by external forces, such as ultraviolet light. Polymerase II is encoded by a gene activated by disruption
of DNA synthesis at the replication fork.

The DNA Pol III Holoenzyme

We conclude this topic by emphasizing the complexity of the DNA Pol III molecule. The active form of DNA Pol III,
referred to as the holoenzyme, is made up of unique polypeptide subunits, ten of which have been identified (Table
8.3). The largest subunit, α, along with subunits ε and θ, form a complex called the core enzyme, which imparts the
catalytic function to the holoenzyme. In E. coli, each holoenzyme contains two, and possibly three, core enzyme
complexes. As part of each core, the a subunit is responsible for DNA synthesis along the template strands, whereas
the e subunit possesses 3' to 5' exonuclease capability, essential to proofreading. The need for more than one core
enzyme will soon become apparent. A second group of five subunits (γ, δ, δ’, χ, and ν) are complexed to form what is
called the sliding clamp loader, which pairs with the core enzyme and facilitates the function of a critical component
of the holoenzyme, called the sliding DNA clamp. The enzymatic function of the sliding clamp loader is dependent on
energy generated by the hydrolysis of ATP. The sliding DNA clamp links to the core enzyme and is made up of
multiple copies of the β subunit, taking on the shape of a donut, whereby it can open and shut, to encircle the
unreplicated DNA helix. By doing so, and being linked to the core enzyme, the clamp leads the way during
synthesis, maintaining the binding of the core enzyme to the template during polymerization of nucleotides. Thus,
the length of DNA that is replicated by the core enzyme before it detaches from the template, a property referred to
as processivity, is vastly increased. There is one sliding clamp per core enzyme. Finally, one τ subunit interacts with
each core enzyme, linking it to the sliding clamp loader.

Table 8.3 Subunits of the DNA Polymerase III Holoenzyme

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The DNA Pol III holoenzyme is diagrammatically illustrated in Figure 8.6. You should compare the diagram to the
description of each component above. Note that we have shown the holoenzyme to contain two core
enzyme complexes, although as stated above, a third one may be present. The components of the DNA Pol III
holoenzyme will be referred to in the topic that follows.

Figure 8.6 The components making up the DNA Pol III holoenzyme, as described in the text. While there may
be three core enzyme complexes present in the holoenzyme, for simplicity, we illustrate only two.

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QUIZ

Quiz for Lecture Topic 10

This is a short quiz to test if you have read and studied the discussions for this topic. You have only
2 minutes to answer this quiz, otherwise it will not be recorded. Good Luck!

Not attempted Due 26 October 2023

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