Download DNA Replication

Molecular Genetics
In eukaryotes, chromosomes bear the genetic information that is passed from
parents to offspring.
The genetic information is stored in molecules of DNA. The DNA, in turn,
codes for enzymes, which, in turn, regulate chemical reactions that direct
metabolism for cell development, growth, and maintenance.
The underlying molecular mechanisms that interpret the information
in DNA to generate these effects is the subject of this section.
The structure of DNA and RNA was presented earlier in the chapter on
chemistry. As a review, both DNA and RNA are polymers of nucleotides.
The nucleotide monomer consists of three parts :
- a nitrogen base
- a sugar
- a phosphate.
1
The Differences in the Structures of DNA and RNA and Summarizes Their
Functions.
Cloverleaf
‫شكل ورقة البرسيم‬
2
3
Thymine instead of
Uracil
4
Uracil instead of
Thymine
DNA Replication
During interphase of the cell cycle, a second chromatid containing a copy
of the DNA molecule is assembled.
(RNA)
(DNA)
a. A chromosome prior to replication contains a single DNA molecule;
b. A chromosome that has been replicated and consists of two chromatids,
each comprising a single DNA double helix molecule.
5
DNA Replication
This process, called DNA
replication, involves separating
(“unzipping”) the DNA molecule
into two strands, each of which
serves as a template to assemble
a new, complementary strand.
The result is two identical
double-stranded molecules of
DNA.
Because each of these doublestranded molecules of DNA
consists of a single strand of old
DNA (the template strand) and a
single strand of new, replicated
DNA (the complementary strand),
the process is called:
semiconservative replication.
6
Semiconservative Replication
7
During DNA replication, the enzyme helicase unwinds the DNA helix,
forming a Y-shaped replication fork.
Single-stranded DNA binding proteins attach to each strand of the
uncoiled
DNA to keep them separate.
8
As helicase unwinds the DNA, it forces the double-helix in front of it to
twist. A group of enzymes, called topoisomerases, break and rejoin the
double helix, allowing the twists to unravel and preventing the formation of
knots.
9
10
Since a DNA double-helix molecule
consists of two opposing DNA strands,
the uncoiled DNA consists of a 3' → 5'
template strand and a 5' → 3' template
strand.
11
The enzyme that assembles the new DNA strand, DNA polymerase,
moves in the 3' → 5' direction along each template strand. The new
(complement) strands grow in the antiparallel, 5' → 3' direction.
12
For the 3' → 5' template strand, replication occurs readily as the DNA
polymerase follows the replication fork, assembling a 5' → 3'
complementary strand. This complementary strand is called the
leading strand.
13
For the 5' → 3' template strand, however, the DNA polymerase moves away from the
uncoiling replication fork.
This is because it can assemble nucleotides only as it travels in the 3' → 5‘ direction. As
the helix is uncoiled, DNA polymerase assembles short segments of nucleotides along
the template strand in the direction away from the replication fork.
After each complement segment is assembled, the DNA polymerase must return back to
the replication fork to begin assembling the next segment.
These short segments of complementary DNA are called Okazaki segments. The
Okazaki segments are connected by DNA ligase, producing a single complement strand.
Because this complementary strand requires more time to assemble than the leading
strand, it is called the lagging strand.
14
DNA polymerase can append
nucleotides only to an
already existing
complementary strand.
The first nucleotides of the
leading strand and each
Okazaki fragment are
initiated by RNA primase
and other proteins.
RNA primase initiates each
complementary segment with
RNA (not DNA) nucleotides
which serve as an RNA
primer for DNA polymerase
to append succeeding DNA
nucleotides.
Later, the RNA nucleotides
are replaced with appropriate
DNA nucleotides.
15
The figure illustrates
the growth of leading
and lagging DNA
complements.
In the figure, the RNA
primer that initiated
the leading strand is
not shown because it
was replaced with
DNA nucleotides
earlier in its synthesis.
The Okazaki fragment
of the lagging strand,
however, still have its
RNA primer attached,
because a primer
must initiate each new
fragment.
16
1. Helicase unwinds the
DNA, producing a
replication fork. Singlestranded DNA binding
proteins prevent the
single strands of DNA
from recombining.
Topoisomerase removes
twists and knots that form
in the double-stranded
template as a result of the
unwinding induced by
helicase. (See 1A, 1B, 1C
and 1D in Figure).
