BTC 313 MICROBIAL GENETICS AND MOLECULAR BIOLOGY (2 Credit Units)

LECTURE NOTES
(Prof. E.O. Ekundayo)
COURSE OUTLINE AND LECTURE SCHEDULE

1. EARLY EXPERIMENTS IN MICROBIAL GENETICS: DISCOVERY OF DNA AS THE CARRIER OF

GENETIC INFORMATION

Genetic studies of microorganism (Griffith’s transformation experiment, Avery’s experiment,

Hershey, and Chase T2 phage life cycle, Beadle, and Tatum hypothesis).

2. GENETIC CODE AND ORGANIZATION OF GENETIC INFORMATION

Chemical composition and structure of DNA (Watson and Crick’s model), evidence for the triple
codes, degeneracy of the genetic code, wobble hypothesis, microbial chromosomes and genes,
extrachromosomal genetic elements and cloning vectors, alterations in genetic information
(mutations and genetic transfers).

3. DNA REPLICATION AND REPLICATION THEORIES (conservative, semi-conservative and dispersive

theories), patterns of DNA replication (bidirectional and rolling circle patterns).

4. GENE EXPRESSION AND REGULATION

The concept of the Central Dogma, transcription and translation, protein synthesis regulation of gene
expression, the concept of operon – lac operon, other operon models.

5. IN-VITRO METHODS IN MICROBIAL GENETICS AND MOLECULAR BIOLOGY

DNA and RNA extraction, synthetic oligonucleotide methods, nucleic acid hybridization, southern
blotting technique, DNA amplification – Polymerase Chain Reaction (PCR), molecular and gene
cloning, DNA profiling and fingerprinting, recombinant DNA technology, gene sequencing and genome
mapping.

6. APPLICATIONS OF MICROBIAL GENETICS AND MOLECULAR BIOLOGY

Locating and isolating genes (from donor to host), improvement of microorganisms through mutation,
hybridization, protoplasm fusion and genetic transfers, microbe-mediated improvement of plants
(Agrobacterium mediated transfer), inserting foreign genes into bacteria, expressing foreign genes in
expression systems.

Lectures 1-3 Prof. E. O Ekundayo

Lectures 4-6 Mr. Danlami Dauda

INTRODUCTION

What is Microbial Genetics?

Microbial Genetics is the study of Genetics in microorganisms. The science of Genetics deals with the
study of heredity and variation i.e. the Science of Biological Inheritance.

Microbial Genetics is a core course in Microbiology. It forms the foundation for understanding,
Molecular Biology, Molecular Genetics, Recombinant DNA Technology/Genetic Engineering. It is a
Prerequisite course to higher level courses in Microbiology. It is also relevant to other disciplines in
Biological Sciences, Medicine, Agriculture.

Microorganisms have relatively simple genetic makeup and are easy to study. Microbial Genetics
provides basis for understanding genetics in higher organisms. Methods and techniques learned in
Microbial Genetics are used for research and applications in Molecular Biology, Genetic Engineering
and Biotechnology. Microorganisms are easy to handle in the laboratory, therefore they have proved
to be of immense importance as research tools in the development and study of the science of
Genetics. Experiments that have helped to elucidate the nature of genetic information, gene structure,
genetic code and mutations were first carried out in microorganisms.

HISTORICAL BACKGROUND

GREGOR MENDEL

Gregor Johann Mendel is regarded as the father of Genetics. He was a monk who studied and
documented the patterns of heredity in pea plants. He stated the general law of Genetics in 1865
Mendel however did not know the mechanisms by which heredity occurs.

FRIEDRICH MIESCHER
Friedrich Miescher first isolated various phosphate-rich chemicals, which he called nuclein from the
nuclei of white blood cells in 1869. Nuclein was later named nucleic acids. The chemical compounds
were called nuclein because they were isolated from nuclei of white blood cells. And nucleic acid
because they have weak acidic properties.
P. A LEVENE
Phoebus Aaron Theodore Levene was an American biochemist who studied the structure and function of
nucleic acids. He elucidated the basic chemical composition of nucleic acids in 1920s. Although his contribution
to the discovery of the basic chemical composition was significant, his proposed structure of nucleic acid was
too simple.

