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Cellular ageing processes
Prof Duncan Shaw
Molecular & Cell Biology
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Lecture 5
Cellular ageing in yeast
Ageing in humans - Werner's syndrome and DNA helicases
Cellular ageing in yeast
Reference: Sinclair & Guarente (1997), Cell 91, 1033-1042.
Ageing is a complex process in higher
organisms, and it is not fully understood.
However ageing also applies to microorganisms such as yeast, and these simple
systems can be used to investigate certain
aspects of the ageing process. In
Saccharomyces cerevisiae, it is wellknown that individual cells do not go on
dividing for ever - they have a limited lifespan and after a certain number of cell
divisions they give up and die. This
cannot just be due to genetic damage
because any mutations would be passed
on to the next generation and eventually
the whole population would be mutated to
death. A clue to the ageing process comes
from observations of the nucleolus, a
region of the nucleus in which the
ribosomal DNA (rDNA) repeats are
located - there are 100-200 copies of a
9.1kb repeat. Normally half of the rDNA
genes are silenced (trancription is
repressed) by the action of the SIR2 gene
product. In ageing yeast the ability to mate
is lost, due to loss of SIR2-mediated
silencing at the mating-type loci. This
may be because all the SIR2 protein
becomes sequestered by the rDNA genes
in the nucleolus. Deletion of another gene,
SGS1, leads to an accleration of these
processes, and SGS1 is a homologue of
the gene for the human premature ageing
disease, Werner's syndrome. As S.
cerevisiae divides by budding it is possible to distinguish between the 2 cells that result from a
division - one comes from the bud and the other is what is left. Each budding event leaves a
"scar" on the cell wall of the mother cell, so it is possible to tell how old a cell is by the number
of scars that are visible. The diagram (left or above) shows analysis of DNA from young and old
yeast, by 2-dimensional gel electrophoresis. The gel has been run first from left to right, in the
presence of the drug chloroquine, and then run from top to bottom with a higher concentration of
the same drug. These treatments have no effect on the mobility of linear DNA, which therefore
forms a diagonal band on the gel (n.b. yeast chromosomes are linear). But the mobility of
circular DNA is affected by the drug and so the presence of strong bands of DNA not on the
diagonal indicates large amounts of circular DNA in the old cells, but not in the young ones.
When this gel was blotted and probed with gene-specific DNA probes, it was shown that the
circular DNA was ribosomal DNA. These circles are called ERC (extra-chromosomal rDNA
circles), and they must have formed by recombination within the tandemly-repeated rDNA genes
on the chromosome. The conclusion is that ageing is associated with the accumulation of ERC in
older cells, but it is not proven that ERC is the cause of ageing. It could be just a side-effect of
the ageing process.
In order for ERCs to accumulate in older
cells, there must be a bias in the
segregation of the ERCs at cell division,
so that more of them end up in the older
(mother) cell than in the bud (daughter)
cell. To investigate this an experiment
was set up in which the segregation of
ERC could easily be followed. A copy
of the ERC was cloned in a plasmid
together with the ade2 gene, which
complements the defect in adenine
biosynthesis in an Ade- yeast host strain.
If the plasmid is lost from the yeast, it
fails to grow on medium lacking
adenine. Yeast containing the plasmid
were grown on agar medium without
adenine, and when they divided, the
mother and daughter were separated by
micro-manipulation into individual
colonies so their fate could be followed.
The "pedigree" in the diagram shows
what typically happened. Red colonies
still contain the plasmid whereas white
ones have lost it and don't grow. The
plasmid tends to stay in the mother cell
rather than segregate to the daughters, except in the case of the daughters of very old mothers
(right-hand part of pedigree) which may retain the plasmid; this correlates with the fact that the
daughters of old mothers have a shorter life-span than daughters of young mothers. Possibly the
ERC becomes attached to something in the mother cell, e.g. the nucleolus, which causes the bias
in segregation.
The above experiment shows that
ERC accumulation could be the
cause of ageing, but still does not
prove it. What was needed was a
means of switching on ERCs at
will. Plasmids containing the yeast
ARS (autonomous replicating
sequence) and centromere
segregate correctly when cells
divide, but if these sequences are
lost, biased segregation will occur
in a similar way to the ERCs in
the above experiment. A 2plasmid system was constructed as
shown in the diagram. The small
plasmid contains the cre
recombinase enzyme gene, under
the control of an inducible
promoter (gal1p). The larger
plasmid contains an rDNA unit, an
ARS + centromere, and 2 copies
of a sequence loxP which is the
substrate for the cre recombinase.
When cells containing both
plasmids are put on a medium
with galactose, the cre gene is
induced and the recombinase
catalyses recombination between
the loxP sites, causing the ARScen sequence to be lost, and the
large plasmid begins to segregate in a biased way and accumulate like an ERC. The survival
curves at the bottom of the diagram show what happens. Cells in which the loss of ARS-cen has
been induced, and ERC accumulates (red curve) show a much reduced life-span compared to
controls in which the plasmid is segregating correctly (black curve). Further experiments showed
that the prematurely-ageing cells were also losing their mating ability, in a similar way to yeast
that are ageing in the normal way; and also that the rDNA gene in the ERC could be replaced
with various other DNA sequences and still have the same effect. Thus the experiment shows
that it is the accumulation of ERCs that causes ageing in yeast.
This diagram shows a model for
the ageing process in yeast.
Recombination within the rDNA
repeats on the chromosome (black
boxes) produces ERCs (red
circles) which then preferentially
accumulate within the mother cell.
Eventually, when perhaps 500 1000 ERCs have built up, the cell
dies. Why do ERCs cause death?
Possibly because of overexpression of the rDNA genes, or
if the ERC contains sequences that
bind transcription or DNA
replication factors, then those
factors could be removed from
their normal functions. The
accumulation of ERCs parallels
the probability of cell death due to ageing, and therefore acts as a "molecular clock" for lifespan.
Variation in lifespan of individual yeast cells could be due to chance variation in the timing of
the first ERC being formed in the young cell.
Human ageing syndromes
Reference: Ellis (1997), Curr. Opin. Genet. Devel. 7, 354-363.
We have now looked at the telomere and ERC mechanisms in cellular ageing. In humans there
are certainly many other processes, one of which is specific, genetic syndromes that cause
premature ageing.
Werner's syndrome
Werner's syndrome (WS) is a rare, autosomal recessive disease. Its symptoms are:

