 Structure of nucleic acids

 Structure of Chromatin - Introduction to

epigenetics

 Molecular biology tecniques

 DNA duplication
prokariotes, eukariotes,
(initiation, elongation, termination)

 DNA/RNA transcription
eukariotes, prokariotes
 (initiation, elongation, termination)

 RNA/protein translation
eukariotes, prokariotes
 (initiation, elongation, termination)
 Genetic code
 mRNA processing
 (capping, splicing, poli A addition)

 DNA mutations and repair

 Regulation of transcription in
prokariotes
 Regulation of transcription in
eukariotes

 Epigenetics
 Regulatory RNAs

 Homologous recombination
 Site specific recombination
The Meselson-Stahl
experiment.

a)Cells were grown for many generations

in a medium containing only heavy
nitrogen, 15N

(b) Once the cells had been transferred to

a medium containing only light nitrogen,
14N, cellular DNA isolated after one

generation equilibrated at a higher position

in the density gradient (purple band).

c) A second cycle of replication yielded a

hybrid DNA band (purple) and another
band (red), containing only [14N]DNA,
confirming semiconservative
replication.
 How do you expect the different models to appear in the
centrifuge experiment?

Original
DNA

First
replication

Second
replication

Conservative model Semiconservative model Dispersive model

Brooker fig 13.2

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
 Visualization of bidirectional
DNA replication

The heaviest grain density (arrows) is at the two ends,

where replication is occurring. The unreplicated part of
the chromosome, outside the bubble, is unlabeled and
thus not visible
 Defining DNA strands at the replication fork

The leading strand is continuously synthesized in the direction

taken by the replication fork. The other strand, the lagging strand, is
synthesized discontinuously in short pieces (Okazaki fragments) in
a direction opposite to that in which the replication fork moves.
REPLICATION FORK :
Red: newly synthesized DNA
Green: RNA primers
Okazaky fragments can vary between 1000-2000
nucleotides in bacteria and 100 to 400 in eukariotes
Template = DNA strand to
be copied
DNA SYNTHESIS REQUIRES TWO SUBSTRATES:

dNTPs
a particular arrangement of primer and template called
primer: template junction

Only the primer is chemically modified during synthesis

DNA is synthetized by extending the 3’ end of the primer

DNA synthesis is initiated when the 3’-OH of the primer mediates the
nucleophilic attack of the -phosphate of the incoming dNTP.
This results is the extension of the primer and the release of
pyrophosphate
The DNA polymerase monitors the ability of the incoming nucleotide to form a
correct A:T or G:C base pair
 Contribution of
base-pair
geometry to the
fidelity of DNA
replication

The standard A=T and G≡C base

pairs have very similar
geometries, and an active site
sized to fit one (blue shading) will
generally accommodate the other

The geometry of incorrectly paired bases

can exclude them from the active site, as
occurs on DNA polymerase
DNA polymerase cannot use rNTP as precursors
Two metal ions bound to DNA polymerase catalyze
nucleotide addition
The two metal ions (shown in
green) are held
in place by interactions with two
highly conserved aspartate
residues.
Metal ion A primarily interacts
with the 3’-OH, resulting in
reduced association between the
O and the H. This leaves a
nucleophilic 3’O-.

Metal ion B interacts with the

triphosphates of the incoming
dNTP to neutralize their negative
charge.
PROOFREADING
ACTIVITY OF
DNA polymerase I

An example of error
correction by the 3′→5′
exonuclease activity of
DNA polymerase I

The error is a C-A

mismatch

The chance to have a

mismatch is 1 every
100000 nucleotides
incorporated (105)
Proofreading 3’5’ exonuclease activity of DNA polymerases

Increased
affinity of
ssDNA for
exonuclease
site

When an incorrect base pair is made, mismached

nucleotide is removed
 How may DNA polimerases are in E.coli
(prokariotes)?
There are five DNA polymerases in Escherichia coli !!!
I, II , III, IV and V

• The main replicative polymerase is Pol III

• Pol I, plays roles in processing Okazaki fragments and also

in gap-filling during excision-repair processes.

• The other three DNA polymerases are involved into DNA

repair and are induced to higher levels of expression by
DNA mutations.
• Two of them, Pol IV and Pol V are very important into SOS
DNA-damage response also called TLS (translesion
synthesis) while the role of Pol II is not well defined yet.
+ pol IV and pol V for repair
Nick translation by
DNA pol I of E.coli
 3D Structure of DNA pol I resemble a right hand

The bound DNA lies on the palm domain: the active site

The fingers domain contains the dNTPs binding site and the
catalytic site is located between the fingers and the thumb
 Holds primer
DNA POL I template in
active site in
dNTP polimerase
binding
site Active site
Catalitic Controls correct
base pairing
site
Single strand DNA region is
bent sharply and does not
pass between thumb and
fingers
Only one base is exposed in
catalitic site

Once the correct base pair is formed, the fingers domain moves to
enclose dNTP (it grips the dNTP and it moves it in close contact with
the catalytic site).