FREDRICK GRIFFITH
Griffith was a British bacteriologist. Griffith conducted experiments which led to the discovery of
Transformation in 1928. His discovery was a significant step in the development of Microbial Genetics. It
provided the foundation for all the experiments that led to the discovery the molecule that carries the genetic
information in heredity.

AVERY, MCLEOD AND MCCARTY

Avery and Colleagues were American scientists who were interested in Genetics. They were searching for the
molecule responsible for heredity like other scientists of their time. The work of Griffith inspired them to look
for the transforming principle. In 1944 Avery, McLeod and McCarty showed DNA as transforming principle.

ERWIN CHARGAFF
In 1950 Erwin Chargaff discovered the variation in base composition of different species and the constant ratio
of the bases. His discovery provided a clue that DNA was not as simple as the scientists thought then. DNA
could have enough complexity to carry the genetic information.

ALFRED HERSHEY AND MARTHA CHASE

Alfred Hershey and Martha Chase were American researchers who sought to settle the question of whether
proteins or DNA was responsible for heredity. In 1952 Alfred Hershey and Martha Chase proved that DNA is
responsible for heredity in T2 Phage. Their findings settled the Protein/DNA debate.

JAMES WATSON AND FRANCIS CRICK

Following the acceptance of DNA as the genetic material, the big question was: How does DNA carry genetic
information?
The answer lies in the structure of DNA. Many scientists were working and proposing various structures. In
1953, James Watson and Francis Crick proposed the double helix structure of DNA which has become widely
accepted.

EARLY EXPERIMENTS IN MICROBIAL GENETICS

1. Fredrick Griffith’s Transformation Experiments
2. Oswald Avery, McCleod and McCarty Experiments
3. Alfred Hershey – Martha Chase Experiments
4. Beadle and Tatum hypothesis

Griffith’s transformation experiments

Fredrick Griffith was studying virulence in the bacterium Streptococcus pneumoniae around 1928.
-He observed that when he injected a combination of heat killed virulent bacteria (Smooth colony) and living
non-virulent bacteria (Rough colony) into a group of mice, the mice died of pneumonia.
-He also recovered living virulent bacteria from the dead mice.
He called this change of non-virulent bacteria into virulent pathogen, transformation.
Figure: Griffith’s transformation experiments.

THE SEARCH FOR THE TRANSFORMING PRINCIPLE- Oswald J. Avery’s Experiments

Around 1940, scientists were searching for the identity of the chemical substance that carries the
genetic information. Proteins were the focus of most scientists. Avery and colleagues reasoned that
the transforming principle in Griffith’s experiment should be the carrier of genetic information.
So, they set out to identify the transforming principle.
 SUMMARY OF AVERY'S EXPERIMENTS

R cells + S cell Purified Polysaccharide R Colonies No transformation

R cells + S cell Purified Protein R Colonies No transformation
R cells + S cells Purified RNA R Colonies No transformation
R cells + S cell Purified DNA S Colonies Transformation

REVERSE EXPERIMENTS
S cell Extract + Proteinase + R cells S colonies Transformation
S cell Extract + RNase + R cells S colonies Transformation
S cell Extract + DNase + R cells R colonies No transformation

CONCLUSION: Transformation only occurred when DNA was present

THE PROTEIN OR DNA DEBATE – Hershey and Chase T2 Phage Experiments

Although the experiments of Avery, McLeod and McCarthy demonstrated that DNA, rather than proteins, is
the carrier of genetic information, there was a strong resistance from much of the scientific community which
favoured proteins. For nearly a decade, the Avery group was forced to repel attacks on the validity of their
experiments, defending both their findings and their reputations.

HERSHEY- CHASE EXPERIMENTS

In 1952, Alfred Hershey and Martha Chase decided to settle the question of whether protein or DNA is the
carrier of genetic information. They employed the reproduction of the T2 phage in Escherichia coli.
 • In Batch 1 the phages were grown in a medium containing radioactive sulfur (35S) that was
incorporated into the phage protein coat.

• In Batch 2 the phages were grown in medium containing radioactive phosphorous(32P) that was
incorporated into the phage DNA

• The difference in substance incorporation was because they wanted to determine which of the two
substances is the genetic material of life.

• First, the batches of radioactively labeled phages were mixed with bacteria. This caused the phages to
infect the bacterial cells and enter the Lysogenic Cycle

• Second, the mixture was agitated in a blender which freed the phage parts that were outside the
bacteria cells and removed them from it.

