Documente Academic
Documente Profesional
Documente Cultură
RECOMBINAREA ADN
Prof. Dr. Marieta COSTACHE
Experiment
MeselsonStahl
Experimentul MeselsonStahl
Replicarea (continuare)
n momentul replicrii unei
molecule de ADN, catenele sale sunt
deprtate pentru a forma una sau
mai multe furci de replicare n
form de Y.
Enzima ADN
polimeraza care se poziioneaz la
nivelul
fiecrei
furci
este
responsabil de sinteza noii catene
de ADN complementar cu fiecare
din catenele parentale, sintetiznd
astfel dou molecule de dublu
helix ;
Majoritatea
replicrii ADN este
bidirecional
Demonstrarea creterii
bidirecionale
prin
autoradiografie: dac celulele
replicative n cultur sunt
expuse unei concentraii mari
de timidin [3H], ADN obinut
va fi puternic marcat (hot) la
nivelul originii de replicare i
mai slab n apropierea acesteia
(warm). Prin autoradiografie
au fost obinute profilele b care
confirm ipoteza din figura a.
Replicarea
la
Drosophila
Sinteza la
nivelul
catenei
ntrziate
Correctly paired
bases are required
for DNA polymerase
catalyzed nucleotide
addition
Correctly paired bases are required for DNA polymerase catalyzed nucleotide
addition. a) Schematic diagram of the attack of a primer; 3'OH end on a correctly base-paired
dNTP.
b) Schematic diagram of the consequence of incorrect base-pairing on catalysis by DNA
polymerase. In the example shown, the incorrect A% base pair displaces the a-phosphate of the
incoming nudeotide. This incorrect alignment reduces the rate of catalysis dramatically resulting
in the DNA polymerase preferentially adding correctly base-paired dNTPs.
Schematic illustration of
the steric constraints
preventing catalysis
using rNTPs by
DNA polymerase.
Schematic illustration of the
steric constraints preventing
catalysis using rNTPs by DNA
polymerase. a) Binding of a
correctly base-paired dNTP to
the DNA polymerase. Under
these conditions, the 3"OH of the
primer and the (x- phosphate of
the dNTP are in close proximity.
b) Addition of a 2'OH results in a
steric dash with amino acids (the
discriminator amino acids) in
the nudeotide binding pocket.
This results in the c -phosphate
of the dNTP being displaced and
a misalignment with the 3'OH of
the
primer,
dramatically
reducing the rate of catalysis
Elongation of a
DNA chain
An example of error correction by the 3'^5' exonuclease activity of DNA polymerase I. Structural analysis has located the exonuclease
activity ahead of the polymerase activity as the enzyme is oriented in its movement along the DNA. A mismatched base (here, a C-A
mismatch) impedes translocation of DNA polymerase I to the next site. Sliding backward, the enzyme corrects the mistake with its
3'-5' exonuclease activity, then resumes its polymerase activity in the 5'>3' direction.
Nick translation
Mechanism of the DNA ligase reaction. In each of the three steps, one phosphodiester bond is formed at the expense
of another. Steps CD and lead to activation of the 5' phosphate in the nick. An AMP group is transferred first to a
Lys residue on the enzyme and then to the 5' phosphate in the nick. In step , the 3'- hydroxyl group attacks this
phosphate and displaces AMP, producing a O Ribose Adenine phosphodiester bond to seal the nick. In the E.
coli DNA ligase reaction, AMP is derived from NAD+. The DNA ligases isolated from a number of viral and
eukaryotic sources use ATP rather than NAD+, and they release pyrophosphate rather than nicotinamide
mononucleotide (NMN) in step .
Chromosome partitioning
in bacteria
DNA polymerases
synthesize DNA in a
processive manner
Action of topoisomerase
at the replication fork
45
49
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51
52
53
54
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Replicarea la drojdii
DNA polymerase
switching during eukaryotic
DNA replication
DNA polymerase switching during
eukaryotic DNA replication. The order
of DNA polymerase function is
illustrated. The length of the DNA
synthesized is shorter than in reality
for
illustrative
purposes.
AlthoTypically the combined DNA Pol
alpha/primase product is between 50100 bp and the further extension by
Pol epsilon or Pol delta is between 100
and 10,000 nucleotides. Both DNA Pol
delta and epsilon can substitute for
DNA Pol alpha/primase, it is likely that
they function in the replication of
specific DNA strands (leading or
lagging). Current studies have yet to
determine which polymerase functions
on which strand, however.