17
The details of DNA replication are
summarized below. Numbers correspond to
events illustrated in Figure below:
2. RNA primase initiates DNA replication at special nucleotide sequences
(called origins of replication) with short segments of RNA nucleotides
(called RNA primers) (see 2A and 2B in Figure).
18
3. DNA polymerase attaches to the RNA primers and begins elongation,
the adding of DNA nucleotides to the complement strand.
19
• 4. The leading complementary strand is assembled continuously as the
double-helix DNA uncoils.
20
5. The lagging complementary strand is assembled in short Okazaki
fragments, which are subsequently joined by DNA ligase.
(see 5A, 5B, and 5C in Figure).
21
6. The RNA primers are replaced by DNA nucleotides.
22
• Energy for elongation is provided by two additional phosphates that are
attached to each new nucleotide (making a total of three phosphates
attached to the nitrogen base).
• Breaking the bonds holding the two extra phosphates provides the
chemical energy for the process.
23
Mutations
The replication process of DNA is extremely accurate. In
bacteria, the DNA polymerase proofreads the pairing process
by checking the newly attached nucleotide to confirm that it is
correct.
If it is not, the polymerase removes the incorrect nucleotide,
backs up, and attaches a new nucleotide. If a mismatch
should escape the proofreading ability of the DNA
polymerase, other, mismatch repair, enzymes will correct the
error. Repair mechanisms occur in eukaryotic cells as well but
are not well understood.
24
Radiation (such as ultraviolet and
x-ray) and various reactive
chemicals can cause errors in DNA
molecules.
One kind of DNA error occurs when
the bases of two adjacent
nucleotides in one DNA strand
bond to each other rather than
make proper pairs with nucleotides
in the complementary DNA strand.
A thymine dimer, for example,
originates when two adjacent
thymine nucleotides in the same
strand base-pair with each other
instead of with the adenine bases in
• the complementary strand. Such
errors can be fixed by excision
repair enzymes that splice out the
error and use the complementary
strand as a pattern, or template, for
replacing
the excised nucleotides.
25
If a DNA error is not repaired, it becomes a mutation.
A mutation is any sequence of nucleotides in a DNA molecule that does
not exactly match the original DNA molecule from which it was copied.
Mutations include:
1. an incorrect nucleotide (substitution),
2. a missing nucleotide (deletion), or
3. an additional nucleotide not present in the original DNA molecule
(insertion).
When an insertion mutation occurs, it causes all the subsequent
nucleotides to be displaced one position, producing a frameshift
mutation.
Radiation or chemicals that cause mutations are called mutagens.
Carcinogens are mutagens that activate uncontrolled cell growth
(cancer).
26
Protein Synthesis
The DNA in chromosomes contains genetic instructions that regulate
development, growth, and the metabolic activities of cells.
The DNA instructions determine whether a cell will be that of a pea plant,
a human, or some other organism, as well as establish specific
characteristics of the cell in that organism.
For example, the DNA in a cell may establish that it is a human cell. If,
during development, it becomes a cell in the iris of an eye, the DNA will
direct other information appropriate for its location in the organism, such
as the production of brown, blue, or other pigmentation.
DNA controls the cell in this manner because it contains codes for
polypeptides.
Many polypeptides are enzymes that regulate chemical reactions, and
these chemical reactions influence the resulting characteristics of the cell.
27
In the study of heredity, the terms gene and genotype are used to
represent the genetic information for a particular trait.
From the molecular viewpoint, traits are the end products of metabolic
processes regulated by enzymes.
When this relationship between traits and enzymes was discovered, the
gene was defined as the segment of DNA that codes for a particular
enzyme (one-gene-one-enzyme hypothesis).
Since many genes code for polypeptides that are not enzymes (such as
structural proteins or individual components of enzymes), the gene has
been redefined as the DNA segment that codes for a particular polypeptide
(one-gene-one- polypeptide hypothesis).
28
The process that describes how enzymes
and other proteins are made from DNA is
called protein synthesis.
There are three steps in protein synthesis:
- Transcription,
- RNA Processing,
- Translation.
In transcription, RNA molecules are
created by using the DNA molecule as a
template.
After transcription, RNA processing
modifies the RNA molecule with deletions
and additions.
In translation, the processed RNA
molecules are used to assemble amino acids
29
into
a polypeptide.
There are three kinds of RNA molecules
produced during transcription, as
follows:
1. Messenger RNA (mRNA)
mRNA is a single strand of RNA that
provides the template used for
sequencing amino acids into a
polypeptide.