• The mixture was centrifuged to separate the bacteria from the free phages and phage parts. The
bacteria settled at the bottom of the test tube as a pellet.

• The supernatant which consisted of free phages and phage parts, was above the pellet.

• The radioactivity of the liquid and the pellet was measured.

Results of The Experiments

• In Batch 1 with the sulfur labeled proteins, radioactivity was found in the supernatant, which
contained the free phage and phage parts.

• None of the sulfur had entered the cells, therefore meaning no protein entered the cell either.

• In Batch 2, only the pellet, which contained the radioactively labeled phosphorous, NOT the
supernatant, was radioactive.

• This result of Batch 2 meant that the phosphorous entered the cell, which means DNA entered the
cell.

Conclusion

Hershey –Chase experiments confirmed the results of Avery and his group.

DNA and not proteins carry the genetic information in bacteria as well as in viruses.

How could a simple compound like DNA do this?

The answer awaited the discovery of the structure of DNA.

BEADLE AND TATUM HYPOTHESIS – ONE GENE- ONE ENZYME

In 1941, Beadle and Tatum built on Garrod’s discovery of the connection between genes and metabolic
pathways. Their research led to the “one gene, one enzyme (or protein)” hypothesis, which states that each
enzyme that acts in a biochemical pathway is encoded by a different gene.

Beadle and Tatum used the fungus Neurospora crassa (a bread mould) for their studies because it had
practical advantages as a laboratory model organism. They knew that Neurospora was prototrophic, meaning
it could grow on minimal medium (MM). Minimal medium lacked most nutrients, except for a few minerals,
simple sugars, and one vitamin (biotin). Prototrophs can synthesize the amino acids, vitamins, etc. necessary
for normal growth.

They isolated a series of mutations known to interrupt the synthesis of arginine, an amino-acid necessary for
growth of the mould. Their hypothesis stated that individual mutations inhibited discrete steps in the pathway
used by the mould to synthesize arginine from precursors in their environmental medium.

They knew that by exposing Neurospora spores to X-rays, they could randomly induce mutations in genes (now
known as damage to the DNA leading to DNA sequence change). Each spore exposed to X-rays potentially
contained a mutation in a different gene. While most mutagenized spores were still able to grow
(prototrophic), some spores had mutations that changed their phenotype from a prototroph into
an auxotrophic strain, which could no longer grow on minimal medium. Instead, these auxotrophs could grow
on complete medium (CM), which was MM supplemented with nutrients, such as amino acids and vitamins,
see Figure 1 below.

In fact, some auxotrophic mutations could grow on minimal medium with only one, single nutrient supplied,
such as the amino acid arginine. This implied that each auxotrophic mutant was blocked at a specific step in a
biochemical pathway and that by adding an essential compound, such as arginine, that block could be
overcome.

Figure 2 gives the results of such experiments, demonstrating the survival (or not) of mutants, depending on
the nutrients supplied, and the perturbation of the biochemical pathway involved, depending on the particular
mutation.

Figure 1: A single mutagenized spore is used to establish a colony of Genetically Identical Fungi,
from which spores are tested for their ability to grow on different types of media. Because
spores of this particular colony are able to grown only on complete medium (CM), or on minimal
medium supplemented with arginine (MM+Arg), they are considered Arg auxotrophs and we
infer that they have a mutation in a gene in the Arg biosynthetic pathway. This type of screen is
repeated many times to identify other mutants in the Arg pathway and in other pathways.

Figure 2: The survival of mutants depends on the nutrients supplied, and the perturbation of the
biochemical lathway involved depends on the particular mutation

Beadle and Tatum linked many nutritional mutants to specific amino acids and vitamin biochemical pathways.
This work demonstrated that individual genes are connected to specific enzymes. This initial discovery which
made the link between genes and enzymes was called the “one gene-one enzyme” hypothesis.

ONE GENE – ONE ENZYME HYPOTHESIS

The one gene, one enzyme hypothesis is the idea that each gene encodes a single enzyme. Sir Archibald
Garrod, a British medical doctor, was the first to suggest that genes were connected to enzymes.

