| M W F; 10:10 - 11:00 pm |
Molecular Biology |
Douglas W. Smith |
| York 2722 |
BIMM 100 |
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
Readings: Brown, 12: 319-327
Outline:
A. Termination of Rounds of DNA Replication in E. coli
Definition: Termination includes all events associated with completion of rounds of DNA replication after all DNA synthesis is completed. These events include Decatenation and Separation of Daughter Chromosomes and Segregation of Daughter Chromosomes into cell regions destined to become parts of the Daughter Cells upon Cell Division.
Basic mechanism: replication forks from bidirectional replication "run into each other" in the E. coli terminus region.
When replication forks begin
to approach each other, the excess of supercoils present in the
DNA ahead of the forks (due to helicase unwinding of parental
DNA strands at the forks) ... will slow down the forks.
The forks are thus thought to "run into each other"
in rather "gentle" fashion ...
1. Ter sites in Terminus Region: ... [Brown, Fig 12.22]
Six specific sites exist in the Terminus Region that dramatically
slow down fork movement.
These are "directionally specific": three slow down
one fork, three slow down the other fork.
Positions of the Ter sites are such that the replication forks
would normally cross each other to reach the requisite
Ter sites
Function of the Ter Sites: thought to be a "backup mechanism":
If one replication fork were to arrive in the Terminus region
"ahead of schedule" and ahead of the other replication
fork, it would be slowed down by the Ter Sites, permitting the
other replication fork to "catch up", resulting in normal
Termination in the Terminus Region.
Tus Protein - Ter Utilization Substance - protein that mediates the slowing down of Replication Forks at the Ter Sites.
2. Decatenation of Daughter Chromosomes:
Daughter chromosomes may be "interlocked". If so, DNA
gyrase could catalyze "decatenation" of the interlocked
daughter chromosomes ... however, bacteria have evolved a separate
'resolvase' or site-specific 'recombinase' system to do this:
XerCD proteins: these proteins are site-specific endonucleases
that act as 'recombinases' to function in the stable maintenance
of E. coli chromosomes.
They recognize a specific site in the terminus region called dif
and catalyze cleavage of a specific pair of DNA strands, exchange
of the strands, and rejoining of the cleaved strands to the parental
molecule.
Both XerC and XerD are required for the recombination reaction,
but each protein can execute the rejoining reaction, possibly
each on its own daughter chromosome.
3. Segregation of Daughter Chromosomes
Daughter chromosomes move toward
the poles of the growing bacterial cell.
The mechanism for this is not clear but probably involves the
bacterial cell surface and the protein MukB may serve as
a "motor force" protein.
Daughter bacterial cells are then produced by asexual fission of the growing parental bacterial cell in a plane at the center of the cell, with each daughter cell receiving one daughter chromosome.
B. Telomeres:... [Brown, Section 12.3.4]
1. Definition:
A Telomere is a DNA sequence forming the ends of eukaryotic
chromosomes that:
1. confers stability to the
chromosome
(without the Telomeric sequence, a chromosome has "sticky
ends" that promote recombination with other chromosomes)
2. provides a mechanism for
DNA replication of the ends without loss of genetic information
... [Brown, Fig 12.23]
This Telomeric sequence is
a series of many Direct Repeats of a simple sequence of the general
form:
Cn(A/T)m, where n > 1 and m is 1 to
4
Examples: ... [Brown, Table 12.3]
Tetrahymena: CCCCAA (the most extensively studied and used)
(or: TTGGGG)
Oxytricha CCCCAAAA Trypanosome: CCCTA; Human: CCCTAA
2. Isolation of Telomeres:
A YAC with an ARS origin and CEN centromere sequence is unstable
in Yeast: it is degraded.
The provides a system for isolation
of Telomeres:
clone fragments onto the ends of the YAC, select those that are
stabilized.
Fragments that work in this
assay come from the ends of natural chromosomes.