A triplet group of three adjacent
nucleotides on the mRNA, called a
codon, codes for one specific amino
acid. Since there are 64 possible ways
that four nucleotides can be arranged
in triplet combinations (4 × 4 × 4 = 64),
there are 64 possible codons.
However, there are only 20 amino
acids,
30
The genetic code, given in the
Figure, provides the “decoding” for
each codon.
That is, it identifies the amino acid
specified by each of the possible 64
codon combinations.
For example, the codon composed
of the three nucleotides cytosineguanine-adenine (CGA) codes for
the amino acid arginine.
This can be found in the Figure by
aligning the C found in the first
column with the G in the center part
of the table and the A in the column
at the far right.
31
2. Transfer RNA (tRNA)
tRNA is a short RNA molecule (consisting of about 80 nucleotides) that is
used for transporting amino acids to their proper place on the mRNA
template.
Interactions among various parts of the tRNA molecule result in basepairings between nucleotides, folding the tRNA in such a way that it forms
a three-dimensional molecule. (In two dimensions, a tRNA resembles the
three leaflets of a clover leaf.)
32
The 3' end of the tRNA (ending with cytosine-cytosine-adenine, or C-C-A-3')
attaches to an amino acid.
Another portion of the tRNA, specified by a triplet combination of
nucleotides, is the anticodon.
33
During translation, the anticodon of the tRNA base pairs with the codon of the mRNA.
Exact base-pairing between the third nucleotide of the tRNA anticodon and the third
nucleotide of the mRNA codon is often not required.
This “wobble” allows the anticodon of some tRNAs to base-pair with more than one kind
of codon. As a result, about 45 different tRNAs base-pair with the 64 different codons.
(look the table in slide 31)
Free amino acids
Growing Protein
chain
mRNA coping
DNA in nucleus
tRNA bringing amino
acid to ribosome
Ribosome incorporation
amino acid into the growing
protein chain
mRNA being translated
34
Anticodon
codon
35
• 3. Ribosomal RNA (rRNA)
• rRNA molecules are the building blocks of ribosomes. The nucleolus is an
assemblage of DNA actively being transcribed into rRNA. Within the
nucleolus, various proteins imported from the cytoplasm are assembled
with rRNA to form large and small ribosome subunits. Together, the two
subunits form a ribosome which coordinates the activities of the mRNA and
tRNA during translation.
• Each ribosome has a binding site for mRNA and three binding sites for
tRNA molecules.
– The P site holds the tRNA carrying the growing polypeptide chain.
– The A site carries the tRNA with the next amino acid.
– The E site exit site (Discharged tRNAs leave the ribosome at this site)
36
A) Transcription
Is the process of making RNA
from a DNA template
Transcription contain three steps:
- Initiation,
- Elongation,
-Termination.
The details follow, with
numbers corresponding to
events:
37
1. Initiation
In initiation, the RNA
polymerase attaches to
promoter regions on the
DNA and begins to unzip
the DNA into two strands.
A promoter region for
mRNA transcriptions
contains the sequence TA-T-A (called the TATA
box).
38
2. Elongation
Elongation occurs as the RNA polymerase unzips the DNA and
assembles RNA nucleotides using one strand of the DNA as a template.
As in DNA replication, elongation of the RNA molecule occurs in the 5' →
3' direction. In contrast to DNA replication, new nucleotides are RNA
nucleotides (rather than DNA nucleotides), and only one DNA strand is
transcribed.
39
3. Termination
Termination occurs when the RNA polymerase reaches a special
sequence of nucleotides that serve as a termination point. In
eukaryotes, the termination region often contains the DNA sequence
AAAAAAA.
40
B) RNA Processing
Before a mRNA molecule leaves
the nucleus, it undergoes two
kinds of alterations:
The first modification (alteration)
adds special nucleotide
sequences to both ends of the
mRNA (see 5 in the Figure). A
modified guanosine triphosphate
(GTP) is added to the 5' end to
form a 5' cap
(-P-P-PG- 5').
41
A GTP molecule is a
guanine nucleotide with two
additional phosphate
groups (in the same way
that ATP is an adenine
nucleotide with two
additional phosphates).
The 5' cap provides
stability to the mRNA and
a point of attachment for
the small subunit of the
ribosome.
42
GTP molecule chemical structure
To the 3' end of the mRNA,
a sequence of 150 to 200
adenine nucleotides is
added, producing a poly-A
tail (-A-A-A . . . A-A-3').
The tail provides stability
and also appears to control
the movement of the mRNA
across the nuclear envelope.
