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Microbial genetics
is a subject area within microbiology and genetic engineering. It studies
the genetics of very small (micro) organisms; bacteria, archaea, viruses
and some protozoa and fungi. This involves the study of the genotype of
microbial species and also the expression system in the form
of phenotypes.
Since the discovery of microorganisms by two Fellows of The Royal
Society, Robert Hooke and Antoni van Leeuwenhoek during the period
1665-1885they have been used to study many processes and have had
applications in various areas of study in genetics. For example:
Microorganisms' rapid growth rates and short generation times are used
by scientists to study evolution.[] Microbial genetics also has applications
in being able to study processes and pathways that are similar to those
found in humans such as drug metabolism.]
Bacter
Bacteria are classified by their shape.
Bacteria have been on this planet for approximately 3.5 billion years, and
are classified by their shape.[5] Bacterial genetics studies the mechanisms
of their heritable information, their chromosomes, plasmids, transposons,
and phages.[6]
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Gene transfer systems that have been extensively studied in bacteria
include genetic transformation, conjugation and transduction.Natural
transformation is a bacterial adaptation for DNA transfer between two
cells through the intervening medium. The uptake of donor DNA and its
recombinational incorporation into the recipient chromosome depends on
the expression of numerous bacterial genes whose products direct this
process. In general, transformation is a complex, energy-requiring
developmental process that appears to be an adaptation for repairing
DNA damage.
Bacterial conjugation
is the transfer of genetic material between bacterial cells by direct cellto-cell contact or by a bridge-like connection between two cells. Bacterial
conjugation has been extensively studied in Escherichia coli, but also
occurs in other bacteria such asMycobacterium smegmatis. Conjugation
requires stable and extended contact between a donor and a recipient
strain, is DNase resistant, and the transferred DNA is incorporated into
the recipient chromosome by homologous recombination. E.
coli conjugation is mediated by expression of plasmid genes, whereas
mycobacterial conjugation is mediated by genes on the bacterial
chromosome
Transduction is the process by which foreign DNA is introduced into a
cell by a virus or viral vector. Transduction is a common tool used by
molecular biologists to stably introduce a foreign gene into a host
cell's genome
Archaea[edit]
Archaea is a domain of organisms that are prokaryotic, single-celled, and
are thought to have developed 4 billion years ago. They share a common
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ancestor with bacteria, but are more closely related to eukaryotes in
comparison to bacteria.] Some Archaea are able to survive extreme
environments, which leads to many applications in the field of genetics.
One of such applications is the use of archaeal enzymes, which would be
better able to survive harsh conditions in vitro.
Gene transfer and genetic exchange have been studied in
the halophilic archaeon Halobacterium volcanii and
the hyperthermophilic archaeons Sulfolobus solfataricus andSulfolobus
acidocaldarius. H. volcani forms cytoplasmic bridges between cells that
appear to be used for transfer of DNA from one cell to another in either
direction.] When S. solfataricus and S. acidocaldarius are exposed to
DNA damaging agents, species-specific cellular aggregation is induced.
Cellular aggregation mediates chromosomal marker exchange and genetic
recombination with high frequency. Cellular aggregation is thought to
enhance species specific DNA transfer between Sulfolobus cells in order
to provide increased repair of damaged DNA by means of homologous
recombination.[14][15][16]
Fungi[edit]
Fungi can be both multicellular and unicellular organisms, and are
distinguished from other microbes by the way they obtain nutrients.
Fungi secrete enzymes into their surroundings, to break down organic
matter. Fungal genetics uses yeast, and filamentous fungi as model
organisms for eukaryotic genetic research, including cell
cycleregulation, chromatin structure and gene regulation.[17]
Studies of the fungus Neurospora crassa have contributed substantially to
understanding how genes work. N. crassa is a type of red bread mold of
the phylum Ascomycota. It is used as a model organism because it is easy
to grow and has a haploid life cycle that makes genetic analysis simple
‫ وفاء عبد الواحد‬.‫د‬
‫المحاضرة االولى والثانية‬
since recessive traits will show up in the offspring. Analysis of genetic
recombination is facilitated by the ordered arrangement of the products
of meiosis in ascospores. In its natural environment, N. crassa lives
mainly in tropical and sub-tropical regions. It often can be found growing
on dead plant matter after fires.
Neurospora was used by Edward Tatum and George Beadle in their
experiments for which they won the Nobel Prize in Physiology or
Medicine in 1958. The results of these experiments led directly to the one
gene-one enzyme hypothesis that specific genes code for
specific proteins. This concept proved to be the opening gun in what
becamemolecular genetics and all the developments that have followed
from that.[19]
Saccharomyces cerevisiae is a yeast of the phylum Ascomycota. During
vegetative growth that ordinarily occurs when nutrients are abundant, S.
cerevisiae reproduces by mitosisas diploid cells. However, when starved,
these cells undergo meiosis to form haploid spores.[20] Mating occurs
when haploid cells of opposite mating types MATa and MATα come into
contact. Ruderfer et al. pointed out that, in nature, such contacts are
frequent between closely related yeast cells for two reasons. The first is
that cells of opposite mating type are present together in the same acus,
the sac that contains the cells directly produced by a single meiosis, and
these cells can mate with each other. The second reason is
that haploid cells of one mating type, upon cell division, often produce
cells of the opposite mating type. An analysis of the ancestry of natural S.
cerevisiae strains concluded that outcrossing occurs very infrequently
(only about once every 50,000 cell divisions).[21] The relative rarity in
nature of meiotic events that result from outcrossing suggests that the
possible long-term benefits of outcrossing (e.g. generation of diversity)
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are unlikely to be sufficient for generally maintaining sex from one
generation to the next. Rather, a short term benefit, such as meiotic
recombinational repair of DNA damages caused by stressful conditions
(such as starvation) may be the key to the maintenance of sex in S.
cerevisiae.
Candida albicans is a diploid fungus that grows both as a yeast and as
a filament. C. albicans is the most common fungal pathogen in humans. It
causes both debilitating mucosal infections and potentially lifethreatening systemic infections. C. albicans has maintained an elaborate,
but largely hidden, mating apparatus. Johnson suggested that mating
strategies may allow C. albicans to survive in the hostile environment of
a mammalian host.
Among the 250 known species of aspergilli, about 33% have an identified
sexual state.] Among those Aspergillus species that exhibit a sexual cycle
the overwhelming majority in nature are homothallic (selffertilizing).] Selfing in the homothallic fungus Aspergillus
nidulans involves activation of the same mating pathways characteristic
of sex in outcrossing species, i.e. self-fertilization does not bypass
required pathways for outcrossing sex but instead requires activation of
these pathways within a single individual.]Fusion of haploid nuclei occurs
within reproductive structures termed cleistothecia, in which the diploid
zygote undergoes meiotic divisions to yield haploid ascospores.
Viruses
Viruses are capsid-encoding organisms composed of proteins and nucleic
acids that can self-assemble after replication in a host cell using the host's
replication machineryThere is a disagreement in science about
whether viruses are living due to their lack
‫ وفاء عبد الواحد‬.‫د‬
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of ribosomes.] Comprehending the viral genome is important not only for
studies in genetics but also for understanding their pathogenic properties.]
Many types of virus are capable of genetic recombination. When two or