3. Telomere length:
Further, fragments from other eukaryotes work in yeast:
yeast Telomeres are ADDED ONTO the ends of the cloned fragments
Thus: Telomeres can be "repaired": Telomeres that are too short are extended...[YACs: J. Art. 1]
This solves the problem of
DNA replication ... the ends do not need to be completely replicated
!! The specific Telomeric "repair" process will complete
the job ...
Why then are Telomeres not extended to very long lengths ??
Another enzyme system, Telomerase Binding Proteins (TBPs),
as yet not well characterized, "shortens" Telomeres
that are too long ... this process is Essential to the cell; mutants
are lethal ... [Brown, Page 323]
Thus: enzyme systems exist
in Yeast which maintain Telomere length within well defined bounds
...
1. Telomerase:
Telomere synthesis: Telomerase
... [Brown, Fig
12.24]
A Tetrahymena enzyme termed Telomerase has been isolated
which:
adds TTGGGG repeats to the 3'-OH end of the G,T strand of Tetrahymena
telomeres
Telomerase is a Ribonucleoprotein:
it contains an RNA component, 159 bases long in Tetrahymena.
This RNA has two repeats of the CCCCAA sequence which serve as
a Template for
synthesis of new TTGGGG repeats.
This is DNA synthesis from an RNA template: Reverse Transcription
How the other strand is synthesized
is not yet known definitively, but probably the end of the TTGGGG
strand transiently loops back, and the 3'-OH of the terminal G
serves as a Primer Terminus for synthesis of the second strand.
Stability of the Telomere end:
NMR and X-ray diffraction studies of d(GGGGTTTTGGGG) - Oxytrichia
indicate a G-Quartet structure. ... [Voet-Voet, Fig 31-35]
although alternative structures have also been proposed ... [Brown,
Fig 12.25]
Telomere-binding proteins promote formation of this structure (Chaperonins) and may themselves contribute to Telomere stability; these structures inhibit Telomerase extention of the Telomere.
2. Possible Roles of Telomerase in Aging and in Cancer:
a. Aging:
In higher eukaryotes, Telomerase activity is present in embryonic
cells
BUT is absent in cells from adult tissue!
It is specifically the Telomerase protein, not the RNA component
that is absent ...
the RNA component remains abundant in adult tissue ...
Thus: during replication of proliferating cells in adult
tissue, Telomeres are progressively shortened, due to the
problem with replication of the Lagging Strand all the way to
its end.
Eventually the Telomere is shortened to the point that
it is removed completely!
And during subsequent replication and cell proliferation, DNA
encoding genes at the ends of chromosomes is lost.
This loss has been implicated in the Aging Process!
However much remains to be done to clarify this ...
b. Cancer:
1) Immortalization of Human Cells cultured in vitro:
When one attempts to establish a new cell line in vitro, one usually
finds that the cells "age" and die after some 30-50
generations ... and Telomerase activity is absent in these cells
This "death" is NOT present in cell lines that are "immortal",
for example, in the tumor cell line called HeLa cells established
from cells from a tumor from Helen Lamar ...
AND Telomerase activity is present in these cells!
Conversely, when immortalized, tumorogenic cell lines are
induced to differentiate, they lose their immortalization ...
and lose their Telomerase activity!
Most importantly: the hTERT gene encoding the human
Telomerase protein has been cloned ... and when inserted into
normal cell lines, the cell lines become 'immortalized' ... This
directly shows that Telomerase alone leads to immortalization
2) Tumorogenesis:
Finally insertion of
the hTERT gene, together with a few oncogenes, into human fibroblasts
or kidney cells converts them into tumorogenic cells.
Overall, about 90% of tumor types reactivate Telomerase (but 10%
don't ...)
Thus: the Telomerase protein appears to be a key protein
in both cell 'immortalization' and in tumorogenesis ...
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
If you have problems or comments, send email to Doug Smith