Lecture
15:
DNA
and
Replication
1.2
DNA
Replication
●
Chargaff’s
Rule:
percentages
of
A/T
and
C/G
are
approximately
equal
DNA
Nucleotides
●
DNA
double
helices
are
composed
of
DNA
nucleotides
w
3
components:
5-carbon
deoxyribose
sugar,
phosphate
group,
and
a
nucleotide
base
(A,
C,
T,
or
G)
●
Nucleotides
are
linked
together
by
phosphodiester
bonds
b/n
5’
phosphate
group
of
one
nucleotide
and
the
3’
hydroxyl
group
of
the
next
nucleotide
○
Alternates
deoxyribose
sugar
and
phosphate
groups
along
strand
→
sugar-phosphate
backbone
●
Nucleotide
bases
are
hydrophobic,
orient
towards
interior
of
helix
●
Hydrogen
bonds
join
the
2
strands
into
a
helix
○
2
hydrogen
bonds
for
A–T
pairs
○
3
hydrogen
bonds
for
G–C
pairs
●
Strand
Polarity
is
established
by
the
5’
phosphate
group
and
3’
hydroxyl
group
at
the
end
of
each
strand
○
Complementary
DNA
strands
run
antiparallel
–
one
is
5’--3’
and
the
other
is
3’--5’
DNA
Replication
●
DNA
replication
generates
2
identical
DNA
duplexes
from
original
strand
●
Semiconservative
Replication:
2
complementary
strands
separate,
each
strand
acts
as
template
for
new
complementary
strand
●
Replication
begins
at
origin
of
replication
,
where
H
bonds
break
●
DNA
Polymerases
are
the
enzymes
active
in
replication
○
Identify
nucleotide
complementary
to
unpaired
nucleotide
in
parent
strands,
catalyze
formation
of
phosphodiester
bond
to
join
new
nucleotide
to
parental
one
●
Strands
only
elongate
in
5’
→
3’
direction
○
Nucleotides
only
added
to
3’
end
of
strand
1=]=Lecture
15
DNA
Review
●
Transformation
(1920s
experiment)
○
Discovered
during
pneumonia
studies ○
2
strains
of
bacteria:
S
strain
w/
polysaccharide
capsules
and
is
quickly
lethal,
R
strain
w/o
capsules
and
high
survival
rate
○
Inactivate
S
strain
by
boiling
it,
then
inject
boiled
strain
in
mouse
→
no
death
(bacteria
is
dead)
○
Mix
R
and
boiled
S
strain,
then
inject
→
mouse
gets
infected
and
dies,
living
S
strain
can
be
extracted
from
mouse
■
Genes
are
in
chemical
molecule
■
Molecule
is
not
destroyed
by
heat
and
can
be
transferred
from
boiled
S
strain
into
living
R
strain
○
Tested
which
molecule
is
transforming/carrying
genetic
info
●
How
does
DNA
encode
genetic
info?
○
Important
to
understand
structure
→
function
●
Building
blocks
of
DNA
○
Nucleotides
■
Deoxyribose
■
Phosphate
group
■
Nitrogenous
base
○
2
types
■
Purines:
A
and
G
■
Pyrimidines:
T
and
C
●
Chargaff’s
Rule:
A
=
T;
G
=
C
○
A
+
G
=
T
+
C
■
Purines
=
pyrimidines
○
A
+
T
/=
G
+
C
CLICKER
QUESTION:
A:
ss,
DNA
B:
ds,
DNA
C:
ds,
RNA
D:
ss,
RNA
E:
ds,
DNA
Distinguishing
ss
vs
ds:
look
at
ratio
of
G
to
C
and
A
to
T
SS:
no
base
pairing,
diff
ratios DNA
Structure
●
Double
helix,
bases
on
insides
connected
by
base
pairing
●
Explains
many
properties
of
how
genes
work
○
DNA
function,
properties
of
life
●
Major
and
minor
groove:
○
Sequence-specific
DNA
binding
proteins
bind
to
the
major
groove
■
Interact
w
nitrogenous
bases
○
Non-specific
DNA
binding
proteins
bind
to
backbone
■
Do
not
interact
●
Impact
of
DNA
structure
○
●
Length
of
human
genome
is
1.9m
long!