After the bond with the new nucleotide is formed, the fingers opens up
and polymerase moves by one pair on the template
DNA Pol bound to DNA
Finger and tumb are
composed of alpha helices

incoming dNTP

template

primer
Palm domain is
composed of beta
sheets
Two metal ions bound to DNA polymerase catalyze
nucleotide addition
The two metal ions (shown in
green) are held
in place by interactions with two
highly conserved aspartate
residues.
Metal ion A primarily interacts
with the 3’-OH, resulting in
reduced association between the
O and the H. This leaves a
nucleophilic 3’O-.

Metal ion B interacts with the

triphosphates of the incoming
dNTP to neutralize their negative
charge.
 Two metal ions (Mg++ or Zn++), in the palm domain
bind to DNA pol and catalize nucleotide addition

Makes –OH Stabilize negative charges of triphosphates

more acidic and of of the leaving pyrophosphate
DNA pol grips both the template and the incoming nucleotide
when a correct base pair is made and stimulates catalysis

O-helix from 40°rotation

finger domain

Incoming
nucleotide
The bent template strand exposes only one base in the
catalytic site: the template base

Single strand
DNA region is
bent sharply
and does not
pass between
thumb and
fingers
Only one base
is exposed in
catalitic site
DNA polymerases synthesize DNA in a processive manner
Replication fork

Okazaki fragments are 1000-2000 nt in bacteria

100-400 nt in eukariotes
 Helicase (DnaB) movement on the ss DNA
DNA helicase separate the two strands of double helix using ATP.
Helicase is directional 5’-> 3’
Only single strand fit the 13 Å diameter (dsDNA = 20 Å)
 Synthesis of Okazaki fragments

DNA synthesis
continues until the
Okazaki fragment
extends as far as the
primer of the previously
added Okazaki
fragment.
A new primer is
synthesized near the
replication fork to begin
the process again
Binding of SSB protein to DNA is cooperative

SSB= Single Stranded Binding protein

As it emerges from helicase,

DNA is coated with SSB
Action of topoisomerase II at replication fork
Structure of a sliding DNA clamp

Sliding clamps have

high affinity for primer
template:junction and
for polymerases
bound to it
08_Figure18b.jpg
08_Figure19.jpg

Sliding clamp increase

processivity of the
associated DNA
polimerase
More than 1000 fold

from 3-200nt
till 500000 nt
Sliding clamps from E.coli, T4, Eukariotes

PCNA
 Composition of the DNA polII enzyme (E.Coli)

Holoenzyme
is made up
the clamp loader, a
by 9-10 pentameric complex
subunits of that uses ATP to open
different type and close the clamp
around DNA
 DNA polymerase III (E.Coli)
Primase
Helicase (dnaB) interacts
with Polimerase III
through subunits.
This interaction increases
helicase rate 10 fold

Intermittently (once per

second) Primase
interacts with helicase
and synthesize 10 nt
primers
This interaction increases
Primase function
1000fold
+ pol IV and pol V for repair
5 polymerases in total:
2 DNA duplication
3 DNA repair

13 polimerases in total
4 DNA duplication
9 repair
08_Figure22.jpg
 In the lagging strand, as the helicase unwinds it , DNA is
spooled out as single strand, looped and bound to SSB

Primase (not shown

here) interacts with
helicase (orange):
synthesis of RNA
primer

Loading of sliding
clamp (blue ring)

DNA polII core

(green) joins clamp
and starts DNA
synthesis
Primase (green) DNA pol III core
interacts with helicase
(orange): synthesis of
RNA primer

Loading of sliding
clamp (blue ring)

DNA pol-lII core (light

green) joins clamp and
starts DNA synthesis
08_Figure22c.jpg
08_Figure23.jpg
08_Figure22d.jpg
08_Figure22e.jpg
Replication fork

Okazaki fragments are 1000-2000 nt in bacteria

100-400 nt in eukariotes
Removal of RNA primers from newly synthesized DNA

Primers from newly synthesized DNA (Okazaki

fragments) can be removed according to two pathways:

• DNA pol I - Nick translation

• RNAse H and 5’ endonuclease

Removal of RNA primers from newly sintesyzed DNA
Nick translation by
DNA pol I of E.coli

Another mechanism
utilized to remove
resistant RNA primers
on lagging strand
 DNA polymerase III Synthesys of
short rRNA
Primase primers
Helicase (dnaB) interacts with
Polimerase III through subunits.
This interaction increases
helicase rate 10 fold

Intermittently (once per second)

Primase interacts with helicase
and synthesize 10 nt primers

This interaction increases

Primase function 1000fold
 Replication initiation

Replicon = all DNA replicated from a particular origin of

replication

Replicator= cis acting DNA sequences that are sufficient to

initate replication (DnaA Box or R sites + AT rich
region or DUE)

Origin of = physical site where DNA is unwound and synthesis

Replication begins. Often part of replicator.

