Chromosomes & Genomes

• Chromosomes
'
– complexes of DNA and proteins – chromatin
– Viruses – linear, circular; DNA or RNA '
– Bacteria – single, circular
~
– Eukaryotes – multiple, linear .

↳
• Genome
– The genetic material that an organism possesses
– Nuclear genome
– Mitochondrial & chloroplasts genome
 Bacterial Chromosomes

c > Proteins
d
binds to chromosomes
1000 fold)
→ copacts the chromosome (
cell
→ fits in the

• Chromosomal DNA is compacted ~ 1000 fold

to fit within cell
 Bacterial Chromosomes
• Size
– Escherichia coli
~ 4.6 million bp base pairs

– E. coli ~6000 genes where

sequence of DNA

www.nu .ip.ep.aein.J
→

– Genes encoding proteins for replication is initiated on a

related functions arranged in

transcribed
chromosome

↳ replication proceeds in both

operons ]m.eu
genes that are
terminates
.
together give
to a single MRNA directions c
then

, →

– Single origin of replication

(Ori)
– Intergenic regions
nontranscribed DNA - ↓
> repetitive
sequence
no
→

genes
genes that
are transcribed
Supercoiling of topologically constrained DNA
The coiling (or wrapping) of duplex DNA around its own axis is called supercoiling.

✓ (solenoidal)
this happens when
>
of wrap
→ to get rid DNA strands
→
!
a super
coil
around histones
to cut
have
you DNA
the
open
c strand

↓
Coil is coiled

A left-handed toroidal (solenoidal) supercoil, at left, will

convert to a right-handed interwound (plectonemic)
supercoil, at right, when the cylinder core is removed.
 Different topological forms of DNA
Topologically closed DNA can be circular (covalently closed circles)
or loops that are constrained at the base.

supervised
fixed

Electron micrographs of circular duplex DNAs. Their conformations

vary from no supercoiling (left) to tightly supercoiled (right).
 Separation of relaxed and supercoiled
when it's less supercoiled
closer to the relaxed
→ goes
↑ end

→ more relaxed
DNA Strand → goes
up

more super coiled

→ goes down
↓
^

Relaxed circles are more energetically stable than supercoiled circles.

Relaxed and supercoiled DNA topoisomers are resolved by gel electrophoresis.

 Supercoiling
Supercoiling results from strain on the molecule caused by under-
or over-winding the double helix. When the ends of a linear DNA molecule are ligated
to produce a covalently closed circle, the two strands
become intertwined like the links of a chain, and will
remain so unless one of the strands is broken.

The equation The number of times one strand is linked with the other
is described by a fundamental property of DNA
supercoiling, the linking number (L).

This is related to two geometrical

←
L=T+W ↓ ↓
properties of the molecule, the twist (T,
rotation of the strands about the helical
linking
twist writhe axis)
number

and the writhe (W, which measures the path of the

helix axis in space).
Understanding of twist and writhe
Twist vs. Writhe

Bent

Twisted

http://www-dna2006.cea.fr/lectures/Roca1.pdf

It takes less energy to bend than to twist DNA.

Supercoiling relaxes a twisting into a bending deformation.
↳ even when twisted , it would start to

bend → less energy

 ←
L=T+W↓
→ writhe
linking twist
Linking number number

The only way to change the linking number is to cut the DNA.
Figures from:
http://www.biophysics.org/education/vologodskii.pdf#search=%22vologodskii%22
 L=T+W
Explaining the equation
L = number of links between two circles

L is a fixed topological property of a closed circle of DNA fixed ! not changed

open !
Unless DNA was cut

Linking number = Twist + Writhe

T = number of times the strands are twisted around

each other along the helical axis

W = number of times the helix axis twists around itself → No . of times it super coiled

→ w=0
T = L when W = 0 ↓
no super coils

Approximation:
10 pairs makes
T = #bp/10.5 always → because 1
base
full turn
Thus ΔW = ΔL - (#bp/10.5)

L can never be changed as long as no topoisomerase is used, and there is no nicks in

DNA. → fixed!! Changes if DNA was cut
 L=T+W
Example Problem #1
÷ to → because 10 bp per turn

Draw a 3150 bp circular plasmid with two negative supercoils.

What are the values of T, L, and W? W=_ 2

T = 3150/10.5 = 300
W = -2
C- 2) 298
L = T + W = 298 →
=
+
300
 L=T+W
Example Problem #2
÷ 10

Start with 210 bp linear DNA. What is the T, W, and L?

T = 210/10.5 = 20 ↳ rsnosupercoils !
W=0 W=O
L = T + W = 20 20+0=20
W= -2

Now, wrap the DNA in two left-handed turns around a solenoid (nucleosome)
-

Join the ends, remove protein. What is T, W, and L now?