The one gene–one enzyme hypothesis postulates that genes act through the production of enzymes, with each
gene responsible for producing a single enzyme that in turn affects a single step in a metabolic pathway.
Today, we know that this idea is not exactly correct. Enzymes are proteins. Some proteins are made of two or
more polypeptides which are coded for by different genes. It is also now known that there are split genes
that code for different polypeptides. Therefore, the one gene- one enzyme hypothesis was later modified to
one gene- one- polypeptide. Later it was discovered that some genes do not code for polypeptides or proteins.
For examples, some genes code for tRNA and rRNA.

In view of this, a gene is more acceptably defined as a segment of polynucleotides which specify or code for an
RNA product. Genes that code for mRNA are translated into polypeptides or proteins while those that code
for other types of RNAs are not translated.

GENETIC CODES AND ORGANIZATION OF GENETIC INFORMATION

CHEMICAL NATURE AND COMPOSITION OF NUCLEIC ACIDS

1. Chemical composition of DNA and RNA

2. Structure of DNA and complimentary base pairing

Historical background

Phoebus Aaron Theodore Levene, an America biochemist studied the structure and function of nucleic

acids. He elucidated the basic chemical composition of nucleic acids in 1920s.

CHEMICAL NATURE OF DNA AND RNA

DNA means Deoxyribonucleic acid.

RNA means Ribonucleic acid.

DNA and RNA are nucleic acids.

A nucleic acid is a polymer made of subunits called Nucleotides.

Each Nucleotides has three parts:

1. Pentose sugar

2 Nitrogenous bases

3 Phosphate group
Pentose sugars

Pentose sugars are five-member ring structures.

They are of two types:

1. Deoxyribose sugar

2. Ribose Sugar
There are two types of Nitrogenous bases.

1. Purines

Adenine (A)

Guanine (G)

2. Pyrimidines

Cytosine (C)

Thymine (T)

Uracil (U)

Phosphate
group

Nucleotide and
nucleoside

Nucleoside = Nitrogenous
base + Pentose Sugar

Nucleotide = Nitrogenous base + Pentose+

Phosphate Group

NUCLEIC ACIDS

Nucleic acids consist of a long chain of

nucleotides joined together by phospho-
diester, and hydrogen bonds.

Structure of DNA and base pairing

James Watson and Francis Crick

proposed the double helix structure of DNA in 1953

DNA consists of two complementary strands coiled together to form a double helix.

DNA is a large polymer made of two long chains of nucleotides joined together by hydrogen bonds.
 Complimentary base pairing rules

5’ A GCCTTCCTT
C 3’
3’ T C G G A A G G A A 5’
Exercise for Practice
Write out the complementary DNA Sequence
3’A G T A T T G G G C T A G C T A C T T A A G T G T C5’
Write out the complementary RNA sequence.
3’G G T G T T G C G C T A T C T A C T G A T G T T A C5’

Differences between DNA and RNA.

DNA and RNA are two different types of nucleic acids.
Nucleic acids are made of:
Nucleotide => Nitrogenous base + Pentose Sugar + Phosphate group
DNA => A, G, T, C + Deoxyribose Sugar + Phosphate Group
RNA => A, G, U, C + Ribose Sugar + Phosphate Group
Structural and functional differences
  STRUCTURAL DIFFERENCES
DNA is double stranded.
RNA is single stranded.

FUNCTIONAL DIFFERENCES
DNA is the primary carrier of the genetic information in organisms except in some RNA viruses.
DNA maintains the permanent genetic information or codes of organisms.
Summary of Differences Between DNA and RNA

Differences Features DNA RNA

Sugar type Deoxyribose Ribose
Chemical
A, C, G, T (Thymine) A, C, G, U (Uracil)
Nitrogenous bases
Structure Double stranded Single stranded
Strand type
Function and Carrier of genetic mRNA – messenger function in
Location Function information protein synthesis
Permanent storage tRNA – transportation function
of genetic codes rRNA – component of ribosomes
Location Nucleus (in Transported out the nucleus in
eukaryotes) eukaryotes, component of
ribosomes