●
DNA
cannot
twist/rotate
due
to
double
helix
structure
○
If
1
end
of
DNA
is
fixed
and
other
is
rotated
→
helix
accumulates
supercoils
○
○
Physical
obstacle
to
many
processes
■
Replication,
repair,
etc
○
Enzyme
responsible
for
resolving
supercoil
issue
during
replication
DNA
Replication
●
Semiconservative:
2
strands
separate,
and
parental
strands
are
used
as
templates
for
new
strands
to
be
built
●
DNA
Replication
experiment: ○
Diff
nitrogen
isotopes
in
DNA
can
be
identified
using
ultracentrifugation
■
14N
is
naturally
occurring,
15N
is
heavier
isotope
○
Put
tube
in
centrifuge,
concentration
of
added
cesium
chloride
gets
very
high
at
bottom
of
the
tube
and
low
at
the
top
○
Put
DNA
sample
on
top
and
centrifuge
■
DNA
sinks
to
density
of
cesium
chloride
that
is
the
same
as
its
own
■
15N
DNA
sinks
to
bottom,
14N
stays
at
the
top
○
First,
put
all
15N
DNA
in
the
tube
→
all
sinks
in
the
bottom
■
Let
it
replicate
+
repeat
○
Newly
created
DNA
molecules
stayed
in
the
middle
→
indicates
half
is
14N
and
half
is
15N
○
Ran
experiment
again
w/
mixed
14/15N,
then
noticed
that
it
was
~½
each
of
middle
density
and
14N
●
Nucleotides
can
ONLY
be
added
to
3’
end
in
replication,
transcription,
any
process!
●
This
means
when
parent
helix
unwinds,
one
strand
synthesizes
away
from
parent
strand
and
one
synthesizes
towards
it
○
If
direction
of
replication
and
adding
is
the
same,
it
is
called
the
leading
strand
○
If
direction
is
the
opposite,
it
is
the
lagging
strand
produced
using
Okazaki
fragments
●
Replication
begins
with
process
of
initiation
at
the
origin
of
replication
(ori)
○
Specific
AT-rich
sequence
■
A–T
are
only
connected
w/
2
hydrogen
bonds
instead
of
3
→
easier
to
break
apart
15:
DNA
and
Replication
1.2
DNA
Replication
●
Chargaff’s
Rule:
percentages
of
A/T
and
C/G
are
approximately
equal
DNA
Nucleotides
●
DNA
double
helices
are
composed
of
DNA
nucleotides
w
3
components:
5-carbon
deoxyribose
sugar,
phosphate
group,
and
a
nucleotide
base
(A,
C,
T,
or
G)
●
Nucleotides
are
linked
together
by
phosphodiester
bonds
b/n
5’
phosphate
group
of
one
nucleotide
and
the
3’
hydroxyl
group
of
the
next
nucleotide
○
Alternates
deoxyribose
sugar
and
phosphate
groups
along
strand
→
sugar-phosphate
backbone
●
Nucleotide
bases
are
hydrophobic,
orient
towards
interior
of
helix
●
Hydrogen
bonds
join
the
2
strands
into
a
helix
○
2
hydrogen
bonds
for
A–T
pairs
○
3
hydrogen
bonds
for
G–C
pairs
●
Strand
Polarity
is
established
by
the
5’
phosphate
group
and
3’
hydroxyl
group
at
the
end
of
each
strand
○
Complementary
DNA
strands
run
antiparallel
–
one
is
5’--3’
and
the
other
is
3’--5’
DNA
Replication
●
DNA
replication
generates
2
identical
DNA
duplexes
from
original
strand
●
Semiconservative
Replication:
2
complementary
strands
separate,
each
strand
acts
as
template
for
new
complementary
strand
●
Replication
begins
at
origin
of
replication
,
where
H
bonds
break
●
DNA
Polymerases
are
the
enzymes
active
in
replication
○
Identify
nucleotide
complementary
to
unpaired
nucleotide
in
parent
strands,
catalyze
formation
of
phosphodiester
bond
to
join
new
nucleotide
to
parental
one
●
Strands
only
elongate
in
5’
→
3’
direction
○
Nucleotides
only
added
to
3’
end
of
strand
1=]=Lecture
15
DNA
Review
●
Transformation
(1920s
experiment)
○
Discovered
during
pneumonia
studies ○
2
strains
of
bacteria:
S
strain
w/
polysaccharide
capsules
and
is
quickly
lethal,
R
strain
w/o
capsules
and
high
survival
rate
○
Inactivate
S
strain
by
boiling
it,
then
inject
boiled
strain
in
mouse
→
no
death
(bacteria
is
dead)
○
Mix
R
and
boiled
S
strain,
then
inject
→
mouse
gets
infected
and
dies,
living
S
strain
can
be
extracted
from
mouse
■
Genes
are
in
chemical
molecule
■
Molecule
is
not
destroyed
by
heat
and
can
be
transferred
from
boiled
S
strain
into
living
R
strain
○
Tested
which
molecule
is
transforming/carrying
genetic
info
●
How
does
DNA
encode
genetic
info?
