PHAR 2811 Dale’s lecture 5
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The differences between Eukaryotes and Prokaryotes
Eukaryotic Replication.
Replication is intimately linked to cell division in all organisms; both prokaryote and
eukaryote. Cell division in eukaryotes is carried out in the context of the cell cycle.
Unlike prokaryotes which can double under optimal conditions in as little as 20 min the
eukaryotic cell cycle takes some 18 to 24 h to complete.
The cell cycle consists of a number of phases: G1, S, G2 and M.
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G1 = growth and preparation of the chromosomes for replication
S = synthesis of DNA (and centrosomes)
G2 = preparation for M
M = mitosis
G1, S and G2 are collectively referred to as interphase. If cells are no longer dividing,
such as with terminally differentiated cells, they are considered to be in G0. As the cell
passes through each phase of the cell cycle certain checks must be satisfied. At critical
stages the level of special cytoplasmic proteins known as cyclins rise then subside once
the cell has passed through the phase. There are also a group of enzymes known as cyclin
dependent kinases (cdks) which phosphorylate protein targets involved in the control of
the cell cycle. The catch is that, although the cdks are present at fairly steady
concentrations in the cell, independent of the cell cycle phase, they only become
activated when they bind the appropriate cyclin.
Certain key indicators are used at each checkpoint. A check for the completion of S phase
(replication) is the presence of Okazaki fragments. If replication is complete there should
be no Okazaki fragments. DNA damage is checked for at G1 before the cell enters S.
Spindles are checked before the cell actually divides (M phase)
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The similarities between prokaryotic and eukaryotic replication:
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Both are bi-directional processes
DNA polymerases work 5’ to 3’
Leading and lagging strands
Primers are required
The unique problems faced by eukaryotes that are not faced by
prokaryotes:
Linear chromosomes with ends,
much more genetic material; the typical animal cell has 50 times more DNA than
the average bacterium (E. coli has 4.6 million base pairs, humans have 3 000
million base pairs or 650 times as much!!),
much more packaging; the nucleosomes (DNA wound around histones) and all
the scaffolds and higher order packing.
Coupled with these factors the DNA polymerases that are found in eukaryotes work
much slower NOT faster!! At the rate they work it would take 30 days to copy the human
genome if it was left to 2 replication forks! The average E. coli replication fork works
around the chromosome at a staggering 105 base per minute. Our eukaryotic counterpart
can only manage somewhere between 500 and 5 000 bases per minute.
The only way to get around such slack work habits is to employ more enzymes! There are
multiple initiation sites scattered some 30 to 300 kilobases apart. Drosophila, alias the
fruit fly, has ~ 5,000 such sites while mammalian cells have ~20,000. This initiation
event has to be tightly coordinated and with so many separate sites the logistics seems
massive. It is regulated but we don’t know how! Most of what we do know about
eukaryotic replication has been gained from studying yeast and SV-40 DNA replication.
Drosophila and toads have also been studied as have human cell cultures.
As with prokaryotes each origin of replication begins with the binding of a large protein
complex; in the case of eukaryotes it is imaginatively named the Origin Recognition
Complex (ORC). This complex will remain on the DNA throughout replication. Other
protein factors then join the ORC (a whole party in fact!) and recruit proteins which coat
the DNA. The next complex to associate is the MCM complex. This complex contains a
helicase amongst other enzymes. This will be required to unravel the DNA. To facilitate
the binding of the MCM complex, a helicase loader must also bind. You can see this party
is getting complicated very quickly. Let’s simplify the story by appreciating that a
number of protein complexes bind in a coordinated fashion and once this has been
completed we have a pre-replication complex. These accessory proteins or licensing
factors accumulate during G1 of the cell cycle. The initiation complex is now described
as licensed to begin replication. These licensing factors also prevent re-initiation. You
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can’t have eager proteins trying to re-initiate replication during one round of replication.
It is essential that only ONE faithful copy of the DNA is made at replication!!
The whole genome is not simultaneously replicated. It appears that clusters of 20 to 80
sites are initiated together. Forks extend from both sides of each initiation site and move
in opposite directions until they fuse with an approaching fork from an adjacent site. New
clusters are activated throughout S phase of the cell cycle and it takes several hours to
copy the whole genome.
The enzymes used by eukaryotes are different.
There are many, named imaginatively as pol α, pol γ, pol δ1 and pol ε(what would we do
without Greek letters). DNA pol α participates in chromosomal replication. It is found
only in the nucleus, extends a DNA strand in a 5’ to 3’ direction; the sequence is directed
by a single stranded DNA template. This enzyme, unlike its prokaryotic equivalent, lacks
exonuclease activity. It is always associated with a primase.
