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DNA Replication (I)
王之仰

Any eukaryotic chromosome
contain three functional elements
to replicate and segregate
correctly: (1) replication origins at
which DNA polymerases and other
proteins initiate synthesis of DNA;
(2) the centromere, the constricted
region required for proper
segregation of daughter
chromosomes; and (3) the two
ends, or telomerases.

Replication of DNA begins from
sites that are scattered throughout
enkaryotic chromosomes.

The yeast genome contains many
~100 bp sequences, called
autonomously replicating
sequences (ARSs), that act as
replication origins.

Up to 20% of progeny cells are
faulty on the mitotic segregation.

A CEN sequence (yeast centromere
sequence) leads to a equal or
nearly equal segregation of yeasts
during mitosis.

If circular plasmids containing an
ARS and CEN sequence are cut, the
resulting linear plasmids do not
replicate unless they contain
special telomeric (TEL)
sequences ligated to their ends.

Three regions (I, II, and III) of
the centromere are conserved
among different chromosomes.

Conserved sequences are
present in the region I and III.
; the region II has a fairly
constant length (rich in A and T
residues) and it contains no
definite consensus sequences.

Regions I and III are bound by
proteins that interact with
more than 30 proteins.
which in turn bind to microtubules.

Region II is bound to a nucleosome
that has a variant form of histone
H3 replacing the usual H3.

Centromeres from all eukaryotes
similarly are bound by nucleosomes
with this specialized, centromerespecific form of histone H3, called
CENP-A.

In human, centromeres contain 2to 4-megabase arrays of a 171-bp
simple-sequence DNA called alphoid
DNA.

The telomere repeat sequence in
vertebrates is TTAGGG; these
simple sequences are repeated at
the very termini of chromosomes.

The 3’end of the G-rich strand
extends 12-16 nucleotides beyond
the 5’-end of the complementary
C-rich strand; The region is bound
by specific proteins that protect the
ends of linear chromosomes from
attacked by exonucleases.

The need for a specialized region at
the ends of eukaryotic chromosome
is apparent when we consider all
DNA polymerases elongate DNA
chain at the 3’-end, all require an
RNA or DNA primer.

Unlike the leading strand, the
lagging-strand template is copied in
a discontinuous fashion, it cannot
be replicated in its entirety.

The telomere shortening is solved
by an enzyme that adds telomeric
(TEL) sequences to the ends of each
chromosome.

Because the sequence of the
telomerase-associated RNA serves
as the template for addition of
dNTPs to the ends of telomeres-the
source of the enzyme and not the
source of the telomeric DNA primer
determines the sequence added.

Telomerase is a specialized form of
a reverse transcriptase that carries
its own internal RNA template to
direct DNA synthesis.

The human genes expressing the
telomerase protein and the
telomerase-associated RNA are
active in germ and stem cells, but
are nearly turned off in most cells of
adult cells.

These genes are activated in most
cancer cells, where telomerase is
required for the multiple cell
divisions necessary to form a tumor.

Telomerase prevents telomere
shortening in most eukaryotes,
some organisms use alternative
strategies; Drosophila species
maintain telomere lengths by the
regulation insertion of non-LTR
retrotransposons into telomeres.

Telomeres: the physical ends of linear
chromosomes, consist of tandem arrays
of a short DNA sequence, TTAGGG in
vertebrates. Telomeres provide the
solution to the end-replication problemthe inability of DNA polymerases to
completely replicate the end of a
double-stranded DNA molecule.

Embryonic cells, germ-line cells,
and stem cells produce telomerase,
but most human somatic cells
produce only a low level of
telomerase as they enter S phase;
their telomeres shorten with each
cell cycle.

Complete loss of telomeres leads to
end-to-end chromosome fusions
and cell death.

Extensive shortening of telomeres
is recognized by the cell as a kind of
DNA damage, with consequent
stabilization and activation of p53
protein, leading to p53-triggered
apoptosis.

Most tumor cells, despite their rapid
proliferation rate, overcome this fate by
producing telomerase.

Specific inhibitors of telomerase have
been used as a cancer therapeutic
agents.

Introduction of telomeraseproducing transgenes into cultured
human cells can extend their
lifespan by more than 20 doublings
while maintaining telomere length.

Treating human tumor cells with
anti-sense RNA against telomerase
caused them to cease growth in
about four weeks.

Dominant-negative telomerases,
such as those carrying a modified
RNA template, can interfere with
cancer cell growth-when such a
mutant was expressed in prostate
or breast cancer cells, the cells
became apoptotic.

Genetic approaches have
demonstrated that mice
homozygous for a deletion of the
RNA subunit of telomerase are
viable and fertile was surprising.
However, after four to six
generations defects began to
appear in the telomerase-null mice
as their very long telomeres (40-60
kb) became significantly shorter;
the defects included depletion of
tissues that require high rates of
cell division, like skin and intestine,
and infertility.

Skin papilloma induced by a
combination of chemical
carcinogens occurs 20 times less
frequently in mice lacking a functional
telomerase than in normal mice,
presumably because p53-triggered
apoptosis is induced in response to the
ever shortening telomeres of cells that
have begun to divide.

If both telomerase and p53 are absent,
there is an increased rate of epithelial
tumors such as squamous-cell
carcinoma, colon, and breast
cancer.

Mice with an APC mutation
normally develop colon tumors,
and these too are reduced if the
mice lack telomerase.