06_01_Hereditary info'jpg

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DNA REPLICATION DNA double helix contain a
sequence of the nucleotides that is complementary
to the nucleotide sequence of its partner
strand. Each strand can be therefore act as
template for the synthesis of a new complementary
strand. The ability of each strand of a DNA
molecule to copy to act as a template for
producing a complementary strand enables a cell
to copy, or replicate itself before passing them
into descendants.
complementary new DNA strand
(daughter strand)
tepmplate
A pairs with T, G pairs with C Billions of
nucleotides. How copying is done in an accurate
and constant speed? --by a cluster of proteins
that form a replication machine.
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Replication machine produces two complete double
helices from the original DNA molecule.
Replication is semiconservative each parental
strand serve as the template for the new strand,
each of the daughter DNA double helices ends up
with one of the original strands plus one strand
that is completely new.
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DNA synthesis begins at replication origins DNA
helix is very stable due to large numbers of
hydrogen bonds between bases of each strand.
Boiling temp. is required to separate strands
for replication . But in the cell, how it is
achieved? --- initiator proteins that saparate
two strands a part by breaking the hydrogen bonds
between bases one by one. hydrogen bonds
collectively make DNA strand stable but
individually hydrogen bond but is
weak. Replication origins the positions at
which DNA is first open.
--- particular sequences that attract the
initiator proteins.
--- streches of the DNA that are especially easy
to open. A-T reach sequences.
--- in eucaryotes multiple replication
origins. (10.000 origins)
--- in bacteria single replication origin.
Approximately 100 bp.
---initiator proteins bind DNA at the
replication origin and locally open up the DNA
double helix,
it attracts a group of proteins that carry out
the replication--- protein machine
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New DNA synthesis occurs at replication
forks Replication forks (Y shaped junctions in
the DNA) move away in both directions from
multiple replication origins in an eucaryotic
chromosome.--- bidirectional
Process being replicated in electron microscope
Replication speed in 1000 nucleotide pairs /sec
in bacteria but 100 nucleotide pairs/sec in
humans. Why? ---due to more complex chromatin
structure in higher organisms. Replication
Machine DNA polymerase which synthesizes new
DNA using one of the old strands as a template.
--- catalyzes the
additionof nucleotides to the 3 end of a growing
DNA strand by forming a
phosphodiester bond between this end and the
5- phosphate group of the incoming
nucleotide. DNA synthesis from
5 to 3.
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DNA is synthesized in the 5 to 3
direction Nucleotides enter the reaction are
energy-rich nucleoside triphosphates (hydrolysis
of one phosphoanhydride bond in the nucleoside)
--- provides energy for the polymerization of the
reaction that links the nucleotide monomer to th
chain and releases pyrophosphate (PPi). PPi is
further hydrolyzed to inorganic phosphate (Pi)
which makes the polymerization reaction
reversible. DNA moves along the template
strand, does not dissociate from DNA each time it
adds a new nucleotide to the chain.
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Replication fork is asymmetrical
5 to 3 synthesis by Dna polymerase --- no
problem. But how is the synthesis of other strand
on another strand is done. 3 to 5 ? Can DNA
polymerse add nucleotides from 5 to 3, is there
such enzyme? No. How problem is solved?
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The DNA strand whose 5 end must grow
discontinuously. Okazaki fragments short DNA
fragments produced as DNA replication
intermediates that are subsequently joined to
form a single continuous strand of DNA --- DNA
polymerase 5 to 3 reaction. DNA strand that is
synthesized discontinuously is called lagging
strand. DNA strand that is synthesized
continuously is called leading strand
animation
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DNA polymerase is self correcting DNA polymerase
can make one error in every 107 nucleotide pairs
it copies. G-T or C-A instead of A-T or
G-C. Accumulation of errors will kill the
organism. So how this errors are corrected? DNA
polymerase has an self error-correcting activity
called proofreading. Before the enzyme adds a
nucleotide to agrowing Dna chain, it checks
whether the previous nucleotide added is
correctly base- paired to the template
strand. If so, the polymerase adds the next
nucleotide. If not, it removes the mismatched
nucleotide by cutting the phosphodiester bond it
has just made, releases the nucleotide and tries
again.
5 to 3 polymerization activity
3 to 5 exonuclease activity
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DNA polymerase contains separate sites for DNA
synthesis and editing ExampleThe structure of
an E. Coli DNA polymerase molecule
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A need for proofreading explains why DNA chains
are synthesized only in the 5 to 3 direction
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Short lengths of RNA act as primers for DNA
synthesis Accuracy of DNA replication depends on
the DNA plymerase activity. But DNA polymerase
can join a nucleotide only to a base-paired
nucleotide in a DNA double-helix, it can not
start a completely new DNA strand. Different
enzyme is required to begin a new strand DNA
primase that synthesizes the RNA primer.
--- can begin a new DNA strand simply
by joining two nucleotides together without the
need for a base- paired
end. --- does not synthesize
DNA, it synthesizes short length (10 bp) of RNA.