By controlling its transport
and subsequent expression,
the poly-A tail may serve to
regulate gene expression.
43
The second alteration of the
mRNA occurs when some mRNA
segments are removed. A
transcribed DNA segment
contains two kinds of
sequences—exons, which are
sequences that express a code
for a polypeptide, and introns,
intervening sequences that are
noncoding.
The original unprocessed mRNA
molecule, called heterogenous
nuclear RNA, contains both the
coding and the noncoding
sequences.
(see 4 in the Figure).
44
Before the RNA moves to
the cytoplasm, small
nuclear
ribonucleoproteins, or
snRNPs, delete out the
introns and splice the exons
(see 6 in the Figure).
45
46
Splicing the exons process
C) Translation
After transcription, the mRNA, tRNA, and
ribosomal subunits are transported across
the nuclear envelope and into the
cytoplasm.
47
In the cytoplasm, amino acids attach to
the 3' end of the tRNAs, forming an
aminoacyl-tRNA. The reaction requires
an enzyme specific to each tRNA
(aminoacyl-tRNA synthetase) and the
energy from one ATP. The amino acidtRNA bond that results is a high-energy
bond, creating an “activated” amino
acid-tRNA complex.
48
As in transcription, translation is categorized into three steps
•
•
•
Initiation,
Elongation, and
Termination.
Energy for translation is provided by several GTP molecules. GTP
acts as an energy supplier in the same manner as ATP.
GTP molecule chemical structure
49
The details of translation
follow, with numbers
corresponding to events
illustrated in the Figure.
50
1. Initiation begins when
the small ribosomal subunit
attaches to a special region
near the 5'
end of the mRNA.
51
2. A tRNA (with anticodon
UAC) carrying the amino
acid methionine attaches to
the mRNA (at the “start”
codon AUG) with hydrogen
bonds.
52
3. The large ribosomal
subunit
attaches to the mRNA,
forming a complete
ribosome with the tRNA
(bearing a methionine)
occupying the P site.
53
4. Elongation begins when the next tRNA (bearing an amino acid) binds
to the A site of the ribosome. The methionine is removed from the first
tRNA and attached to the amino acid on the newly arrived tRNA.
54
The Figure shows
elongation after several
tRNAs have delivered
amino acids. The
growing polypeptide is
shown at 4.
55
5. The first tRNA,
which no longer carries
an amino acid, is
released. After its
release, the tRNA can
again bind with its
specific amino acid,
allowing repeated
deliveries to the mRNA
during translation.
56
6. The remaining
methionine tRNA
(together with the mRNA to
which it is bonded)
moves from the A site to
the P site.
Now the A site is
unoccupied and a new
codon is exposed. This is
analogous to the ribosome
moving over one codon.
57
7. A new tRNA carrying a
new amino acid enters the
A site. The two amino
acids on the tRNA in the P
site are transferred to the
new amino acid, forming a
chain of three amino
acids.
58
mRNA
movement
Stop codon
59
The Figure shows a
chain of four amino acids.
8. As in step 5, the
tRNA in the P site
is released, and
subsequent steps
are repeated. As
each new tRNA
arrives, the
polypeptide chain
is elongated by
one new amino
acid, growing in
sequence and
length as dictated
by the codons on
the mRNA.
60
9. Termination occurs when the ribosome encounters one of the three
“stop” codons (see the Figure). At termination, the completed polypeptide, the
last tRNA, and the two ribosomal subunits are released. The ribosomal
subunits can now attach to the same or another mRNA and repeat the
process.
61
Once the polypeptide is completed, interactions among the amino acids
give it its secondary and tertiary structures.
Subsequent processing by the endoplasmic reticulum or a Golgi body
may make final modifications before the protein functions as a
structural element or as an enzyme.
DNA Organization
In eukaryotes, DNA is
packaged with proteins to
form a matrix called
chromatin.
The DNA is coiled around
bundles of eight or nine
histone proteins to form
DNA-histone complexes
called nucleosomes.
Through the electron
microscope, the
nucleosomes appear like
beads on a string.
During cell division, DNA is
compactly organized into
chromosomes.
During cell division, DNA is
compactly organized into
chromosomes. In the
nondividing cell, the DNA is
arranged as either of two
types of chromatin, as
follows:
1.
2.
Euchromatin describes
regions where the DNA is
loosely bound to
nucleosomes. DNA in these
regions is actively being
transcribed.