DNA pol δ is also a nuclear enzyme with 5’ to 3’ polymerase activity and a 3’
exonuclease function. It does not, however, have a primase partner. It does have greater
processivity (the ability to stick to the template and keep copying) than DNA pol α. Our
DNA pol α has a processivity of ~100 nucleotides whereas good old pol δ will replicate
the whole template provided it is associated with a protein PCNA (proliferating cell
nuclear antigen). The complex formed from DNA pol δ and PCNA is necessary for both
leading and lagging strand synthesis. It seems that pol α/primase synthesizes RNA
primers of 7 -10 nt then extends these another 15 nt with DNA before switching to PCNA
and pol δ to finish the job.
Then we have pol ε. It can polymerase DNA and sticks to the job very well but may have
a regulatory role rather than a serious catalytic one. DNA pol γ is restricted to the
mitochondrion and is thus thought to be responsible the replication of its genome. The
chloroplast also has DNA pol γ. On top of the pols α, δ and ε eukaryotes have lots of
repair enzymes: pols β, η, ι, κ and ζ.
Not only do we have different enzymes but eukaryotic cells have more copies of these
enzymes than do prokaryotes. Our typical animal cell has between 20,000 and 60,000
molecules of pol α whereas our regular bacterium, E. coli has 10 to 20 molecules of
DNA pol III.
Once the DNA has replicated in eukaryotes it must be packaged. This happens rapidly
after replication. The cell must synthesise the required histones as replication proceeds (it
doesn’t have enough stored histones to do the packing).
Summary of Proteins associated with DNA Replication in both Prokaryotes and
Eukaryotes
PHAR 2811 Dale’s lecture 5
Protein
DNA Polymerase I
DNA Polymerase III
DNA Polymerase α
DNA Polymerase δ
DNA Polymerase ε
DNA Polymerase γ
Primase
DNA helicase
Single stranded Binding
Protein (SSBP)
Topoisomerases (type I and
II)
DNA gyrase
DNA ligase
Initiator proteins
Telomerase
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Prokaryotic/
Activity/role
eukaryotic
5’ to 3’ polymerase, 3’ to 5’ exonuclease,
Prokaryotic
5’ to 3’ exonuclease
Prokaryotic
5’ to 3’ polymerase, 3’ to 5’ exonuclease,
5’ to 3’ polymerase, complexes with
primase then begins DNA synthesis from
Eukaryotic
RNA primers, low processivity (~100 nt),
no exonuclease activity
5’ to 3’ polymerase, 3’ to 5’ exonuclease
Eukaryotic
(proof reading), high processivity when
complexed with PCNA
5’ to 3’ polymerase, high processivity,
Eukaryotic
probable regulatory role
Eukaryotic
Mitochondrial DNA polymerase (5’ to 3’)
Both
RNA polymerase (5’ to 3’) makes primers
Both
Untwists DNA
Coats DNA to prevent strands reBoth
annealing
Relieves stress of supercoiling (type I) and
Both
introduces negative supercoiling (type II)
Prokaryotic
Type II topoisomerase
Seals breaks in the DNA backbone
Both
between 3’OH and 5’ PO4 requires energy
source
Both
Bind at the origin of replication
Reverse transcriptase activity (5’ to 3”)
Eukaryotic
using an endogenous RNA template
What to do with the ends!
The ends of the DNA are known as telomeres. As each chromosome is one continuous
double stranded length of DNA it has 2 telomeres (not rocket science!). Replicating the
ends and what to do with the ends are problems. There is a particular problem with the
lagging strand. The 3’ to 5’ parent strand is copied no trouble as the leading strand. The
lagging strand (parent 5’ to 3’) has small primers made every so often and filled in. There
is no template at the end to bind to so when the primers are removed there is an overhang.
This can’t be filled in by normal lagging strand mechanisms because a 3’ Oh is required
and there is none at the end. This is not good!! It exposes the DNA to digestion which
would eventually eat into the genes at the end (gene erosion). The other problem is that
with each round of cell division the DNA would become shorter. This is known as the
“end-replication” problem.
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This problem was solved by the discovery of a group of reverse transcriptases known as
telomerases. (This discovery was made by an Australian scientist Elizabeth
Blackburn).The end DNA, the telomeres, have an unusual sequence; several thousand
copies of a tandem repeat which is species specific, but is G rich. The vertebrate version
is TTAGGG. This repeating sequence is added onto the 3’ end by telomerases and is
sometimes tucked in to the end forming a cap. Capping proteins associate to prevent
nuclease erosion of the ends.
The telomerases contain an integral RNA component which has a sequence
complementary to the repeat sequence and this acts as a template for a reverse
transcriptase reaction. The telomerase then moves to the new 3’ end once the repeat has
been added and does it all again. The telomerase protein component is a type of reverse
transcriptase. The other strand then copies the sequence by normal lagging strand
mechanisms. The 3’ end of telomeres has an overhang and this mechanism accounts for
this.
See Powerpoint diagrams.
Telomeres and aging, cancer and immortality.