DNA REPLICATION
In molecular biology, DNA replication is the biological process of producing two identical replicas of
DNA from one original DNA molecule. DNA Replication simply means the reproduction of DNA. The
Replication of DNA is an extraordinarily important and highly coordinated process. The continuity of
life depends on it. Since the sequence of bases in the chromosomal DNA is a coded message
specifying the cell’s structure and functions, the DNA must be duplicated precisely so that each
daughter cell will receive an exact copy(replica) of the molecule.
DNA replication occurs in all living organisms acting as the most essential part for biological
inheritance. This is essential for cell division during growth and repair of damaged tissues, while it
also ensures that each of the new cells receives its own copy of the DNA.
The Replication of DNA involves the synthesis of a replica of the molecule. Shortly after their
description of the DNA structure in 1953, Watson and Crick also published another article in which
they suggested the mechanism by which the DNA might be replicated based on the structure they
had proposed.
In their hypothesis, the two strands of the double helix unwind from one another and separate. Free
nucleotides then line up along the two parental strands through complementary base-paring (A with
T, and G with C). When these nucleotides are linked together by one or more enzymes, two replicas
of the DNA molecule are produced, each containing a parental DNA strand and a newly formed
strand. This is referred to as Semi-Conservative Replication. Research in subsequent years has
validated the hypothesis of Watson and Crick.
Hypotheses of DNA Replication
Hypothesis 1: Semi- Conservative replication – One parental strand and a newly formed strand.
Hypothesis 2: Conservative replication – both parental (old) strands remain together, and two newly
formed strands will form a second double strand.
Hypothesis 3: Dispersive replication -the parental and newly synthesized strands are randomly mixed
during replication.
MESELSON AND STAHL EXPERIMENTS
In 1958, Mathew Meselson and Franklin Stahl performed centrifugation experiments that showed
that DNA replication is by semi-conservative mechanism.
The Meselson-Stahl experiment used density gradient to follow the behaviour of newly-synthesized
DNA strands. The experimental results are shown in the diagrams below:
The Meselson – Stahl experiment: Proof of semi-conservative replication
Meselson & Stahl first grew bacteria for several generations in a medium containing only 15N
(“heavy” nitrogen). When examined in an analytical centrifuge, DNA isolated from these bacteria
produced a single “heavy” band. Meselson and Stahl then transferred a portion of the culture to a
new medium that contained only 14N (“light” nitrogen). When DNA was isolated from these bacteria
after one generation, they observed a single band that was “lighter” than the one obtained before;
the “heavy” band was not observed in these bacteria. When DNA was isolated from the same culture
after two generations, they observed two distinct bands of equal intensity, one with the same weight
as seen in the previous experiment, and a new one still “lighter.” When DNA was isolated from the
same culture after three generations, this lightest band became the predominant one, and the
middle band faded.

Meselson & Stahl reasoned that these experiments showed that DNA replication was semi-
conservative: the DNA strands separate and each makes a copy of itself, so that each daughter
molecule comprises one “old” and one “new” strand. Bacteria grown in “heavy” Nitrogen have been
labeled on both strands entirely with “heavy” Nitrogen. After one generation in “light” Nitrogen, all
of the DNA molecules comprise one “old heavy” and one “new light” strand, and have the same
“heavy / light” molecular weight, which is less than that of “heavy / heavy” molecules. After two
generations in “light” medium, the “heavy” and “light” strands separate, and both replicate with
“light” nitrogen. Half therefore become “light / light”, and half become “heavy / light” as in the
previous experiment. In each successive generation, the proportion of “heavy” strands is reduced by
half, and the “heavy / light” band gradually fades.
Alternative Modes of DNA replication