✓

still ← T = 20, W = -2, L = 18 → left -

20

↓
Get a negative (right-handed) plectonemic supercoil handed turns

because ↓
18 → Positive
we took the 20 + C-2) =

number because
same DNA
it's piectonemic →" ~

wrapped
Right handed
strand 3. -
"

it around a becomes -

f Piectonemic

É¥
nucleosome
-
Effect of Ethidium Bromide on DNA Supercoiling

intercalated <

middle of base pairs

unwind
↓
T↑ W↓

Ethidium intercalation unwinds the double helix by about 26 degrees.

 Topoisomerases enzymes for
crucial

many fundamental
aspects

↳ they cleave DNA strands

Functions of topoisomerases in order to provide torsional -
stress
DNA replication
>
is carried out relief → or to untangle DNA
in both directions

sometimes ,
>
while it 's untangling
the DNA it, gets stuck .

(can't get rid of the tangle)

↳ topoisomerases go to the sight
of the tangle } untangles it !

Topoisomerases can relieve negative superhelicity behind polymerases

and helicases, and can relieve positive superhelicity in front of
polymerases and helicases. → they can get rid of super coils }
the
return them back to
were
way they
.
 Type I Topoisomerases
L changes
↓
because you're
the DNA
cutting
the other
strand goes ↓
/ the space
seals it
if L is

- changing

/ the 73W
back

are changing

cuts 1- Strand
of DNA

@ ⊖
Bacterial Type I (Type IA) topoisomerases are specific for relaxing negatively
supercoiled DNA only. W= → -

Eukaryotic Type I (Type IB) topoisomerases have a different mechanism and

are specific for relaxing either negatively or positively supercoiled DNA.
 Type II topoisomerases

7
r

/ Right
left handed
handed
<
cuts
through
BOTH
DNA Strands

Type II topoisomerases require ATP hydrolysis, and can

introduce negative supercoiling into DNA.
↳ it turned a left handed supercoil into
a right handed supercoil
 Type II topoisomerases (DNA Gyrase)

+
> cannot generate
super coils → cannot
make a left handed
Plectonemic Supercoil

using topoisomerases

↓
↓ has to do with the
it requires way that the active
ATP hydrolysis site of the enzyme is

Positioned 3 how it
→ you don't need interacts with a

relax a C- )
energy to relaxed circle to

supercoil
→ but YOU this model
generate
need energy to return
it to the way it was → w = +

(Negative Supercoiled DNA) → w= -

why is it negative if 1-
it is

right handed ?

because → when
wrapped around turns into
µ a nucleosome (solenoid) ↳Piectonemicsupercoil
right handed
,
↳ 2 left handed turns (w
-
= -

a) .É¥¥
Example of topoisomerase activity
righthanded
/Plectonomic super -

coil → w= -

→
1 relaxed
double stranded
,
DNA

7
✓ to poisoner was
linking number
not used to
is changed
↓ DNA
> relax the
DNA is
being strands
cut .

linking number
< >
is changed

3¥ → linking no .

back
not changed
L goes ←

to 32 be caused
it is in the
W decreased by
y
↓ relaxed ↓
because it's
when W stated
in a relaxed
is decreased
↓ state
by 4 Lis
T↓W↑-
,

decreasedby
4!
 Clinical Relevance
> it 's inhibited

↳ cell dies (only )

cancer cells

➢The cancer drug Etoposide (VP-16) inhibits

topoisomerases and is widely used in the treatment of
lung, ovarian, testicular, and prostate cancer.

➢Quinolone antibiotics, such as ciprofloxacin, inhibit

DNA gyrase, a topoisomerases found only in bacteria,
and are used for numerous infections, including
complicated urinary tract infections and lower
respiratory tract infections. ↳ that's they prescribe why
with
antibiotics to patients
bacterial infections
!
Homework
1.What is DNA supercoiling? the amount of strain
amount of twist in a particular DNA strand → determines
on it .

2. What is the relationship between the linking number and the twist of
DNA ? any change in the twist of the DNA will result in an equal and opposite change in the writhe (if the linking number is
fixed) and any change in linking number results in a change in the twist and/or writhe

3. A plasmid is in a closed circular double-stranded DNA conformation

and has 4,200 base pairs.
A. How many helical turns (twist) are there in this relaxed molecule? 4%-0=420
B. What is the linking number of the molecule when it is relaxed B-DNA? W=O 420+0
420 1=420
=

C. How do T and L change if this plasmid changes its structure from a B-DNA to I

an A-DNA form? form B- form B form

42¥ 420 L 4201-0
T=
a- =
= =

420 =

Aform =
4Z 382 1=3821-0
T =
=

4. What effect does ethidium bromide intercalation have on supercoiled=3 82

DNA? intercalated
TA W↓
middle of base pairs 7
unwind -

5. What is the function of Topoisomerases?

enzymes for
crucial
fundamental aspects
many
↳ they cleave DNA strands
in order to provide torsional -
Stress
relief → or to untangle DNA