more individual viruses of the same type infect a cell, their genomes may
recombine with each other to produce recombinant virus progeny. Both
DNA and RNA viruses can undergo recombination. When two or more
viruses, each containing lethal genomic damage infect the same host cell,
the virus genomes often can pair with each other and undergo
homologous recombinational repair to produce viable progeny. This
process is known as multiplicity reactivation. Enzymes employed in
multiplicity reactivation are functionally homologous to enzymes
employed in bacterial and eukaryotic recombinational repair. Multiplicity
reactivation has been found to occur with pathogenic viruses including
influenza virus, HIV-1, adenovirus simian virus 40, vaccinia virus,
reovirus, poliovirus and herpes simplex virus as well as numerous
bacteriophages.]
Applications of microbial genetics
Taq polymerase which is used in Polymerase Chain Reaction(PCR)
Microbes are ideally suited for biochemical and genetics studies and have
made huge contributions to these fields of science such as the
demonstration that DNA is the genetic material,[37][38] that the gene has a
‫ وفاء عبد الواحد‬.‫د‬
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simple linear structure,] that the genetic code is a triplet code,] and that
gene expression is regulated by specific genetic processes.] Jacques
Monod and François Jacob used Escherichia coli, a type of bacteria, in
order to develop the operon model of gene expression, which lay down
the basis of gene expression and regulation.] Furthermore
the hereditary processes of single-celled eukaryotic microorganisms are
similar to those in multi-cellular organisms allowing researchers to gather
information on this process as well.] Another bacterium which has greatly
contributed to the field of genetics is Thermus aquaticus, which is a
bacterium that tolerates high temperatures. From this microbe scientists
isolated the enzyme Taq polymerase, which is now used in the powerful
experimental technique, Polymerase chain reaction(PCR).] Additionally
the development of recombinant DNA technology through the use of
bacteria has led to the birth of modern genetic
engineering andbiotechnology.]
Using microbes, protocols were developed to insert genes into
bacterial plasmids, taking advantage of their fast reproduction, to make
biofactories for the gene of interest. Such genetically engineered bacteria
can produce pharmaceuticals such as insulin, human growth
hormone, interferons and blood clotting factors.[5] Microbes synthesize a
variety of enzymes for industrial applications, such as fermented foods,
laboratory test reagents, dairy products (such as renin), and even in
clothing (such as Trichoderma fungus whose enzyme is used to give
jeans a stone washed appearance).[5]
Bibliography[edit]
‫ وفاء عبد الواحد‬.‫د‬
‫المحاضرة االولى والثانية‬
Genetic
recombination
contributes
to
population
diversity:
recombinations more likely than mutations to provide beneficial
change since it tends not to destroy gene function Eukaryotes:
recombination during meiosis for sexual reproduction -creates
diversity in offspring but parent remains unchanged -vertical gene
transfer = genes passed from organism to offspring Prokaryotes:
recombination via gene transfer between cells or within cell by
transformation, conjugation, or transduction -original cell is altered horizontal gene transfer = genes passed to neighboring microbes of
same generation -transfer involves donor cell that gives portion of
DNA to recipient cell -when donor DNA incorporated into recipient,
recipient now called recombinant cell -if recombinant cell acquired
new function/characteristic as result of new DNA, cell has been
transformed Generation of recombinant cells is very low frequency
event (less than 1%): very few cells in population are capable of
exchanging and incorporating DNA Three methods of prokaryotic
gene transfer: 1. Bacterial Transformation -genes transferred as
naked DNA -can occur between unrelated genus/species -discovered
by F. Griffith 1928 who studied Streptococcus pneumoniae -virulent
strain had capsule -non-virulent stain did not -in mouse, dead
virulent strain could pass “virulence factor” to live nonvirulent strain
-competent cells can pick up DNA from dead cells and incorporate it
into genome by recombination (e.g. antibiotic resistance) transformed cell than passes genetic recombination to progeny2.
Conjugation -genes transferred between two live cells via sex pilus
(Gram -) or surface adhesion molecules (Gram +) -transfer mediated
by a plasmid: small circle of DNA separate from genome that is self
replicating but contains no essential genes -plasmid has genes for its
‫ وفاء عبد الواحد‬.‫د‬
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own transfer -Gram negative plasmids have genes for pilus -Gram
positive plasmids have genes for surface adhesion molecules
Conjugation requires cell to cell contact between two cells of opposite
mating type, usually the same species, During conjugation plasmid is
replicated and single stranded copy is transferred to recipient.
Recipient synthesizes complementary strand to complete plasmid plasmid can remain as separate circle or -plasmid can be integrated
into host cell genome Transformation (genetics)
From Wikipedia, the free encyclopedia
Not to be confused with an unrelated process called malignant
transformation which occurs in the progression of cancer.
In this image, a gene from bacterial cell 1 is moved from bacterial cell 1
to bacterial cell 2. This process of bacterial cell 2 taking up new genetic
material is called transformation.
In molecular
a cell resulting
biology, transformation is
from
the
direct
uptake
the genetic alteration
and
incorporation
of
of
exogenousgenetic material (exogenous DNA) from its surroundings
through the cell membrane(s). Transformation occurs naturally in some
species of bacteria, but in biotechnology transformation is a basic
technique, and can be made to occur in many kinds of cells. For
transformation to happen in nature, bacteria must be in a state
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of competence, which might occur as a time-limited response to
environmental conditions such as starvation and cell density.
Transformation is one of three processes by which exogenous genetic
material may be introduced into a bacterial cell, the other two
beingconjugation (transfer of genetic material between two bacterial cells
in direct contact) and transduction (injection of foreign DNA by
abacteriophage virus into the host bacterium).
"Transformation" may also be used to describe the insertion of new
genetic material into nonbacterial cells, including animal and plant cells;
however, because "transformation" has a special meaning in relation to
animal cells, indicating progression to a cancerous state, the term should
be avoided for animal cells when describing introduction of exogenous
genetic material. Introduction of foreign DNA intoeukaryotic cells is
often called "transfection".[1]
resulting in permanent chromosomal ch
Transduction
Transduction is the process by which DNA is transferred from one
bacterium to another by a virus . It also refers to the process whereby
foreign DNA is introduced into another cell via a viral vector.
Transduction does not require physical contact between the cell donating
the DNA and the cell receiving the DNA (which occurs in conjugation),
and it is DNAase resistant (transformation is susceptible to DNAase).
Transduction is a common tool used by molecular biologists to stably
introduce a foreign gene into a host cell's genome.
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Transduction
Transduction is the process by which DNA is transferred from one
bacterium to another by a virus. It also refers to the process whereby
foreign DNA is introduced into another cell via a viral vector.
When bacteriophages (viruses that infect bacteria) infect a bacterial cell,
their normal mode of reproduction is to harness the replicational,
transcriptional, and translation machinery of the host bacterial cell to
make numerous virions, or complete viral particles, including the viral
DNA or RNA and the protein coat.
Transduction is especially important because it explains one mechanism
by which antibiotic drugs become ineffective due to the transfer of
antibiotic-resistance genes between bacteria. In addition, hopes to create
medical methods of genetic modification of diseases such as
Duchenne/Becker Muscular Dystrophy are based on these methodologies.