○
Important
to
understand
structure
→
function
●
Building
blocks
of
DNA
○
Nucleotides
■
Deoxyribose
■
Phosphate
group
■
Nitrogenous
base
○
2
types
■
Purines:
A
and
G
■
Pyrimidines:
T
and
C
●
Chargaff’s
Rule:
A
=
T;
G
=
C
○
A
+
G
=
T
+
C
■
Purines
=
pyrimidines
○
A
+
T
/=
G
+
C
CLICKER
QUESTION:
A:
ss,
DNA
B:
ds,
DNA
C:
ds,
RNA
D:
ss,
RNA
E:
ds,
DNA
Distinguishing
ss
vs
ds:
look
at
ratio
of
G
to
C
and
A
to
T
SS:
no
base
pairing,
diff
ratios DNA
Structure
●
Double
helix,
bases
on
insides
connected
by
base
pairing
●
Explains
many
properties
of
how
genes
work
○
DNA
function,
properties
of
life
●
Major
and
minor
groove:
○
Sequence-specific
DNA
binding
proteins
bind
to
the
major
groove
■
Interact
w
nitrogenous
bases
○
Non-specific
DNA
binding
proteins
bind
to
backbone
■
Do
not
interact
●
Impact
of
DNA
structure
○
●
Length
of
human
genome
is
1.9m
long!
●
DNA
cannot
twist/rotate
due
to
double
helix
structure
○
If
1
end
of
DNA
is
fixed
and
other
is
rotated
→
helix
accumulates
supercoils
○
○
Physical
obstacle
to
many
processes
■
Replication,
repair,
etc
○
Enzyme
responsible
for
resolving
supercoil
issue
during
replication
DNA
Replication
●
Semiconservative:
2
strands
separate,
and
parental
strands
are
used
as
templates
for
new
strands
to
be
built
●
DNA
Replication
experiment: ○
Diff
nitrogen
isotopes
in
DNA
can
be
identified
using
ultracentrifugation
■
14N
is
naturally
occurring,
15N
is
heavier
isotope
○
Put
tube
in
centrifuge,
concentration
of
added
cesium
chloride
gets
very
high
at
bottom
of
the
tube
and
low
at
the
top
○
Put
DNA
sample
on
top
and
centrifuge
■
DNA
sinks
to
density
of
cesium
chloride
that
is
the
same
as
its
own
■
15N
DNA
sinks
to
bottom,
14N
stays
at
the
top
○
First,
put
all
15N
DNA
in
the
tube
→
all
sinks
in
the
bottom
■
Let
it
replicate
+
repeat
○
Newly
created
DNA
molecules
stayed
in
the
middle
→
indicates
half
is
14N
and
half
is
15N
○
Ran
experiment
again
w/
mixed
14/15N,
then
noticed
that
it
was
~½
each
of
middle
density
and
14N
●
Nucleotides
can
ONLY
be
added
to
3’
end
in
replication,
transcription,
any
process!
●
This
means
when
parent
helix
unwinds,
one
strand
synthesizes
away
from
parent
strand
and
one
synthesizes
towards
it
○
If
direction
of
replication
and
adding
is
the
same,
it
is
called
the
leading
strand
○
If
direction
is
the
opposite,
it
is
the
lagging
strand
produced
using
Okazaki
fragments
●
Replication
begins
with
process
of
initiation
at
the
origin
of
replication
(ori)
○
Specific
AT-rich
sequence
■
A–T
are
only
connected
w/
2
hydrogen
bonds
instead
of
3
→
easier
to
break
apart
