An origin of replication (ori) is a site at
which DNA replication begins. Origins of
replication are often AT rich sequences, which allows them to be
more easily pulled apart than GC rich sequences. Without an origin
of replication, DNA cannot be replicated and will not be passed on
to the next generation.
|Eukaryotic chromosomes contain numerous
replication origins, allowing for DNA synthesis to proceed
in parallel at many sites simultaneously. Each pair of
replication forks is referred to as a "replicon". Replicons
can be visualized as bubbles of replicated DNA that expand
in both directions. Eventually, all replicons merge into a
single large bubble, until replication terminates at the
The electron micrograph at right shows several replicons, each originating from a different origin (arrows).
Each replicon has two replication forks, moving in opposite directions. Ultimately, replication forks meet, until replication of each template strand is complete.
Displayed by hypertext link to http://bio3400.nicerweb.net/Locked/media/ch11/11_14-replication_bubbles.jpg
A series of experiments from Jack
Szostak's lab helped to define the critical components of
chromosomes. These experiments all had the same steps:
1. Transform yeast, deficient in Leucine production (Leu-) with an artificial construct
2. Isolate a colony on complete media (permissive conditions)
3. Grow in complete media for several generations
4. Transfer to minimal media without Leucine
5. Identify clones that grow without Leucine
CHROMOSOMAL ELEMENTS: EXPERIMENT A - Origins of
If you transfect into yeast
a plasmid containing a selectable marker, in this case a
LEU gene (Leucine biosynthesis) into Leu-
yeast, the cells will not grow, even though you have
given them the correct gene. But if you randomly
clone yeast sequences into this same plasmid and
select on minimal media, you can recover a few clones
that are able to grow (ie. synthesize leucine).
The inserts contained in these surviving plasmids are
origins of replication, referred to in yeast as autonomously
replicating sequences (ARS).
CHROMOSOMAL ELEMENTS: EXPERIMENT B - Centromeres
You might have noticed in the previous diagram that in the case of the ARS plasmid transformants, not all the daughter cells received the construct. Furthermore, in the absence of selection, even those cell lines may lose the construct. When yeast genomic DNA is randomly cloned into plasmids containing both a LEU gene and an ARS, some are stably inherited in the absence of selection. These plasmids happened to receive a fragment of DNA containing a centromere.
|Centromeres are sequences of DNA to which the
kinetochore proteins attach, and therefore to which spindle
fibres attach during mitosis. Without a centromere, the
construct has no guarantee of being passed on to both
daughter cells during cell division. They tend to include
repetitive sequences (satellite DNA), and also to not have
many nucleosomes. Presumably, that is to make spindle fibre
attachment during mitosis easier.
Detail of a positive staining centromere region of a C-banded human chromosome. (C banding preferentially stains constitutive heterochromatin). The fibrous organization of this region is still apparent but is either covered by, or embedded in, an amorphous matrix. Magnification x 64,000. Chromosomes and Chromatin, Vol. II (1988) Ed. K.W.Adolph. CRC Press. Fig. 14 pg. 67
CHROMOSOMAL ELEMENTS: EXPERIMENT C - Telomeres
In the first two experiments, the researchers created what are now known as Yeast Artificial Chromosomes (YACs). Up to now, we've been getting away with creating artificial chromosomes by making them circular. In yeast, they function as completely independent chromosomes. But eukaryotic chromosomes are linear, and Experiment C below illustrates that linear chromosomes can't normally replicate without telomeres at each end.
Why is that? What function do telomeres serve that make them so necessary for linear chromosomes?
|The basic problem with linear chromosomes are the ends - specifically, the 5' ends of a new strand. DNA polymerases can synthesize from 5' to 3' only. While the leading strand can read right to the end of the template, the lagging strand cannot. What ends up happening is this:||Parent strands are in dark blue.
As you can see, the 5' ends of the daughter strands are short.
Now, for one replication, this might not matter so much. The
daughter cells would lose some DNA off the chromosome ends, but
not too much. Probably, function would not be affected. But what
happens in the next division, and the next? More and more DNA is
lost and the chance of losing part of an important gene is much
more likely. Put this way, a species with linear
chromosomes and without telomeres is unlikely to survive for more
than a few generations.
Circular chromosomes satisfy the requirement that there
must be an upstream DNA polymerase complex to remove RNA primers
and fill in gaps.
IN-CLASS EXERCISE: LINEAR AND CIRCULAR REPLICATION
Linear molecules will always have an unfilled gap upstream from the origin of replication closest to each end of the chromosome. Since this unfilled gap will always be a 3' protruding end, there is no way for DNA polymerase to "fill it in". Given that eukaryotes evolved so long ago, there must be a mechanism for dealing with this problem.
That solution is the telomere. Telomeric
sequences are composed of repeated motifs and extend the linear
eukaryotic chromosome. Think of telomeric sequences like a buffer
for loss of sequence. As long as you add roughly as many
nucleotides to the ends of chromosomes as they lose during each
round of DNA replication, there is no net loss of DNA at the ends.
Telomeric sequence data was first obtained in Tetrahymena
thermophilus, a cilliated protozoan. Tetrahymena
has two separate nuclei, a germinal micronucleus, which carries a
diploid complement of chromosomes, and a somatic macronucleus, in
which particular chromosomes or parts of chromosomes are present
in very high copy numbers. The rDNA genes are present on separate
chromosomes with about 104 copies. Because the rDNA
genes are located near the ends of the chromosomes, it was
therefore possible to directly sequence the termini of chromosomal
DNA using rDNA-specific primers. These studies revealed that
individual rDNA termini carry a variable number (~120-420bps) of C4A2/T2G4
Other examples:These telomeric sequences are oriented so that the C-rich strand always runs 5'-->3' from the end towards the interior of the chromosome. In some cases, there are nicks, or gaps in the C-rich strand.