1. Conservative
If DNA Replication is Conservative, then the two parental strands will always be together and the new
two daughter strands will be newly synthesized. Then there will be two bands, one heavy and the
other light.
2. Dispersive
If DNA Replication is dispersive, then there will be only one band of intermediate weight with old and
new segments mixed up in the two strands.
The results of Meselson and Stah’s experiment showed that DNA Replication was not Conservative
or Dispersive but Semi-Conservative.
MECHANISM OF DNA REPLICATION
In a cell, DNA replication begins at specific locations, or origins of replication, in the genome which
contains the genetic material of an organism. Unwinding of DNA at the origin and synthesis of
newstrands, is facilitated by an enzyme known as helicase. At the point of unwinding of the two
strands, replication forks are created. As the separated strands serve as templates for synthesis of
new strands, the replication forks are growing bidirectional from the origin.
A number of proteins are associated with the replication fork to help in the initiation and
continuation of DNA synthesis. Most prominently, DNA polymerase synthesizes the new strands by
adding nucleotides that complement each (template) strand.
DNA polymerases are a family of enzymes that carry out all forms of DNA replication. DNA
polymerases in general cannot initiate synthesis of new strands but can only extend an existing DNA
or RNA strand paired with a template strand.
To begin synthesis, a short fragment of RNA, called a primer, must be created, and paired with the
template DNA strand. DNA polymerase adds a new strand of DNA by extending the 3’end of an
existing nucleotide chain, adding new nucleotides matched to the template strand one at a time via
the creation of phosphodiester bonds. The energy for this process of DNA polymerization comes from
hydrolysis of the high-energy phosphate (phosphoanhydride) bonds between the three phosphates
attached to each unincorporated base. Free bases with their attached phosphate groups are called
nucleotides; in particular, bases with three attached phosphate groups are called nucleoside
triphosphates. When a nucleotide is being added to a growing DNA strand, the formation of a
phosphodiester bond between the proximal phosphate of the nucleotide to the growing chain is
accompanied by hydrolysis of a high-energy phosphate bond with release of the two distal
phosphates as a pyrophosphate. Enzymatic hydrolysis of the resulting pyrophosphate into inorganic
phosphate consumes a second high-energy phosphate bond and renders the reaction effectively
irreversible.
In general, DNA polymerases are highly accurate, with an intrinsic error rate of less than one mistake
for every 107 nucleotides added. In addition, some DNA polymerases also have proofreading ability;
they can remove nucleotides from the end of a growing strand in order to correct mismatched bases.
Finally, post-replication mismatch repair mechanisms monitor the DNA for errors, being capable of
distinguishing mismatches in the newly synthesized DNA strand from the original strand sequence.
Together, these three discrimination steps enable replication fidelity of less than one mistake for
every 109 nucleotides added.
PROCESS OF REPLICATION
DNA replication, like all biological polymerization processes, proceeds in three enzymatically
catalyzed and coordinated steps:
1. initiation.
2. elongation.
3. termination.
Initiation
For a cell to divide, it must first replicate its DNA. DNA replication is an all-or none process; once
replication begins, it proceeds to completion. Once replication is complete, it does not occur again in
the same cell cycle. This is made possible by the division of initiation of the pre-replication complex.
Pre-replication complex
In late mitosis and early G1 phase, a large complex of initiator proteins assembles into the pre-
replication complex at particular points in the DNA, known as "origins". In E. coli the primary initiator
protein is DnaA; in yeast, this is the origin recognition complex. Sequences used by initiator proteins
tend to be "AT-rich" (rich in adenine and thymine bases), because A-T base pairs have two hydrogen
bonds (rather than the three formed in a C-G pair) and thus are easier to strand separate. In
eukaryotes, the origin recognition complex catalyzes the assembly of initiator proteins into the pre-
replication complex. Cdc6 and Cdt1 then associate with the bound origin recognition complex at the
origin in order to form a larger complex necessary to load the Mcm complex onto the DNA. The Mcm
complex is the helicase that will unravel the DNA helix at the replication origins and replication forks
in eukaryotes. The Mcm complex is recruited at late G1 phase and loaded by the ORC-Cdc6-Cdt1
complex onto the DNA via ATP-dependent protein remodeling. The loading of the Mcm complex onto
the origin DNA marks the completion of pre-replication complex formation.
If environmental conditions are right in late G1 phase, the G1 and G1/S cyclin-Cdk complexes are
activated, which stimulate expression of genes that encode components of the DNA synthetic
machinery. G1/S-Cdk activation also promotes the expression and activation of S-Cdk complexes,
which may play a role in activating replication origins depending on species and cell type. Control of