--- short length of RNA
base-paired to the template provides a
base-paired 3 end as a starting point for
DNA synthesis --- primer for
DNA synthesis. --- leading
strand--only one primer at replication origion.
lagging strand new
primers are required for each okazaki
fragment. Since RNA is chemically similar to DNA
but different sugar is ribose not deoxiribose,
contains base U instead of T. Removal of RNA and
replacement with DNA for continuous replication
requires three enzymes Nuclease removes RNA
primer Repair polymerase (a DNA
polymerase)replaces RNA with DNA (using the
adjecent okazaki fragment as a primer. DNA
ligase joins the 5 phosphate end of one new DNA
fragment tothe 3-hydroxyl end of the next.
ATP or NADH is required for
ligase activity. It is not known yet that these
three enzymes are the part of the replication
machine. But could be.
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On the lagging strand, DNA is synthesized in
fragments
nuclease repair polymerase
DNA ligase
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Proteins at a replication fork cooperate to form
a replication machine DNA polymerase Helicase a
protein that uses the energy of ATP hydrolysis
and unwinds the DNA helix into single strands
during replication. Single-strand binding
protein clings to the single-stranded Dna
exposed bythe hlicase and transiently prevents it
from re-forming base pairs. Sliding clamp keeps
DNA polymerase firmly attached to the DNA
template.---releases DNA polymerase at the end of
each okazaki fragment in lagging strand.
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06_18_telomeres.jpg
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TELOMERES
During mitosis, cells make copies of their
genetic material. Half of the genetic material
goes to each new daughter cell. To make sure that
information is successfully passed from one
generation to the next, each chromosome has a
special protective cap called a telomere located
at the end of it's "arms". Telomeres are
controlled by the presence of the enzyme
telomerase. Now that we have covered some basics,
let's explore telomeres, telomerase, and their
importance to you! (view animation)      A
telomere is a repeating DNA sequence (TTAGGG) at
the end of the body's chromosomes. The telomere
can reach a length of 15,000 base pairs.
Telomeres function by preventing chromosomes from
losing base pair sequences at their ends. They
also stop chromosomes from fusing to each other.
However, each time a cell divides, some of the
telomere is lost (usually 25-200 base pairs per
division). When the telomere becomes too short,
the chromosome reaches a "critical length" and
can no longer replicate. This means that a cell
becomes "old" and dies by a process called
apoptosis. Telomere activity is controlled by two
mechanisms erosion and addition. Erosion, as
mentioned, occurs each time a cell divides.
Addition is determined by the activity of
telomerase.
Telomerase, also called telomere terminal
transferase, is an enzyme made of protein and RNA
subunits that elongates chromosomes by adding
TTAGGG sequences to the end of existing
chromosomes. Telomerase is found in fetal
tissues, adult germ cells, and also tumor
cells. Telomerase activity is regulated during
development and has a very low, almost
undetectable activity in somatic (body) cells.
Because these somatic cells do not regularly use
telomerase, they age. The result of aging cells
is an aging body. If telomerase is activated in a
cell, the cell will continue to grow and divide.
This "immortal cell" theory is important in two
areas of research aging and cancer.
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DNA REPAIR Mutations can have severe
consequences for an organism Mutation A
permanent change in the DNA due to a failure in
DNA replication or DNA repair mechanisms. Even a
single nucleotide change may cause severe
diseases. example a life threatening disease,
sickle cell anemia. --- amino acid
sequence change in ?-globin chain.
--- red blood cells are more fragile and break
frequently in blood stream, reduced number of
cells --- can cause weakness,
dizziness, pain etc.
Cells from sickle cell anemia patient
Normal red blood cells
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Mutations in the DNA accumulate during the years
and incidence of cancer increase as a function of
age. example incidence of colon cancer in
women in England.
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Replication machine corrects the mismatches
durin replication but some times may
fail Replication machine corrects the errors but
may fail miss some errors --- approximately one
error per 107 nucleotides copied. If mismatch is
not corrected, it remains as a permanent mutation
No mutation
Permanent mutation Both DNA molecules will
have
mutations
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DNA mismatch repair system removes replication
errors that escape the replication machine during
replication DNA mismatch repair mech. correct
99 of these mistakes.---- increase the accuracy
to one mistake in 109 nucleotides. A complex of
mismatch repair proteins recognizes the DNA
mismatches that escaped from replication machine,
removes (excises) one of the two strands of DNA
involved in the mismatch, and resynthesizes the
missing strand. Only newly synthesized strand
must be excised. How? --- Nicks (single-stranded
breaks) in the newly synthyesized lagging and
leading strand as signals.
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DNA is continuesly suffering damge in
cells Other mechanisms are required for other
various damages which occur on the
DNA. Depurination purine bases (A and G) are
lost spontaneously due to thermal collision of
molecules with DNA ---
approximately 1012 purine bases will be lost
--- depurination does not break
the phosphodiester bond, but loss of a puirne
from the DNA. Deamination spontaneous loss of
an amino group from from cytosinein DNA to to
produce uracil due to thermal
collisions of molecules with DNA.
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Tymine dimer covalent linkage of adjecent
tymine bases in DNA due to reaction of
chemically by products of metabolism or sunlight
. They alter them in such a way that their
base-pairing properties are chaged.

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If these damages are not repaired, they will lead
either the substitution of one nucleotide pair by
another as a result of incorrect base pairing
during replication or to deletion of one or more
nucleotide pairs in daughter DNA strand after DNA
replication.
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The basic mechanism of DNA repair Three steps
excision (nuclease)
resynthesis ( DNA polymerase)
ligation (DNA ligase)
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DNA RECOMBINATION
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