The Lytic Cycle and the Lysogenic Cycle
Transduction happens through either the lytic cycle or the lysogenic
cycle. If the lysogenic cycle is adopted, the phage chromosome is
integrated (by covalent bonds) into the bacterial chromosome, where it
can remain dormant for thousands of generations. If the lysogen is
induced (by UV light for example), the phage genome is excised from the
bacterial chromosome and initiates the lytic cycle, which culminates
in lysis of the cell and the release of phage particles. The lytic cycle leads
to the production of new phage particles which are released by lysis of
the host.
Transduction is a method for transferring genetic material. The packaging
of bacteriophage DNA has low fidelity and small pieces of bacterial
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DNA, together with the bacteriophage genome, may become packaged
into the bacteriophage genome. At the same time, some phage genes are
left behind in the bacterial chromosome.
There are generally three types of recombination events that can lead to
this incorporation of bacterial DNA into the viral D
Transduction is especially important because it explains one mechanism
by which antibiotic drugs become ineffective due to the transfer of
antibiotic-resistance genes between bacteria. In addition, hopes to create
medical methods of genetic modification of diseases such as
Duchenne/Becker Muscular Dystrophy are based on these methodologies.
The Lytic Cycle and the Lysogenic Cycle
Transduction happens through either the lytic cycle or the lysogenic
cycle. If the lysogenic cycle is adopted, the phage chromosome is
integrated (by covalent bonds) into the bacterial chromosome, where it
can remain dormant for thousands of generations. If the lysogen is
induced (by UV light for example), the phage genome is excised from the
bacterial chromosome and initiates the lytic cycle, which culminates
in lysis of the cell and the release of phage particles. The lytic cycle leads
to the production of new phage particles which are released by lysis of
the host.
Transduction is a method for transferring genetic material. The packaging
of bacteriophage DNA has low fidelity and small pieces of bacterial
DNA, together with the bacteriophage genome, may become packaged
into the bacteriophage genome. At the same time, some phage genes are
left behind in the bacterial chromosome.
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There are generally three types of recombination events that can lead to
this incorporation of bacterial DNA into the viral DNA, leading to two
modes of recombination.
Generalized transduction is the process by which any bacterial gene may
be transferred to another bacterium via a bacteriophage, and typically
carries only bacterial DNA and no viral DNA. In essence, this is the
packaging of bacterial DNA into a viral envelope. This may occur in two
main ways, recombination and headful packaging.
If bacteriophages undertake the lytic cycle of infection upon entering a
bacterium, the virus will take control of the cell's machinery for use in
replicating its own viral DNA. If by chance bacterial chromosomal DNA
is inserted into the viral capsid which is usually used to encapsulate the
viral DNA, the mistake will lead to generalized transduction.
If the virus replicates using "headful packaging," it attempts to fill th
Fates of DNA Inserted into the Recipient Cell
When the new DNA is inserted into this recipient cell it can fall to one
of three fates: the DNA will be absorbed by the cell and be recycled
for spare parts; if the DNA was originally a plasmid, it will recirculate
inside the new cell and become a plasmid again; if the new DNA
matches with a homologous region of the recipient cell's chromosome,
it will exchange DNA material similar to the actions in conjugation.
This type of recombination is random and the amount recombined
depends on the size of the virus being used.
Specialized transduction is the process by which a restricted set of
bacterial genes are transferred to another bacterium. The genes that
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get transferred (donor genes) depend on where the phage genome is
located on the chromosome. Specialized transduction occurs when
the prophage excises imprecisely from the chromosome so that
bacterial genes lying adjacent to the prophage are included in the
excised DNA. The excised DNA is then packaged into a new virus
particle, which can then deliver the DNA to a new bacterium, where
the donor genes can be inserted into the recipient chromosome or
remain in the cytoplasm, depending on the nature of the
bacteriophage.
When the partially encapsulated phage material infects another cell
and becomes a "prophage" (is covalently bonded into the infected
cell's chromosome), the partially coded prophage DNA is called a
"heterogenote. " Example of specialized transduction is λ phages
in Escherichia coli, which was discovered by Esther Lederberg.
Genetics of prokaryotes and viruses[change | change source]
The genetics of bacteria, archaea and viruses is a major field or research.
Bacterial mostly divide by asexual cell division, but do have a kind of sex
by horizontal gene transfer.Bacterial
conjugation, transduction and transformation are their methods. In
addition, the complete DNA sequence of many bacteria, archaea and
viruses is now known.
Although many bacteria were given generic and specific names,
like Staphylococcus aureus, the whole idea of a species is rather
meaningless for an organism which does not have sexes and crossing-
‫ وفاء عبد الواحد‬.‫د‬
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over of chromosomes.[27] Instead, these organisms have strains, and that
is how they are identified in the laboratory.
Genes and development[change | change source]
Gene expression[change | change source]
Gene expression is the process by which the heritable information in
a gene, the sequence of DNA base pairs, is made into a functional
gene product, such as protein or RNA. The basic idea is that DNA
is transcribed into RNA, which is then translated into proteins. Proteins
make many of the structures and all the enzymes in a cell or organism.
Several steps in the gene expression process may be modulated (tuned).
This includes both the transcription and translation stages, and the final
folded state of a protein. Gene regulation switches genes on and off, and
so controls cell differentiation, and morphogenesis. Gene regulation may
also serve as a basis for evolutionary change: control of the timing,
location, and amount of gene expression can have a profound effect on
the development of the organism. The expression of a gene may vary a lot
in different tissues. This is called pleiotropism, a widespread
phenomenon in genetics.
Alternative splicing is a modern discovery of great importance. It is a
process where from a single gene a large number of variant proteins can
be assembled. One particularDrosophila gene (DSCAM) can be
alternatively spliced into 38,000 different mRNA.[28]
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DN
The structure of part of a DNA double helix
Chemical structure of DNA. The phosphate groups are yellow, the deoxyribonucleicsugars are
orange, and
thenitrogen bases are green,purple,pink, and blue.
Theatoms shown are:
P=phosphorus O=oxygenN=nitrogen H=hydrogen
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DNA being copied
DNA, short for deoxyribonucleic acid, is the molecule that contains
the genetic code of organisms. This
includes animals, plants, protists,archaea and bacteria.
DNA is in each cell in the organism and tells cells what proteins to make.
Mostly, these proteins are enzymes. DNA is inherited by children from
their parents. This is why children share traits with their parents, such as
skin, hair and eye color. The DNA in a person is a combination of the
DNA from each of their parents.
Part of an organism's DNA is "non-coding DNA" sequences. They do not
code for protein sequences. Some noncoding DNA is transcribed intononcoding RNA molecules, such as transfer RNA, ribosomal RNA,
and regulatory RNAs. Other sequences are not transcribed at all, or give
rise to RNA of unknown function. The amount of non-coding DNA
varies greatly among species. For example, over 98% of the human
genome is non-coding DNA,[1] while only about 2% of a
typical bacterial genome is non-coding DNA.
Viruses use either DNA
or RNA to infect organisms.[2] The genome replication of most DNA
viruses takes place in the cell's nucleus, whereas RNA viruses usually
replicate in the cytoplasm.
Contents
[hide]

