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DNA, Chromosomes and
DNA Replication
Dr.Aida Fadhel Biawi
DNA REPLICATION
DNA replication is a biological process that occurs in
all living organisms and copies their DNA ;it is the
basis for biological inheritance .
The process starts when one double-stranded DNA
molecule produces two identical copies of the
molecule .
How does DNA replicate?
DNA Replication is a semiconservative process that results in a doublestranded molecule that synthesizes to produce two new double
stranded molecules such that each original single strand is paired with
one newly made single strand .
Semiconservative replication would produce two
copies that each contained one of the original
strands and one new strand .
replication begins at specific sites on DNA molecule called
"origins of replication, "
origins are specific sequence of bases
Genetic studies (in prokaryotic) suggested that initiation of replication
at oriC most likely depended on the protein encoded by a gene
designated dnaA., dnaB, dnaC.
Eukaryotic DNA have many origins of replication
We have seen how the activities of helicase and primase solve two of
the problems inherent to DNA replication — unwinding of the duplex
template and the requirement of DNA polymerases for a primer
Remember, though, that both strands of the DNA template are copied
as the replication bubble enlarges. Each end of the bubble represents a
growing fork where both new strands are synthesized.
Growing fork
Site in double-stranded DNA at which the template strands
are separated and addition of deoxyribonucleotides to each
newly formed chain occurs; also called ( 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.
Replication fork is where the parental DNA strands hasn't untwist. Replication
bubbles allow DNA replication to speed up therefore the untwisted DNA would
not be attacked by enzymes while replicating .. ( Which enzymes can attack
DNA?? )
Specific enzymes & proteins recognize origins & bind DNA :
1- primase and DNA polymerase will find these specific
portions and will bind to the template DNA at the
correct location .
( DNA replication requires a RNA primer, primer
synthesized by the enzyme primase , primer is a short
strand RNA about 5 bases and RNA primer is
complementary to DNA )
- new DNA synthesized by DNA polymerase , DNA
polymerase binds to parent DNA strand with primer.
- DNA polymerase sequentially adds
deoxyribonucleotides to RNA primer ,
deoxyribonucleotides added have bases
complementary to parent strand DNA .
- The rate nucleotide additions in bacteria add about
500 bases/second while in mammels add about 50
bases/second ??!!
2-replication requires strand separation
a. strand separation begins at origin of
replication (Helicase)
b. specific proteins prevent the two separated
DNA strands from coming back together
(single strand binding protein)
At origin of replication ,one strand of DNA is made in a continuous
manner (the leading strand) and the other in a discontinuous manner (the
lagging strand(.
DNA is made in only the 5-prime to 3-prime direction and the replication
bubble opens the original double stranded DNA to expose both a 3-prime
to 5-prime template (Leading strand template) and it complement .
The lagging strand must be synthesized as a series of discontinuous
segments of DNA .??
These small fragments are called Okazaki fragments and they are joined
together by an enzyme known as DNA ligase .
As synthesis of the leading strand progresses, sites
uncovered on the single-stranded template of the lagging
strand are copied into short RNA primers (<15
nucleotides) by primase .
.Each of these primers is then elongated by addition of
deoxyribonucleotides to its 3′ end. In E. coli ,this reaction
is catalyzed by DNA polymerase III (Pol III), one of three
DNA polymerases produced by E. coli .
Thus each lagging strand grows in a direction opposite to
that in which the growing fork is moving. The resulting short
fragments, containing RNA covalently linked to DNA, are
called Okazaki fragments
After their discoverer Reiji Okazaki. In bacteria and
bacteriophages ,Okazaki fragments contain 1000 – 2000
nucleotides, and a cycle of Okazaki-strand synthesis takes
about 2 seconds to complete. In eukaryotic cells, Okazaki
fragments are much shorter (100 – 200 nucleotides.
As each newly formed segment of the lagging strand
approaches the 5′ end of the adjacent Okazaki fragment
(the one just completed ,)E. coli DNA polymerase I takes
over.
Unlike polymerase III, polymerase I has 5′ → 3′
exonuclease activity ,which removes the RNA primer of
the adjacent fragment; the polymerization activity of
polymerase I simultaneously fills in the gap between the
fragments by addition of deoxyribonucleotides. Finally,
another critical enzyme ,DNA ligase ,joins adjacent
completed fragments .
DNA Polymerase in Pro and
Eukaryotic
See attached word file
Enzyme
DNA
Helicase
DNA
Polymeras
e
SingleStrand
Binding
(SSB)
Proteins
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 proof-reading and error
correction.
Bind to ssDNA and prevent the DNA double helix from reannealing after DNA helicase unwinds it thus maintaining the
strand separation.
Topoisome
Relaxes the DNA from its super-coiled nature.
rase
DNA
Ligase
Primase
Re-anneals the semi-conservative strands and joins Okazaki
Fragments of the lagging strand.
Provides a starting point of RNA (or DNA) for DNA polymerase
to begin synthesis of the new DNA strand.