H. sapiens CCCTAA
Example: Oxytricha (ciliated protozoan):
end of chromosome to centromere -->
Telomeres actually have two functions:
DNA fragments containing telomeric sequences were
hybridized to metaphase chromosomes. Telomeric probe is
visualized in white. Chromosomal DNA is counterstained
with DAPI (blue).
Image displayed by hypertext link to The New Genetics, Chapter 2
RNA and DNA Revealed: New Roles, New Rules
Natl. Inst. of General Medical Science
Telomeres prevent degradation, repair and recombination of chromosome ends
The overhang ends are still a problem, though. Typically, the end
of a linear DNA molecule would be recognized as a damaged piece of
DNA and "repaired", resulting in loss of sequence from the ends of
chromosomes, or fusion with other double-stranded DNAs. Eukaryotic
cells have mechanisms for protecting chromosome ends from repair
enzymes, which vary among eukaryotes. For example, telomeres of
ciliates and fungi are protected by telomere binding proteins
which effectively hide the telomeres from repair machinery.
|Mammals have a more elaborate D-loop structure, in which the double-stranded telomere DNA opens up to form a single-stranded D-loop (or T-Loop). The 3' protruding end can loop back to form a T-loop. The end of the T-loop can base pair with internal repeat units by non-Watson-Crick base pairing. The D-loop is maintained through additional proteins that bind both the T-loop and the D-loop seen in the figure.|
Telomerases carry a small RNA molecule which
acts as a template for elongation of telomeric repeats.
the telomere is a 3' protruding end which can not be
elongated by DNA polymerase. Telomere terminal transferase
(telomerase) allows the telomere to function as a 3'
recessed end in the following way: The telomerase enzyme
carries an RNA molecule (blue) whose sequence is the
complement of the telomeric repeat. When the telomere (red) base pairs with this RNA,
the 5' end of the RNA extends beyond the end of the
telomere. This allows the RNA to act as template for
the addition of nucleotides (green)to
the telomeric 3' end, which acts as a primer. In the
example, an RNA template with the sequence 5'(ccccaaaa)n3'
codes for the actual telomeric repeat 5'(ttttgggg)n3'.
So, by extending the 3' ends of linear chromosomes with each round of DNA synthesis, telomerase provides a longer template for the lagging strand, which offsets the inevitable loss of DNA from the ends.
Is telomerase active in somatic cells? As it turns out, the answer depends on species is being observed, and stage of development. Below is an incomplete list of organisms along with telomerase activity in their somatic cells. Clearly there is much variation between eukaryotes when it comes to somatic telomerase activity.
|Is telomerase active in
Unicellular eukaryotes - Telomerase is required in each cell division to maintain telomere length
Humans1 - Telomerase activity is usually only seen in stem cells or germline cells, and telomerase activity is usually not found in somatic cells. It is hypothesized that because of the long human lifespan, somatic suppression of telomerase activity occurs as a check on cell proliferation, which could otherwise result in cancer. This is not a perfect control, because ultimately as telomeres are lost, oncogenes near the telomeres begin to be lost as well, resulting in cancer.
Mice1 - Telomerase activity is often found in somatic cells in mice. This observation makes sense in contrast to the lack of telomerase activity in human somatic cells, because mice have very short life spans, and therefore would have less need for suppressing telomerase activity as a way of suppressing cancer.
Drosophila3 - doesn't use traditional short telomeric repeats elongated by telomerase. Instead, two retrotransposons, HeT-A and TART transpose specifically to chromosome ends, elongating the array of transposon repeats at the telomeres. (Weird or what?)
Tobacco2 - High levels of telomerase activity was seen in actively dividing cells (roots and flowers), with low levels of activity in stems, and no detectable activity in mature leaves. This is consistent with the hypothesis that telomerase activity is needed in rapidly dividing tissues.
1 Wong JMY and Collins K (2003) Telomere maintanence and disease. The Lancet 362:983-988.
2 Yang SW, Jin ES, Chung IK, Kim WT (2001) Expression of telomerase activity is closely correlated with the capacity for cell division in tobacco plants. J. Plant Biol. 44:168.
3 Danilevskaya ON, Traverse KL, Hogan NC, DeBaryshe GP and Pardue ML (1999) The two Drosophila telomeric transposable elements have very different patterns of transcription. Mol. Cell. Biol. 19:873-881.
Hinnebusch J, Tilly K (1993)
Linear plasmids and chromosomes in bacteria. Mol. Microbiol.
Volff J-N, Altenbuchner J
(2000) A new beginning with new ends: linearisation of
circular chromosomes during bacterial evolution. FEMS Microbiology
Surprising, right? Why are we talking about prokaryotes - don't
they have circular chromosomes and therefore have no need for
telomeres? In biology, every rule has exceptions. There are a
number of cases in which prokaryotes have either linear
chromosomes, or linear plasmids. One well-known
example is bacteriophage Lambda, which is linear in its
encapsidated form, but circularizes, by annealing of its "sticky
ends", prior to replication as a circular molecule. However, there
are now many examples of linear chromosomes and plasmids,
including the spirochete Borrelia, the actinomycete Streptomyces,
and the plant pathogen Agrobacterium.
see Figure 1 from Volff and
While linear chromosomes or plasmids are rare amongst prokaryotes, their occurence in widely diverse species suggests that linearity has arisen independently numerous times over the course of microbial evolution. Two common schemes have been seen for linear chromosome replication: hairpins and invertrons.