1Structure of DNA
o
1.1Nucleotides
‫ وفاء عبد الواحد‬.‫د‬
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o

1.2Chromatin
2Copying DNA
o
2.1Mutations

3Protein synthesis

4History of DNA research

5Related pages

6References

7Other websites
Structure of DNA
DNA has a double helix shape, which is like a ladder twisted into a spiral.
Each step of the ladder is a pair of nucleotides.
Nucleotides
A nucleotide is a molecule made up of:

deoxyribose, a kind of sugar with 5 carbon atoms,

a phosphate group made of phosphorus and oxygen, and

nitrogenous base
DNA is made of four types of nucleotide:

Adenine (A)

Thymine (T)

Cytosine (C)

Guanine (G)
The 'rungs' of the DNA ladder are each made of two bases, one base
coming from each leg. The bases connect in the middle: 'A' only pairs
with 'T', and 'C' only pairs with 'G'. The bases are held together
by hydrogen bonds.
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Adenine (A) and thymine (T) can pair up because they make two
hydrogen bonds, and cytosine (C) and guanine (G) pair up to make three
hydrogen bonds. Although the bases are always in fixed pairs, the pairs
can come in any order (A-T or T-A; similarly, C-G or G-C). This way,
DNA can write 'codes' out of the 'letters' that are the bases. These codes
contain the message that tells the cell what to do.
Chromatin
On chromosomes, the DNA is bound up with proteins called histones to
form chromatin. This association takes part in epigenetics and gene
regulation. Genes are switched on and off during development and cell
activity, and this regulation is the basis of most of the activity which takes
place in cells.
Copying DNA
When DNA is copied this is called DNA replication. Briefly, the
hydrogen bonds holding together paired bases are broken and the
molecule is split in half: the legs of the ladder are separated. This gives
two single strands. New strands are formed by matching the bases (A
with T and G with C) to make the missing strands.
First, an enzyme called DNA helicase splits the DNA down the middle by
breaking the hydrogen bonds. Then after the DNA molecule is in two
separate pieces, another molecule called DNA polymerase makes a new
strand that matches each of the strands of the split DNA molecule. Each
copy of a DNA molecule is made of half of the original (starting)
molecule and half of new bases.
Mutations
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When DNA is copied, mistakes are sometimes made – these are
called mutations. There are three main types of mutations:

Deletion, where one or more bases are left out.

Substitution, where one or more bases are substituted for another base
in the sequence.

Insertion, where one or more extra base is put in.