Heterochromatin represents
areas where the
nucleosomes are more
tightly compacted, and where
DNA is inactive. Because of
its condensed arrangement,
heterochromatin stains
darker than euchromatin.
DNA
Nucleosome
Heterochromatin
Euchromatin
Histone modefications
Some DNA segments within a DNA molecule are able to move to new
locations. These transposable genetic elements, called transposons (or
“jumping genes”) can move to a new location on the same chromosome or
to a different chromosome.
Some transposons consist only of DNA that codes for an enzyme that
enables it to be transported.
Other transposons contain genes that invoke replication of the transposon.
After replication, the new transposon copy is transported to the new
location.
Wherever they are inserted, transposons have the effect of a mutation.
They may change the expression of a gene, turn on or turn off its
expression, or have no effect at all.
Recombinant DNA
Recombinant DNA is DNA that contains DNA segments or genes from
different sources.
DNA transferred
• from one part of a DNA molecule to another,
• from one chromosome to another chromosome, or
• from one organism to another
all constitute recombinant DNA.
The transfer of DNA segments can occur,
Transduction is the process by
which DNA is transferred from one
bacterium to another by a virus .
• Naturally through viral transduction, bacterial conjugation, or transposons
or
• Artificially through recombinant DNA technology
Recombinant DNA technology uses
restriction enzymes to cut up
DNA. Restriction enzymes are
obtained from bacteria that
manufacture these enzymes to
combat invading viruses.
Restriction enzyme
recognition sequence
DNA
1
Restriction enzyme
cuts the DNA into
fragments
2
Restriction enzymes are very
specific, cutting DNA at specific
recognition sequences of
nucleotides.
The cut across a double-stranded
DNA is usually staggered,
producing fragments
that have one strand of the DNA
extending beyond the
complementary strand. The
unpaired extension is called a
sticky end.
Sticky end
Addition of a DNA
fragment from
another source
3
Two (or more)
fragments stick
together by
base-pairing
4
DNA ligase
pastes the strand
5
Recombinant DNA molecule
1+2+3) Fragments produced by a
restriction enzyme are often
inserted into a plasmid because
plasmids can subsequently be
introduced into bacteria by
transformation.
This is accomplished by first
treating the plasmid with the same
restriction enzyme as was used to
create the DNA fragment.
The restriction enzyme will cut the
plasmid at the same recognition
sequences, producing the same
sticky ends carried by the
fragments.
4) Mixing the fragments with the
cut plasmids allows base-pairing at
the sticky ends. Application of DNA
ligase stabilizes the attachment.
5+6) The recombinant plasmid is
then introduced into a bacterium
by transformation. By following
this procedure, the human gene
for insulin has been inserted into
E. coli.
The transformed E. coli produce
insulin which is isolated and used
to treat diabetes.
Transformation, the taking
up of DNA from the fluid
surrounding the cell
Restriction fragments can be separated by gel electrophoresis.
In this process, DNA fragments of different lengths are separated as they
diffuse through a gelatinous material under the influence of an electric
field.
Since DNA is negatively charged (because of the phosphate groups), it
moves toward the positive electrode.
Shorter fragments migrate further through the gel than longer, heavier
fragments.
Gel electrophoresis is often used to compare DNA fragments of
closely related species in an effort to determine evolutionary relationships.
Mixture of DNA
molecules of
different size
Longer
molecules
Shorter
molecules
Completed gel
When restriction fragments between
individuals of the same species are
compared, the fragments differ in
length because of polymorphisms,
which are slight differences in DNA
sequences.
These fragments are called restriction
fragment length polymorphisms, or
RFLPs.
In DNA fingerprinting, RFLPs
produced from DNA left at a crime
scene are compared to RFLPs from
the DNA of suspects.
Problem in recombinant DNA
When foreign genes are inserted
into the genome of a bacterium with
recombinant DNA technology, introns
often prevent their transcription.
Also the presence of introns (non
coding sequence) in the gene of
interest make this gene too large to
be cloned easily.
To avoid this problem, the DNA
fragment bearing the required gene is
obtained directly from the mRNA that
codes for the desired polypeptide.
Reverse transcriptase (obtained from
retroviruses) is used to make a DNA
molecule directly from the mRNA.
DNA obtained in this manner is called
complementary DNA, or cDNA, and
lacks the introns
Instead of using a bacterium to
clone DNA fragments, some
fragments can be copied millions of
times by using DNA polymerase
directly.
This method, called polymerase
chain reaction, or PCR, uses
synthetic primers that initiate
replication at specific nucleotide
sequences.