these Cdks vary depending on cell type and stage of development. This regulation is best understood
in budding yeast, where the S cyclins Clb5 and Clb6 are primarily responsible for DNA replication.
Clb5,6-Cdk1 complexes directly trigger the activation of replication origins and are therefore required
throughout S phase to directly activate each origin.
In a similar manner, Cdc7 is also required through S phase to activate replication origins. Cdc7 is not
active throughout the cell cycle, and its activation is strictly timed to avoid premature initiation of
DNA replication. In late G1, Cdc7 activity rises abruptly because of association with the regulatory
subunit Dbf4, which binds Cdc7 directly and promotes its protein kinase activity. Cdc7 has been found
to be a rate-limiting regulator of origin activity. Together, the G1/S-Cdks and/or S-Cdks and Cdc7
collaborate to directly activate the replication origins, leading to initiation of DNA synthesis.
Preinitiation complex
In early S phase, S-Cdk and Cdc7 activation led to the assembly of the preinitiation complex, a
massive protein complex formed at the origin. Formation of the preinitiation complex displaces Cdc6
and Cdt1 from the origin replication complex, inactivating and disassembling the pre-replication
complex. Loading the preinitiation complex onto the origin activates the Mcm helicase, causing
unwinding of the DNA helix. The preinitiation complex also loads α-primase and other DNA
polymerases onto the DNA. After α-primase synthesizes the first primers, the primer-template
junctions interact with the clamp loader, which loads the sliding clamp onto the DNA to begin DNA
synthesis. The components of the preinitiation complex remain associated with replication forks as
they move out from the origin.
Elongation
DNA polymerase has 5’ – 3’ activity. All known DNA replication systems require a free 3’ hydroxyl
group before synthesis can be initiated (note: the DNA template is read in 3’ to 5’ direction whereas a
new strand is synthesized in the 5’ to 3’ direction). Four distinct mechanisms for DNA synthesis are
recognized:
1. All cellular life forms and many DNA viruses, phages and plasmids use a primase to synthesize a
short RNA primer with a free 3’-OH group which is subsequently elongated by a DNA polymerase.
2. The retroelements (including retroviruses) employ a transfer RNA that primes DNA replication
by providing a free 3’-OH that is used for elongation by the reverse transcriptase.
3. In the adenoviruses and the ϕ29 family of bacteriophages, the 3’OH group is provided by the
side chain of an amino acid of the genome attached protein (the terminal protein) to which
nucleotides are added by the DNA polymerase to form a new strand.
4. In the single stranded DNA viruses, a group that includes the circoviruses, the geminiviruses,
the parvoviruses and others, and also the many phages and plasmids that use the rolling circle
replication (RCR) mechanism, the RCR endonuclease creates a nick in the genome strand (single
stranded viruses) or one of the DNA strands (plasmids). The 5’ end of the nicked strand is transferred
to a tyrosine residue on the nuclease and the free 3’OH group is then used by the DNA polymerase to
synthesize the new strand.
The first is the best known of these mechanisms and is used by the cellular organisms. In this
mechanism, once the two strands are separated, primase adds RNA primers to the template strands.
The leading strand receives one RNA primer while the lagging strand receives several. The leading
strand is continuously extended from the primer by a DNA polymerase with high processivity, while
the lagging strand is extended discontinuously from each primer forming Okazaki fragments. RNase
removes the primer RNA fragments, and a low processivity DNA polymerase distinct from the
replicative polymerase enters to fill the gaps. When this is complete, a single nick on the leading
strand and several nicks on the lagging strand can be found. Ligase works to fill these nicks in, thus
completing the newly replicated DNA molecule.
Multiple DNA polymerases take on different roles in the DNA replication process. In E. coli, DNA Pol III
is the polymerase enzyme primarily responsible for DNA replication. It assembles into a replication
complex at the replication fork that exhibits extremely high processivity, remaining intact for the
entire replication cycle. In contrast, DNA Pol I is the enzyme responsible for replacing RNA primers
with DNA. DNA Pol I has a 5’ to 3’exonuclease activity in addition to its polymerase activity and uses
its exonuclease activity to degrade the RNA primers ahead of it as it extends the DNA strand behind
it, in a process called nick translation. Pol I is much less processive than Pol III because its primary
function in DNA replication is to create many short DNA regions rather than a few very long regions.
As DNA synthesis continues, the original DNA strands continue to unwind on each side of the bubble,
forming a replication fork with two prongs. In bacteria, which have a single origin of replication on
their circular chromosome, this process creates a "theta structure" (resembling the Greek letter
theta: O. In contrast, eukaryotes have longer linear chromosomes and initiate replication at multiple
origins within these.
The Replication forks.