Duplication, where a sequence of bases pairs are repeated.
Mutations may also be classified by their effect on the structure and
function of proteins, or their effect on fitness. Mutations may be bad for
the organism, or neutral, or of benefit. Sometimes mutations are fatal for
the organism – the protein made by the new DNA does not work at all,
and this causes the embryo to die. On the other hand, evolution is moved
forward by mutations, when the new version of the protein works better
for the organism.
Protein synthesis
A section of DNA that contains instructions to make a protein is called
a gene. Each gene has the sequence for at least
one polypeptide.[3] Proteins form structures, and also form enzymes. The
enzymes do most of the work in cells. Proteins are made out of
smaller polypeptides, which are formed of amino acids. To make a
protein to do a particular job, the correct amino acids have to be joined up
in the correct order.
Proteins are made by tiny machines in the cell called ribosomes.
Ribosomes are in the main body of the cell, but DNA is only in the
nucleus of the cell. The codon is part of the DNA, but DNA never leaves
the nucleus. Because DNA cannot leave the nucleus, the cell makes a
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copy of the DNA sequence in RNA. This is smaller and can get through
the holes – pores – in the membrane of the nucleus and out into the cell.
Genes encoded in DNA are transcribed into messenger RNA (mRNA) by
proteins such as RNA polymerase. Mature mRNA is then used as
a template for protein synthesis by the ribosome. Ribosomes read codons,
'words' made of three base pairs that tell the ribosome which amino
acid to add. The ribosome scans along an mRNA, reading the code while
it makes protein. Another RNA called tRNA helps match the right amino
acid to each codon
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History of DNA research
James D. Watson and Francis Crick (right), with Maclyn McCarty (left)
DNA was first isolated (extracted from cells)
by Swiss physician Friedrich Miescher in 1869, when he was working on
bacteria from the pus in surgical bandages. The molecule was found in
the nucleus of the cells and so he called it nuclein.[4]
In 1928, Frederick Griffith discovered that traits of the "smooth" form
of Pneumococcus could be transferred to the "rough" form of the same
bacteria by mixing killed "smooth" bacteria with the live "rough"
form.[5] This system provided the first clear suggestion that DNA carries
genetic information.
The Avery–MacLeod–McCarty experiment identified DNA as
the transforming principle in 1943.[6][7]
DNA's role in heredity was confirmed in 1952, when Alfred
Hershey and Martha Chase in the Hershey–Chase experiment showed
that DNA is the genetic material of the T2 bacteriophage.[8]
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‫المحاضرة االولى والثانية‬
In the 1950s, Erwin Chargaff [9] found that the amount of thymine (T)
present in a molecule of DNA was about equal to the amount of adenine
(A) present. He found that the same applies to guanine (G) and cytosine
(C). Chargaff's rules summarises this finding.
In 1953, James D. Watson and Francis Crick suggested what is now
accepted as the first correct double-helix model of DNA structure in the
journal Nature.[10] Their double-helix, molecular model of DNA was then
based on a single X-ray diffraction image "Photo 51", taken by Rosalind
Franklin and Raymond Gosling in May 1952.[11]
Experimental evidence supporting the Watson and Crick model was
published in a series of five articles in the same issue of Nature.[12] Of
these, Franklin and Gosling's paper was the first publication of their own
X-ray diffraction data and original analysis method that partly supported
the Watson and Crick model;[13] this issue also contained an article on
DNA structure by Maurice Wilkins and two of his colleagues, whose
analysis and in vivo B-DNA X-ray patterns also supported the presence in
vivo of the double-helical DNA configurations as proposed by Crick and
Watson for their double-helix molecular model of DNA in the previous
two pages of Nature. In 1962, after Franklin's death, Watson, Crick, and
Wilkins jointly received the Nobel Prize in Physiology or
Medicine.[14] Nobel Prizes were awarded only to living recipients at the
time. A debate continues about who should receive credit for the
discovery.[15]
In 1957, Crick explained the relationship between DNA, RNA, and
proteins, in the central dogma of molecular biology.[16]
How DNA was copied (the replication mechanism) came in 1958 through
the Meselson–Stahl experiment.[17] More work by Crick and coworkers
showed that the genetic code was based on non-overlapping triplets of
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bases, called codons.[18] These findings represent the birth of molecular
biology.
How Watson and Crick got Franklin's results has been much debated.
Crick, Watson and Maurice Wilkins were awarded the Nobel Prize in
1962 for their work on DNA – Rosalind Franklin had died in 1958.
DNA replication
From Wikipedia, the free encyclopedia
DNA replication. The double helix is unwound and each strand acts as a template (blue) for the
next strand. Bases are matched to synthesize the new partner strands (green).
In molecular biology, DNA replication is the biological process of
producing two identical replicas of DNA from one
original DNA molecule. This process occurs in all living organisms and is
the basis for biological inheritance. DNA is made up of a double helix of
two complementary strands. During replication, these strands are
separated. Each strand of the original DNA molecule then serves as a
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template for the production of its counterpart, a process referred to
as semiconservative replication. Cellular proofreading and error-checking
mechanisms ensure near perfect fidelity for DNA replication.[1][2]
In a cell, DNA replication begins at specific locations, or origins of
replication, in the genome.[3] Unwinding of DNA at the origin and
synthesis of new strands results in replication forks growing bidirectionally 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 addingnucleotides that complement each (template) strand.
DNA replication occurs during the S-stage of interphase.
DNA replication can also be performed in vitro (artificially, outside a
cell). DNA polymerases isolated from cells and artificial DNA primers
can be used to initiate DNA synthesis at known sequences in a template
DNA molecule. The polymerase chain reaction (PCR), a common
laboratory technique, cyclically applies such artificial synthesis to
amplify a specific target DNA fragment from a pool of DNA.
DNA structures[edit]
DNA usually exists as a double-stranded structure, with both strands
coiled together to form the characteristic double-helix. Each single strand
of DNA is a chain of four types ofnucleotides. Nucleotides in DNA
contain a deoxyribose sugar, a phosphate, and a nucleobase. The four
types of nucleotide correspond to the
four nucleobases adenine,cytosine, guanine, and thymine, commonly
abbreviated as A,C, G and T. Adenine and guanine are purine bases,
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‫المحاضرة االولى والثانية‬
while cytosine and thymine are pyrimidines. These nucleotides
form phosphodiester bonds, creating the phosphate-deoxyribose
backbone of the DNA double helix with the nuclei bases pointing inward
(i.e., toward the opposing strand). Nucleotides (bases) are matched
between strands through hydrogen bonds to form base pairs. Adenine
pairs with thymine (two hydrogen bonds), and guanine pairs with
cytosine (stronger: three hydrogen bonds).
DNA strands have a directionality, and the different ends of a single
strand are called the "3' (three-prime) end" and the "5' (five-prime) end".
By convention, if the base sequence of a single strand of DNA is given,
the left end of the sequence is the 5' end, while the right end of the
sequence is the 3' end. The strands of the double helix are anti-parallel
with one being 5' to 3', and the opposite strand 3' to 5'. These terms refer
to the carbon atom in deoxyribose to which the next phosphate in the
chain attaches. Directionality has consequences in DNA synthesis,