The really interesting thing about telomerases is not so much their existence as their lack
of activity in certain cells, most somatic cells to be exact. While germ-line cells and some
highly proliferative stem cells have telomerase activity and thus the zygote will have full
length telomeres, the telomeres of somatic cells shorten with each mitotic cell division.
Eventually they become too short and the cell stops dividing. It is this phenomenon that
explains the observation that normal cells in culture only survive ~30 cell divisions
before they die out.
In contrast, immortal cell lines, which are derived from cancers can be kept in culture
indefinitely. The most famous cell line is the HeLa cell. HeLa cells are derived from
Henrietta Lacks who died in 1951 of cervical cancer:
Here is the extract from the following website with a brief history of these famous cells:
http://www.madsci.org/posts/archives/may97/860431113.Cb.r.html
“In 1951 a physician removed cells from the cervix of Henrietta Lacks, a 31 year
old black woman from Baltimore, and sent the cells to a lab to determine if they
were malignant. The cells were malignant and Henrietta Lacks died eight months
later from cervical cancer. Henrietta Lacks' physician provided George and
Margaret Gey of Johns Hopkins University with a sample of these cervical cancer
cells. The sample of Henrietta Lacks' cells was code-named HeLa, for the first
two letters of her first and last name. The HeLa cell cultures survived and
multiplied so well in culture, that they were soon being shipped to research labs
around the world for study.
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Over the past 45 years, research on HeLa cells has provided scientists with an
enormous amount of basic knowledge about the physiology and genetics of cells.
However, HeLa cells have been responsible for generating a great deal of bogus
scientific data as well. It turns out that HeLa cells grow very aggressively in
culture and can easily invade other cell cultures during routine lab transfer
procedures, when proper precautions are not taken. As a result, numerous
research papers have been published on the biology of a variety of cultured cell
types which have subsequently been shown to be HeLa cells.”
Immortal cells i.e. cancer cells, have active telomerases (90%) or other mutated strategies
to keep their telomeres long (the other 10%). This is not the reason they have become
malignant BUT cancer cells with active telomerases are selected out as the malignancy
establishes itself. If they were not the malingnancy would soon die as the cells divide so
rapidly they would very quickly reach the damage control point and the cells would die.
The lack of telomerase activity in most somatic cells, in particular terminally
differentiated cells, means they age and die out. This is ultimately the cause of aging. But
before we get too negative about telomerases or the lack of them, bear in mind that this
may be a protection against cancer. To have cells undergo a program of cell divisions
before senescence is a good strategy against uncontrolled proliferation. So maybe as we
get old we should remember this process is protecting us from cancer.
Other evidence supporting the role of telomeres in aging
Progeria
Progeria is a premature aging disease which actually has two forms: Werners syndrome,
the adult onset form with an average life span of 47 years and Hutchinson-Gilford
progeria, the juvenile onset form with an average life span of 13 years.
It is the Hutchinson-Gilford progeria which is of particular interest on the topic of
telomeres. Only certain cells age; in particular the cells of the skin, bone and
cardiovascular system. These cells show significantly shorter telomeres than the rest of
the unaffected cells at birth. Skin fibroblasts of progeria sufferers at birth have telomeres
the length of an 85-year-old. This website below is very interesting to follow up.
http://www.bookrags.com/sciences/genetics/accelerated-aging-progeria-gen-01.html
“Cellular data, particularly regarding structures called telomeres, suggests that
some of the cells from Hutchinson-Gilford patients are prone to early cell
senescence. Telomeres are special DNA structures at the tips of the
chromosomes. These telomeres gradually shorten over time, and this shortening
is associated with some aspects of cellular aging. Skin fibroblasts from
Hutchinson-Gilford patients have shorter than normal telomeres and
consequently undergo early cell senescence. At birth, the mean telomere length
of these children is equivalent to that of a normal eighty-five-year-old.
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Introduction of human telomerase into such cells leads to reextension of the
telomeres and results in normal immortalization of these progeric cell cultures.
Clinical interventional studies using this strategy in humans are pending.
Predictably, circulating lymphocytes of Hutchinson-Gilford children have normal
telomere lengths, in keeping with their normal immune function. Research thus
far suggests that progeria may not be so much a genetic disease as it is an
"epigenetic mosaic disease." In progeria, this means that the genes are normal,
but the abnormally short telomere length in only certain cells lines causes an
abnormal pattern of gene expression. The senescent pattern of gene expression
in specific tissues results in the observed clinical disease of progeria.”
Dolly the sheep.
You may recall that Dolly the first cloned sheep was born in 1996. She was cloned by,
essentially, removing the genome from an egg and replacing it with the genome from the
mammary cells (somatic cells) of another sheep. Although Dolly, incidentally named
after Dolly Parton since her genome origin was mammary tissue, developed normally,
was able to have a lamb, named Bonnie, but died prematurely of a lung infection typical
of old sheep. She was euthanased at 6 years old suffering arthritis and showing all the
classic signs of sheep age. The explanation is the DNA was “OLD”; it had shortened
telomeres.