Telomerase Prevents Progressive
Shortening of Lagging Strands during
Eukaryotic DNA Replication
Unlike bacterial chromosomes, which are
circular, eukaryotic chromosomes are
linear and carry specialized ends called
telomeres. Telomeres consist of repetitive
oligomeric sequences; for example, the
yeast telomeric repeat sequence is 5′-G1 –
3 T-3′.
The need for a specialized region at the ends of
eukaryotic chromosomes is apparent when we
consider that all known DNA polymerases
elongate DNA chains from the 3′ end, and all
require an RNA or DNA primer.
As the growing fork approaches the end of a
linear chromosome, synthesis of the leading
strand continues to the end of the DNA template
strand; the resulting completely replicated
daughter DNA double helix then is released.
However, because the lagging-strand template is
copied in a discontinuous fashion, it cannot be
replicated in its entirety .
When the final RNA primer is removed, there is
no upstream strand onto which DNA polymerase
can build to fill the resulting gap. Without some
special mechanism, the daughter DNA strand
resulting from lagging-strand synthesis would be
shortened at each cell division.
The enzyme that prevents this progressive
shortening of the lagging strand is a modified
reverse transcriptase called telomerase, which
can elongate the lagging-strand template from
its 3′-hydroxyl end.
This unusual enzyme contains a catalytic site
that polymerizes deoxyribonucleotides directed
by an RNA template, and the RNA template
itself, which is brought to the site of catalysis as
part of the enzyme (Figure 12-13).
The repetitive sequence added by telomerase is
determined by the RNA associated with the
enzyme, which varies among telomerases from
different sources.
Once the 3′ end of the lagging-strand template is
sufficiently elongated, synthesis of the lagging
strand can take place, presumably from
additional primers.
Cell Cycle in Prokaryotic and
Eukaryotic
The Prokaryotic Cell Cycle
1- The prokaryotic cell cycle is a relatively
straightforward process. Essentially, unicellular
prokaryotic organisms grow until reaching a
critical size, and synthesize more cytoplasm,
cell membrane, ribosomes, cell wall, and other
cell constituents. They then replicate their DNA,
segregate copies of the chromosome, and divide
by a process called binary fission to produce two
new genetically identical daughter cells.
Binary fission in prokaryotic
Binary fission in a prokaryotic
1- The bacterium before binary fission is when the DNA
tightly coiled.
2- The DNA of the bacterium has replicated.
3- The DNA is pulled to the separate poles of the bacterium
as it increases size to prepare for splitting.
4- The growth of a new cell wall begins to separate the
bacterium.
5- The new cell wall fully develops, resulting in the
complete split of the bacterium.
6- The new daughter cells have tightly coiled DNA,
ribosomes, and plasmids
2- Most research suggests that the rate of
fission in prokaryotic organisms is largely
controlled by environmental conditions.
For example, most prokaryotic organisms
have an optimum temperature range for
cell growth. When environmental
temperatures are above or below the
optimum, cell division tends to decrease.
3-Under ideal environmental conditions,
many prokaryotic species undergo binary
fission at a fairly rapid rate with generation
times of one to several hours. This can
lead to an astonishing growth in population
size over a relatively short period of time.
In some instances, populations of
prokaryotes may increase by a million or
even a billion fold in a matter of days.
Phases of the Cell Cycle in Eukaryotic
• Interphase
– G1 - primary growth
– S - genome replicated
– G2 - secondary growth
• M - mitosis
• C - cytokinesis
Cell cycle begins with the formation of two cells from the division of a parent cell
and ends when the daughter cell does so as well.
Observable under the microscope, M phase consists of two events, mitosis
(division of the nucleus) and cytokinesis (division of the cytoplasm).
As replication of the DNA occurs during S-phase, when condensation of the
chromatin occurs two copies of each chromosome remain attached at the
centromere to form sister chromatids.
After the nuclear envelope fragments, the microtubules of the mitotic spindle
separate the sister chromatids and move them to opposite ends of the cell.
Cytokinesis and reformation of the nuclear membranes occur to complete the
cell division.
-Most of the time, cells are in interphase, where growth occurs and cellular
components are made. DNA is manufactured during S phase.
-To prepare the cell for S phase (DNA synthesis), G1 phase occurs (the
preparation of DNA synthesis machinery, production of histones).
-In an analogous manner, the cell prepares for mitosis in the G2 phase by
producing the machinery required for cell division.
-The length of time spent in G1 is variable. In growing mammalian cells often
spend ??? hours in G1 phase. G2 is usually shorter than G1 and is usually ???
hours. And S phase ???
Interphase
• G1 - Cells undergo majority of growth
• S - Each chromosome replicates (Synthesizes)
to produce sister chromatids
– Attached at centromere
– Contains attachment site (kinetochore)
• G2 - Chromosomes condense - Assemble
machinery for division such as centrioles
Mitosis
 Some haploid & diploid cells divide by mitosis.
 Each new cell receives one copy of every
chromosome that was present in the original cell.
 Produces 2 new cells that are both genetically
identical to the original cell.
DNA duplication
during interphase
Mitosis
Diploid Cell