because DNA polymerase can synthesize DNA in only one direction by
adding nucleotides to the 3' end of a DNA strand.
The pairing of complementary bases in DNA (through hydrogen bonding)
means that the information contained within each strand is redundant.
Phosphodiester (intra-strand) bonds are stronger than hydrogen (interstrand) bonds. This allows the strands to be separated from one another.
The nucleotides on a single strand can therefore be used to reconstruct
nucleotides on a newly synthesized partner strand.[4]
DNA polymerase[edit]
Main article: DNA polymerase
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DNA polymerases adds nucleotides to the 3' end of a strand of DNA.[5] If
a mismatch is accidentally incorporated, the polymerase is inhibited from
further extension. Proofreading removes the mismatched nucleotide and
extension continues.
DNA polymerases are a family of enzymes that carry out all forms of
DNA replication.[6] 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
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‫المحاضرة االولى والثانية‬
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.[Note 1]
In general, DNA polymerases are highly accurate, with an intrinsic error
rate of less than one mistake for every 107 nucleotides added.[7] 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.[7]
The rate of DNA replication in a living cell was first measured as the rate
of phage T4 DNA elongation in phage-infected E. coli.[8]During the
period of exponential DNA increase at 37 °C, the rate was 749
nucleotides per second. The mutation rate per base pair per replication
during phage T4 DNA synthesis is 1.7 per 108.[9]
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Replication process[edit]
Main articles: Prokaryotic DNA replication and Eukaryotic DNA
replication
DNA replication, like all biological polymerization processes, proceeds in
three enzymatically catalyzed and coordinated steps: initiation, elongation
and termination.
Initiation[edit]
Role of initiators for initiation of DNA replication.
Formation of pre-replication complex.
For a cell to divide, it must first replicate its DNA.[10] This process is
initiated at particular points in the DNA, known as "origins", which are
targeted by initiator proteins.[3] In E. coli this protein is DnaA; in yeast,
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this is the origin recognition complex.[11] 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 decouple.[12] Once the origin
has been located, these initiators recruit other proteins and form the prereplication complex, which unzips the double-stranded DNA.
Elongation[edit]
DNA polymerase has 5'-3' activity. All known DNA replication systems
require a free 3' hydroxyl group before synthesis can be initiated
(Important note: the DNA template is read in 3' to 5' direction whereas a
new strand is synthesized in the 5' to 3' direction—this is often confused).
Four distinct mechanisms for initiation of 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
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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 atyrosine 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.
The primase used in this process differs significantly
between bacteria and archaea/eukaryotes. Bacteria use a primase
belonging to the DnaG protein superfamily which contains a catalytic
domain of the TOPRIM fold type.[13] The TOPRIM fold contains an α/β
core with four conserved strands in a Rossmann-like topology. This
structure is also found in the catalytic domains of topoisomerase Ia,
topoisomerase II, the OLD-family nucleases and DNA repair proteins
related to the RecR protein.
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‫المحاضرة االولى والثانية‬
The primase used by archaea and eukaryotes, in contrast, contains a
highly derived version of the RNA recognition motif (RRM). This
primase is structurally similar to many viral RNA-dependent RNA
polymerases, reverse transcriptases, cyclic nucleotide generating cyclases
and DNA polymerases of the A/B/Y families that are involved in DNA
replication and repair. In eukaryotic replication, the primase forms a
complex with Pol α.[14]
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.
In eukaryotes, the low-processivity enzyme, Pol α, helps to initiate
replication. The high-processivity extension enzymes are Pol δ and Pol ε.
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: θ). In contrast, eukaryotes have longer linear
chromosomes and initiate replication at multiple origins within these.>[15]
Replication fork[edit]
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‫المحاضرة االولى والثانية‬
Scheme of the replication fork.
a: template, b: leading strand, c: lagging strand, d: replication fork, e:
primer, f:Okazaki fragments
Many enzymes are involved in the DNA replication fork.
The replication fork is a structure that forms within the nucleus during
DNA replication. It is created by helicases, which break the hydrogen
bonds holding the two DNA strands together. 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 always 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 nascent
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‫المحاضرة االولى والثانية‬
(new) lagging strand DNA, whose direction of synthesis is opposite to the
direction of the growing replication fork.
Leading strand[edit]
The leading strand is the strand of nascent DNA which is being
synthesized in the same direction as the growing replication fork. A
polymerase "reads" the leading strand template and adds
complementary nucleotides to the nascent leading strand on a continuous
basis.
The polymerase involved in leading strand synthesis is DNA polymerase
III (DNA Pol III) in prokaryotes.[16] In eukaryotes, leading strand
synthesis is thought to be conducted by Pol ε; however, this view has
recently been challenged, suggesting a role for Pol δ.[17]
Lagging strand[edit]
The lagging strand is the strand of nascent 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. As a consequence,
the DNA polymerase on this strand is seen to "lag behind" the other
strand.
The lagging strand is synthesized in short, separated segments. On the
lagging strand template, a primase "reads" the template DNA and initiates
synthesis of a short complementary RNA primer. A DNA polymerase
extends the primed segments, forming Okazaki fragments. The RNA
primers are then removed and replaced with DNA, and the fragments of
DNA are joined together by DNA ligase.
DNA polymerase III (in prokaryotes) or Pol δ (in eukaryotes) is
responsible for extension of the primers added during replication of the
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lagging strand. Primer removal is performed by DNA polymerase I (in
prokaryotes) and Pol δ (in eukaryotes).[18] Eukaryotic primase is intrinsic
to Pol α.[19] In eukaryotes, pol ε helps with repair during DNA replication.
Dynamics at the replication fork[edit]
The assembled human DNA clamp, a trimer of the protein PCNA.
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.[20] 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.[21]
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.[22]
Clamp proteins form a sliding clamp around DNA, helping the DNA
polymerase maintain contact with its template, thereby assisting with
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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.[2]:274-5
DNA replication proteins[edit]
At the replication fork, many replication enzymes assemble on the DNA
into a complex molecular machine called the replisome. The following is
a list of major DNA replication enzymes that participate in the
replisome:[23]
Enzyme
DNA Helicase
Function in DNA replication
Also known as helix destabilizing enzyme. Unwinds
the DNA double helix at the Replication Fork.
Builds a new duplex DNA strand by adding
nucleotides in the 5' to 3' direction. Also performs
DNA Polymerase
proof-reading and error correction. There exist many
different types of DNA Polymerase, each of which
perform different functions in different types of cells.
A protein which prevents elongating DNA
DNA clamp
polymerases from dissociating from the DNA parent
strand.