The -subunit of the clamp loader interads with both the DNA helicase and the DNA polymerase at the
replication fork. (a) When this interaction is made, the DNA helicase unwinds the DNA at
approximately the same rate as the DNA polymerases replicate the DNA. (b) If the DNA helicase is not
associated with DNA Pol III holoenzyme, DNA unwinding slows by tenfold. Under these conditions, the
DNA polymerases can replicate faster than the DNA helicase can separate the strands of unreplicated
DNA. This allows the DNA Pol III holoenzyme to "catch up" to the DNA helicase and the reformation
of a full replisome.
SeqAbound
to hemimethylated DNA
inhibits reinitiation from
recently replicated
daughter origins.
SeqAbound
to hemimethylated DNA
inhibits reinitiation from
recently replicated
daughter origins
(d) Bound SeqA protein inhibits the
full methylation of these sequences
and the binding of onC by DnaA
protein (for simplidly, only one of
the daughter molecules is illustrated
in parts d, e, and f). (e) When SeqA
infrequently disassodates from the
GATC sites, the sequences can
become fully methylated by Dam
DNA methyl transferase, preventing
rebinding by SeqA. (f) When the
GATC
sites
become
fully
methylated, DnaA can bind and
direct a new round of replication
from the daughter oriC replicators.
The Replication
Factory Hypothesis
Chromosome breakage
as a result of incomplete DNA replication
Replicators are
inactivated by DNA
replication
A chromosome with five replicators is
shown. The replicators labeled 3 and 5
are the first to be activated, leading to the
formation of two pairs of bidirectional
replication forks. Activation of the
parental replicator results in the
inactivation of the copies of each
replicator on both daughter DNA
molecules until the next cell cycle
(indicated by a red X). Further extension
of the resulting replication forks replicates
the DNA overlapping with the number 2
and 4 replicators. When a replicator is
copied by a fork derived from an adjacent
origin prior to initiation, it is said to have
been passively replicated. Although these
replicators have not initiated, they are
nevertheless inactivated by the act of
replicating their DNA.
Structura ADN in
telomeri
Baze modificate
chimic: dimeri timintimin
Mecanismul UvrABC
de excizie-repair a
ADN la E. coli
Excision Repair
Thymine dimers can be removed from DNA by excising them (cutting them out),
using a complex of proteins called the uvrABC system. The sequence of events is
shown in the figure below:
Modaliti de ndeprtarea
a Uracilului
Photoreactivation
Pyrimidine dimers can be
eliminated by photoreactivation,
using the energy of light to split
the dimer apart. The enzyme
reaction is diagrammed in this
figure:
The enzyme is called
photolyase and uses visible light
(l = 370 nm) to oxidize the
cyclobutane ring. The light
energy is harvested by a
chromophore and that energy is
used to produce a free radical
(FADH) that carries out the
oxidation, returning the DNA to
its normal state. Examples of
these enzymes are found in both
prokaryotic and eukaryotic
systems.
Repararea capetelor
ADN neomolog
Prezentare (1)
n timpul recombinrii omologe, dou molecule ADN duplex sunt
scindate i catenele sunt schimbate. Acest proces, care apare aleator pe toat
lungimea genoamelor tuturor organismelor, joac un rol important n generarea
diversitii genetice;
Ruperea dublu-catenelor iniiaz n majoritatea cazurilor recombinare
omolog. Ruptura devine se lrgete devenind bre formnd capete 3 de
recombinare care invadeaz alte duplexuri. Sinteza reparatorie a regiunilor lips
formeaz un intermediar care conine dou jonciuni Holliday ncruciate.
Rezolvarea acestui intermediar apare prin rotaie urmat de clivarea i ligarea
celor dou catene ale fiecrei jonciuni Holliday.
La E. coli captul de recombinare creat de complexul enzimatic RecBCD
este stabilizat prin legarea proteinei RecA. Catalizat de RecA, captul
monocatenar 3 se mperecheaz apoi i invadeaz un segment de duplex omolog,
formnd un intermediar coninnd dou regiuni de heteroduplex ADN i dou
jonciuni Holliday. Dup migrarea ramurii, catalizat de ctre RuvA i RuvB,
dou catene ale fiecrei jonciuni sunt tiate de ctre endonucleaz RuvC i apoi
ligate pentru a forma dou molecule ADN duplex
Prezentare (2)