Growth is deficient post-puberty

Predisposition to arteriosclerosis, diabetes, non-epithelial cancers

Premature ageing - wizened appearance, greying hair, hair loss
Cultured cells from patients show:

Poor division

Telomere shortening

Karyotype changes

Increased mutation rate
The gene was mapped in Japanese families by looking for regions of the genome that were
homozygous in patients (because it is a recessive disease). The gene was isolated in 1996. It
codes for a DNA helicase enzyme.
DNA helicases
The main features of DNA helicases are:

Role is to unwind DNA for replication, recombination, repair, etc. in the 5' -> 3' or 3'->5'
direction

Highly conserved in all organisms

Defined by presence of 7 motifs within the protein sequence

Many types (>30 in humans)

Variation in biochemical properties, e.g. processivity (speed of unwinding), type of DNA
acted on, direction, subunit structure

Information about helicases from the GeneCards database
Six DNA helicase genes are known to cause human diseases, and all of these diseases have the
common features of being recessive and pleiotropic (having several different effects). Two
examples are briefly described below.
Bloom's syndrome
The main features are:

Immunodeficiency

Growth deficiency from birth

Sun sensitivity, altered pigmentation

General predisposition to cancer

Chromosomal instability with multiple breaks and sister chromatid exchanges
It was mapped by homozygosity in Ashkenazi Jewish families. The Bloom phenotype can be
observed in cultured cells as DNA instability, and this phenotype was exploited in isolating the
Bloom's gene.
ATR-X syndrome
The main features are:

Developmental delay

Severe mental retardation

Facial and genital abnormalities

Alpha thalassemia

X-linkage
The helicase gene mutated in this disease, called XH2, is homologous to the yeast swi/snf family
of helicases, whose function is restructuring chromatin prior to transcription.
Further information on these diseases, and recent references, can be found at the Online
Mendelian Inheritance in Man database (OMIM). Links are in the table below.
Disease
OMIM link
Werner's syndrome
277700
ATR-X
300032
Bloom's syndrome
210900
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