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‫المحاضرة االولى والثانية‬
Single-Strand
Binding (SSB)
Proteins
Topoisomerase
DNA Gyrase
DNA Ligase
Bind to ssDNA and prevent the DNA double helix
from re-annealing after DNA helicase unwinds it,
thus maintaining the strand separation, and
facilitating the synthesis of the nascent strand.
Relaxes the DNA from its super-coiled nature.
Relieves strain of unwinding by DNA helicase; this
is a specific type of topoisomerase
Re-anneals the semi-conservative strands and
joins Okazaki Fragments of the lagging strand.
Provides a starting point of RNA (or DNA) for DNA
Primase
polymerase to begin synthesis of the new DNA
strand.
Lengthens telomeric DNA by adding repetitive
Telomerase
nucleotide sequences to the ends of eukaryotic
chromosomes. This allows germ cells and stem cells
to avoid the Hayflick limit on cell division.[24]
Replication machinery[edit]
Replication machineries consist of factors involved in DNA replication
and appearing on template ssDNAs. Replication machineries include
primosotors are replication enzymes; DNA polymerase, DNA helicases,
DNA clamps and DNA topoisomerases, and replication proteins; e.g.
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single-stranded DNA binding proteins (SSB). In the replication
machineries these components coordinate. In most of the bacteria, all of
the factors involved in DNA replication are located on replication forks
and the complexes stay on the forks during DNA replication. These
replication machineries are called replisomes or DNA replicase systems.
These terms are generic terms for proteins located on replication forks. In
eukaryotic and some bacterial cells the replisomes are not formed.
Since replication machineries do not move relatively to template DNAs
such as factories, they are called a replication factory.[25] In an
alternative figure, DNA factories are similar to projectors and DNAs are
like as cinematic films passing constantly into the projectors. In the
replication factory model, after both DNA helicases for leading stands
and lagging strands are loaded on the template DNAs, the helicases run
along the DNAs into each other. The helicases remain associated for the
remainder of replication process. Peter Meister et al. observed directly
replication sites in budding yeast by monitoring green fluorescent
protein(GFP)-tagged DNA polymerases α. They detected DNA
replication of pairs of the tagged loci spaced apart symmetrically from a
replication origin and found that the distance between the pairs decreased
markedly by time.[26] This finding suggests that the mechanism of DNA
replication goes with DNA factories. That is, couples of replication
factories are loaded on replication origins and the factories associated
with each other. Also, template DNAs move into the factories, which
bring extrusion of the template ssDNAs and nascent DNAs. Meister’s
finding is the first direct evidence of replication factory model.
Subsequent research has shown that DNA helicases form dimers in many
eukaryotic cells and bacterial replication machineries stay in single
intranuclear location during DNA synthesis.[25]
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The replication factories perform disentanglement of sister chromatids.
The disentanglement is essential for distributing the chromatids into
daughter cells after DNA replication. Because sister chromatids after
DNA replication hold each other by Cohesin rings, there is the only
chance for the disentanglement in DNA replication. Fixing of replication
machineries as replication factories can improve the success rate of DNA
replication. If replication forks move freely in chromosomes, catenation
of nuclei is aggravated and impedes mitotic segregation.[26]
Termination[edit]
Eukaryotes initiate DNA replication at multiple points in the
chromosome, so replication forks meet and terminate at many points in
the chromosome; these are not known to be regulated in any particular
way. Because eukaryotes have linear chromosomes, DNA replication is
unable to reach the very end of the chromosomes, but ends at
the telomereregion of repetitive DNA close to the ends. This shortens the
telomere of the daughter DNA strand. Shortening of the telomeres is a
normal process in somatic cells. 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
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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 through the
use of 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.[27]
Regulation[edit]
The cell cycle of eukaryotic cells.
Eukaryotes[edit]
Within eukaryotes, DNA replication is controlled within the context of
the cell cycle. As the cell grows and divides, it progresses through stages
in the cell cycle; DNA replication takes place during the S phase
(synthesis phase). The progress of the eukaryotic cell through the cycle is
controlled by cell cycle checkpoints. Progression through checkpoints is
controlled through complex interactions between various proteins,
including cyclins and cyclin-dependent kinases.[28] Unlike bacteria,
eukaryotic DNA replicates in the confines of the nucleus.[29]
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The G1/S checkpoint (or restriction checkpoint) regulates whether
eukaryotic cells enter the process of DNA replication and subsequent
division. Cells that do not proceed through this checkpoint remain in the
G0 stage and do not replicate their DNA.
Replication of chloroplast and mitochondrial genomes occurs
independently of the cell cycle, through the process of D-loop replication.
Replication focus[edit]
In vertebrate cells, replication sites concentrate into positions
called replication foci.[26] Replication sites can be detected by
immunostaining daughter strands and replication enzymes and
monitoring GFP-tagged replication factors. By these methods it is found
that replication foci of varying size and positions appear in S phase of cell
division and their number per nucleus is far smaller than the number of
genomic replication forks.
P. Heun et al.(2001) tracked GFP-tagged replication foci in budding yeast
cells and revealed that replication origins move constantly in G1 and S
phase and the dynamicsdecreased significantly in S
phase.[26] Traditionally, replication sites were fixed on spatial structure of
chromosomes by nuclear matrix or lamins. The Heun’s results denied the
traditional concepts, budding yeasts don't have lamins, and support that
replication origins self-assemble and form replication foci.
By firing of replication origins, controlled spatially and temporally, the
formation of replication foci is regulated. D. A. Jackson et al.(1998)
revealed that neighboring origins fire simultaneously in mammalian
cells.[26] Spatial juxtaposition of replication sites brings clustering of
replication forks. The clustering do rescue of stalled replication
forks and favors normal progress of replication forks. Progress of
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replication forks is inhibited by many factors; collision with proteins or
with complexes binding strongly on DNA, deficiency of dNTPs, nicks on
template DNAs and so on. If replication forks stall and the remaining
sequences from the stalled forks are not replicated, the daughter strands
have nick obtained un-replicated sites. The un-replicated sites on one
parent's strand hold the other strand together but not daughter strands.
Therefore, the resulting sister chromatids cannot separate from each other
and cannot divide into 2 daughter cells. When neighboring origins fire
and a fork from one origin is stalled, fork from other origin access on an
opposite direction of the stalled fork and duplicate the un-replicated sites.
As other mechanism of the rescue there is application of dormant
replication origins that excess origins don't fire in normal DNA
replication.
Bacteria[edit]
Dam methylates adenine of GATC sites after replication.
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‫المحاضرة االولى والثانية‬
Most bacteria do not go through a well-defined cell cycle but instead
continuously copy their DNA; during rapid growth, this can result in the
concurrent occurrence of multiple rounds of replication.[30] In E. coli, the
best-characterized bacteria, DNA replication is regulated through several
