3. Replication - DNA
replica,on‐computer
anima,on
...

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Unformatted text preview: DNA
replica,on‐computer
anima,on
 Replica,on:
 I:
Ge7ng
started
 II:
Elonga,on
 II:
Telomerase
and
the
end
game
 The
replica,on
machinery
 What
is
an
origin?
 In
bacteria
it
is
a
specific
DNA
sequence,
oriC
and
 is
unique
in
the
chromosome.
 What
specific
proper,es
does
it
need?
 Figure 06-04 Separa,ng
strands
of
duplex
DNA
requires
energy
 Energy
 What
features
of
an
origin
facilitate
stand
separa,on
in
vivo,
ie,
 how
do
cells
reduce
the
energy
barrier
of
strand
separa,on?
 DnaA
is
a
AAA+
ATPase
that
binds
DNA.
 DnaA
filament
 DnaA
monomer
 DNA
unwinding
element
 It
uses
the
energy
of
ATP
binding
to
wrap
DNA
in
posi,ve
 supercoils.
This
induces
local
nega,ve
supercoiling
in
the
A:T‐rich
 DNA
unwinding
element,
facilita,ng
“mel,ng”
of
the
origin.
 DnaC
then
recruits
DnaB,
the
replica,ve
helicase
 Figure 06-21 The
replica,ve
helicase
from
Papilloma
virus,
 another
AAA+
ATPase
 The
central
channel
can
 accommodate
only
a
single
strand
 of
DNA
 ATP
binding
site
 Figure 06-11 Biochemical assay for DNA helicase activity ©1997 by American Society for Biochemistry and Molecular Biology Karow J K et al. J. Biol. Chem. 1997;272:30611-30614 The
replica,on
machinery
 Finding
a
needle
in
a
hay
stack
 How
do
you
figure
out
what
defines
a
eukaryo,c
origin
of
replica,on?
 Seeing
replica,on
forks
in
vivo
 but….…how
do
we
go
from
here
to
iden,fying
the
DNA
sequence?
 Figure 5-29 Molecular Biology of the Cell (© Garland Science 2008) Iden,fying
physical
regions
of
a
genome
that
 contain
origins
using
DNA
microarrays
 Box Figure 06-01-03 Eukaryo,c
genomes
have
mul,ple
origins
on
 each
chromosome
 Yeast
chromosome
III
has
19
origins;
green=origins
used
90%
of
S
phases
 The
replica,on
machinery
 Iden5fying
an
origin
by
func5on
 The
replica,on
machinery
 In
budding
yeast
origins
have
defined
sequence.
 But
mammalian
origins
are
s5ll
poorly
understood
 LCR
 The
Locus
Control
Region
(LCR)
controls
transcrip,on
of
the
 globin
locus,
50,000
nt
away.
Dele,on
of
the
LCR
is
found
in
an
 inherited
Hispanic
β‐thalassemia
in
which
β‐glogin
is
not
 expressed
and
the
origin
in
this
locus
fails
to
fire.
 Origin
“firing”
in
eukaryotes
is
coupled
to
the
cell
cycle
 The
clamp
increases
DNAP
processivity
and
speed
 E. coli ß dimer human PCNA trimer The prokaryotic and eukaryotic versions are almost identical in structure Loading
the
clamp:
how
ATP
binding
and
hydrolysis
drive
 unidirec,onal
conforma,onal
changes
 ATP-bound form has binding site for RNA primer-template jtn Only ATP-bound conformation can bind clamp 1.  ATP binding is generally associated with the assembly stage 2.  ATP hydrolysis is typically involved in the disassembly step ATP hydrolysis releases both clamp and DNA Cells can control the timing of complex events by requiring that the trigger for ATP hydrolysis be the formation of a key intermediate In
clamp
loaders,
a
class
of
AAA+
ATPases,
an
arginine
residue
is
cri,cal
 to
complete
the
ac,ve
site
of
the
ATPase.
It
is
poised
just
out
of
range
 un,l
the
complex
of
clamp
loader,
open
clamp
and
ATP
recognizes
the
 end
of
a
primer.
Similar
poised
mechanisms
work
in
GTPases.
 DNA
replica,on‐computer
anima,on
 Trombone
model
for
replica,on
fork
 Two
polymerases
are
tethered
to
the
fork
bycomplex
 Clamp
releases
DNAP
as
soon
as
it
runs
into
a
 double
stranded
region
 How
fast,
in
revolu,ons
(turns)
per
minute,
 does
helicase
spin
the
DNA?
 Replica,on
of
covalently
closed
circles
produces
 posi,vely
supercoiled
interlocked
circles
 Finishing the ends: Linear chromosomes pose challenges for completion of replication Eukaryotic telomere sequences are not random and are species-specific • Not
random
implies
that
sequence
is
templated
somehow
 • Species‐specificity
implies
the
template
source
is
different
between
 organisms‐can
mutate
and
evolve.
 Telomerase turns out to contain an essential RNA-the sequence of which varies btw species Telomerase RNA is the template for DNA synthesis What
type/class
 of
enzyme
is
this?
 Completion of the lagging strand requires removal of the RNA primer by a special repair polymerase
 The last step in lagging strand synthesis is ligation of the DNA pieces by DNA ligase DNAP cannot initiate DNA synthesis from 2 nucleotides: Primers are required for re-inititation on the lagging strand Primase is an RNAP
 ...
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This note was uploaded on 12/05/2011 for the course BIO 344 taught by Professor Herrin during the Spring '08 term at University of Texas at Austin.

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