The replication fork is a structure that forms within the long helical DNA during DNA replication. It is
created by helicases, which break the hydrogen bonds holding the two DNA strands together in the
helix. The resulting structure has two branching "prongs", each one made up of a single strand of
DNA. These two strands serve as the template for the leading and lagging strands, which will be
created as DNA polymerase matches complementary nucleotides to the templates; the templates
may be properly referred to as the leading strand template and the lagging strand template.
DNA is read by DNA polymerase in the 3’ to 5’ direction, meaning the new strand is synthesized in the
5' to 3' direction. Since the leading and lagging strand templates are oriented in opposite directions
at the replication fork, a major issue is how to achieve synthesis of new lagging strand DNA, whose
direction of synthesis is opposite to the direction of the growing replication fork.
Leading strand
The leading strand is the strand of new DNA which is synthesized in the same direction as the
growing replication fork. This sort of DNA replication is continuous.
Lagging strand
The lagging strand is the strand of new DNA whose direction of synthesis is opposite to the direction
of the growing replication fork. Because of its orientation, replication of the lagging strand is more
complicated as compared to that of the leading strand. Consequently, the DNA polymerase on this
strand is seen to "lag behind" the other strand.
Dynamics at the replication fork
In all cases the helicase is composed of six polypeptides that wrap around only one strand of the DNA
being replicated. The two polymerases are bound to the helicase heximer. In eukaryotes the helicase
wraps around the leading strand, and in prokaryotes it wraps around the lagging strand.
As helicase unwinds DNA at the replication fork, the DNA ahead is forced to rotate. This process
results in a build-up of twists in the DNA ahead. This build-up forms a torsional resistance that would
eventually halt the progress of the replication fork. Topoisomerases are enzymes that temporarily
break the strands of DNA, relieving the tension caused by unwinding the two strands of the DNA
helix; topoisomerases (including DNA gyrase) achieve this by adding negative supercoils to the DNA
helix.
Bare single-stranded DNA tends to fold back on itself forming secondary structures; these structures
can interfere with the movement of DNA polymerase. To prevent this, single-strand binding proteins
bind to the DNA until a second strand is synthesized, preventing secondary structure formation.
Double-stranded DNA is coiled around histones that play an important role in regulating gene
expression so the replicated DNA must be coiled around histones at the same places as the original
DNA. To ensure this, histone chaperones disassemble the chromatin before it is replicated and
replace the histones in the correct place.
Clamp proteins form a sliding clamp around DNA, helping the DNA polymerase maintain contact with
its template, thereby assisting with processivity. The inner face of the clamp enables DNA to be
threaded through it. Once the polymerase reaches the end of the template or detects double-
stranded DNA, the sliding clamp undergoes a conformational change that releases the DNA
polymerase. Clamp-loading proteins are used to initially load the clamp, recognizing the junction
between template and RNA primers.
Termination
Eukaryotes initiate DNA replication at multiple points in the chromosome, so replication forks meet
and terminate at many points in the chromosome. Because eukaryotes have linear chromosomes,
DNA replication is unable to reach the very end of the chromosomes. Due to this problem, DNA is lost
in each replication cycle from the end of the chromosome. Telomeres are regions of repetitive DNA
close to the ends and help prevent loss of genes due to this shortening. Shortening of the telomeres
is a normal process in somatic cells. This shortens the telomeres of the daughter DNA chromosome.
As a result, cells can only divide a certain number of times before the DNA loss prevents further
division. (This is known as the Hayflick limit.) Within the germ cell line, which passes DNA to the next
generation, telomerase extends the repetitive sequences of the telomere region to prevent
degradation. Telomerase can become mistakenly active in somatic cells, sometimes leading to cancer
formation. Increased telomerase activity is one of the hallmarks of cancer.
Termination requires that the progress of the DNA replication fork must stop or be blocked.
Termination at a specific locus, when it occurs, involves the interaction between two components:
(1) a termination site sequence in the DNA, and (2) a protein which binds to this sequence to
physically stop DNA replication. In various bacterial species, this is named the DNA replication
terminus site-binding protein, or Ter protein. Because bacteria have circular chromosomes,
termination of replication occurs when the two replication forks meet each other on the opposite
end of the parental chromosome. E. coli regulates this process using termination sequences that,
when bound by the Tus protein, enable only one direction of replication fork to pass through. As a
result, the replication forks are constrained to always meet within the termination region of the
chromosome.