mechanisms, including: the hemimethylation and sequestering of the
origin sequence, the ratio of adenosine triphosphate (ATP) toadenosine
diphosphate (ADP), and the levels of protein DnaA. All these control the
binding of initiator proteins to the origin sequences.
Because E. coli methylates GATC DNA sequences, DNA synthesis
results in hemimethylated sequences. This hemimethylated DNA is
recognized by the protein SeqA, which binds and sequesters the origin
sequence; in addition, DnaA (required for initiation of replication) binds
less well to hemimethylated DNA. As a result, newly replicated origins
are prevented from immediately initiating another round of DNA
replication.[31]
ATP builds up when the cell is in a rich medium, triggering DNA
replication once the cell has reached a specific size. ATP competes with
ADP to bind to DnaA, and the DnaA-ATP complex is able to initiate
replication. A certain number of DnaA proteins are also required for
DNA replication — each time the origin is copied, the number of binding
sites for DnaA doubles, requiring the synthesis of more DnaA to enable
another initiation of replication.
Polymerase chain reaction[edit]
Main article: Polymerase chain reaction
Researchers commonly replicate DNA in vitro using the polymerase
chain reaction (PCR). PCR uses a pair of primers to span a target region
in template DNA, and then polymerizes partner strands in each direction
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from these primers using a thermostable DNA polymerase. Repeating this
process through multiple cycles amplifies the targeted DNA region. At
the start of each cycle, the mixture of template and primers is heated,
separating the newly synthesized molecule and template. Then, as the
mixture cools, both of these become templates for annealing of new
primers, and the polymerase extends from these. As a result, the number
of copies of the target region doubles each round, increasing
exponentially.[32]
Notes[edit]
1. Jump up^ The energetics of this process may also help explain
the directionality of synthesis—if DNA were synthesized in the 3'
to 5' direction, the energy for the process would come from the 5'
end of the growing strand rather than from free nucleotides. The
problem is that if the high energy triphosphates were on the
growing strand and not on the free nucleotides, proof-reading by
removing a mismatched terminal nucleotide would be problematic:
Once a nucleotide is added, the triphosphate is lost and a single
phosphate remains on the backbone between the new nucleotide
and the rest of the strand. If the added nucleotide were
mismatched, removal would result in a DNA strand terminated by
a monophosphate at the end of the "growing strand" rather than a
high energy triphosphate. So strand would be stuck and wouldn't
be able to grow anymore. In actuality, the high energy
triphosphates hydrolyzed at each step originate from the free
nucleotides, not the polymerized strand, so this issue does not
exist.
References[edit]
‫ وفاء عبد الواحد‬.‫د‬
‫المحاضرة االولى والثانية‬
1. Jump up^ Imperfect DNA replication results in mutations. Berg
JM, Tymoczko JL, Stryer L, Clarke ND (2002). "Chapter 27: DNA
Replication, Recombination, and Repair". Biochemistry. W.H.
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Walter P (2002). "5DNA Replication, Repair, and
Recombination". Molecular Biology of the Cell. Garland
Science. ISBN 0-8153-3218-1. External link in |chapter= (help)
3. ^ Jump up to:a b Berg JM, Tymoczko JL, Stryer L, Clarke ND
(2002). "Chapter 27, Section 4: DNA Replication of Both Strands
Proceeds Rapidly from Specific Start Sites". Biochemistry. W.H.
Freeman and Company. ISBN 0-7167-3051-0. External link
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4. Jump up^ Alberts, B., et al., Molecular Biology of the
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5. Jump up^ Allison, Lizabeth A. Fundamental Molecular Biology.
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6. Jump up^ Berg JM, Tymoczko JL, Stryer L, Clarke ND
(2002). Biochemistry. W.H. Freeman and Company. ISBN 0-71673051-0. Chapter 27, Section 2: DNA Polymerases Require a
Template and a Primer
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translesion synthesis polymerases". Cell Research. 18 (1): 148–
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‫ وفاء عبد الواحد‬.‫د‬
‫المحاضرة االولى والثانية‬
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Mutation. Holden-Day, San Francisco ISBN 0816224501 ISBN
978-0816224500
10.Jump up^ Alberts B, Johnson A, Lewis J, Raff M, Roberts K,
Walter P (2002). Molecular Biology of the Cell. Garland
Science. ISBN 0-8153-3218-1. Chapter 5: DNA Replication
Mechanisms
11.Jump up^ Weigel C, Schmidt A, Rückert B, Lurz R, Messer W
(November 1997). "DnaA protein binding to individual DnaA
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Baltimore D, Darnell J (2000). Molecular Cell Biology. W. H.
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Features of Chromosomal Replication: Three Common Features of
Replication Origins
13.Jump up^ Aravind, L.; Leipe, D. D.; Koonin, E. V.
(1998). "Toprim—a conserved catalytic domain in type IA and II
topoisomerases, DnaG-type primases, OLD family nucleases and
RecR proteins". Nucleic Acids Research. 26 (18): 4205–
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‫المحاضرة االولى والثانية‬
4213. doi:10.1093/nar/26.18.4205.PMC 147817
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14.Jump up^ Frick, David; Richardson, Charles (July 2001). "DNA
Primases". Annual Review of Biochemistry. 70: 39–
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15.Jump up^ Huberman, Joel A.; Riggs, Arthur D. (March 1968).
"On the mechanism of DNA replication in mammalian
chromosomes". Journal of Molecular Biology. 32 (2): 327–
341.doi:10.1016/0022-2836(68)90013-2. PMID 5689363.
16.Jump up^ Johnson, RE; Klassen, R; Prakash, L; Prakash, S (July
2015). "A Major Role of DNA Polymerase δ in Replication of Both
the Leading and Lagging DNA Strands.". Molecular Cell. 59 (2):
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17.Jump up^ Stillman, Bruce (July 2015). "Reconsidering DNA
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18.Jump up^ Distinguishing the pathways of primer removal during
Eukaryotic Okazaki fragment maturation Contributor Author
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Date Available: 2009-02-23T17:05:09Z. Date Issued: 2009-0223T17:05:09Z. Identifier Uri: http://hdl.handle.net/1802/6537.
Description: Dr. Robert A. Bambara, Faculty Advisor. Thesis
(PhD) – School of Medicine and Dentistry, University of
Rochester. UR only until January 2010. UR only until January
2010.
‫ وفاء عبد الواحد‬.‫د‬
‫المحاضرة االولى والثانية‬
19.Jump up^ Barry, Elizabeth R.; Bell, Stephen D. (8 December
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20.Jump up^ Alberts B, Johnson A, Lewis J, Raff M, Roberts K,
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DNA Topoisomerases Prevent DNA Tangling During Replication
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(26 September 2008). "DNA Gyrase: Structure and
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‫المحاضرة االولى والثانية‬
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2. Eukaryotes vs Prokaryotes
3. There is much conservation between the two systems, in as much
as the enzymology, the replication fork geometry, the basic
fundamental features and the use of multi-protein machinery are all
very much the same in both. However, there are more protein
components in the Eukaryotic replication machinery. In
prokaryotes, the replication form moves 10x faster than in
eukaryotes. Prokaryotic replication Eukaryotic replication
semiconservative replication semiconservative replication single
origin replication (oriC) multiple origins of replication (ARS)
primer synthesized by primase primer synthesized by subunits of
DNA polymerase α processing enzyme: DNA polymerase III
processing enzymes: DNA polymerases α and δ removal of primer:
DNA polymerase I removal of primer: DNA polymerase β DNA
free in cytoplasm as nucleoid chromatin structure, chromosomes,
histones circular DNA linear DNA: problem of replication of
chromosome ends → telome