Initiator = Protein that recognize DNA elements in replicator

and activates the initiation of replication. Sequence
specific (DnaA)
 Arrangement of sequences in the E. coli replication
origin, oriC.

DnaA protein binds to R and I sites and hydrolyzes ATP

This interaction DNA+DnaA causes unwinding of the nearby
DUE sequence rich in AT base pairs
 Origin of Replication
E. coli
chromosome
AT-rich region=
oriC DUE
AT-rich region
5′ –GGA T CC T GGGT A T T AAAAAGAAGA T C T AT T T A T T T AGAGA T C T G T T C T AT
CC T AGGACCC A T A A T T T T T C T T C T AGAT AA AT AAA T CTCT AGAC AAGAT A
1 DnaA box 50
T G T GA T C T CT T A T T AGGA T CGC A C T GCCCT GT GGA T AACA AGGA T CGGCT
AC AC T AGAGA A T A ATCCT AGCGT GACGGGACACCT AT TGT T CC T AGCCGA
51 DnaA box 100
T T T A AGA T CA A CA A CCTGGA AAGGA T C AT T AA CTG T GAAT GA T CGG T GAT
DnaA box= R sites A A AT T C T AGT T GT T GGACC T T T CC T AGT AAT T GAC ACT T AC T AGCC AC T A
101 DnaA box 150
CC T GGACCGT A T A AGCTGGGA T C AGA A TGAGGGT T A TACA CAGC TC A A AA
GGACC T GGCA T AT T CGACCC T AGT C T T ACT CCCAA T ATGT GT CGAGT T T T
151 DnaA box 200
AC T GA AC AACGGT T GT TCT T TGGA T A ACTACCGGT T GA T CCA AGCT T CCT
T GAC T T GT T GCC A ACAAGA A ACCT A T T GAT GGCCA ACT AGGT T CGA AGGA
201 DnaA box 250
GAC AGAGT TA T CCA CAGTAGA T CGC –3′
CT GT C T C A AT AGGT GTCAT C T AGC G
Brooker, fig 13.5 251 275

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Origin of replication in E.coli, the virus SV40 and the
yeast S.cerevisiae

AT-rich region =DUE DnaA box= R sites

Model for initiation of replication at the E. coli origin, oriC.

DnaA protein binds to R and I sites and hydrolyzes ATP

This interaction DNA+DnaA causes unwinding of the nearby
DUE sequence rich in AT base pairs

Dna A recruits DnaB (helicase) protein, loaded on each strand

with the aid of DnaC protein.
 Helicase (DnaB) movement on the ss DNA
DNA helicase separate the two strand of double helix using ATP.
Helicase is directional 5’-> 3’
Only single strand fit the 13 Å diameter (dsDNA = 20 Å)
 Replication initiation

Replicon = all DNA replicated from a particular origin of

replication

Replicator= cis acting DNA sequences that are sufficient to

initate replication (DnaBox or R sites + AT rich
region or DUE)

Origin of = physical site where DNA is unwound and synthesis

Replication begins. Often part of replicator.

Initiator = Protein that recognize DNA elements in replicator

and activates the initiation of replication. Sequence
specific (DnaA)
Basic principle of origin in of
replication
Model E. Coli initiation of replication
Model E. Coli initiation of replication (step f)

RNA primers are

synthetized on
leading strand first

lagging leading

leading lagging
 Model E. Coli initiation of replication (step g)

lagging

lagging

After helicase has moved 1000 bp, a RNA primer is

synthetized on lagging strand, and DNApol III together
with beta clamp initiate synthesis on lagging strand
In molecular biology, a primosome is a protein complex responsible for creating RNA
primers on single stranded DNA during DNA replication.
The primosome consists of : DnaG primase, DnaB helicase, DnaC
+ pol IV and pol V for repair
DNA replication in eukariotes
5 polymerases in total:
2 DNA duplication
3 DNA repair

13 polimerases in total
4 DNA duplication
9 repair
DNA polymerase switching during eukaryotic DNA replication
Chromosome breakage as a results of incomplete DNA
replication during S phase of the cell cycle
Eukariotic chromosomes have each many origins of
replication

In eukaryotes
polymerases are slow:
only 30-50 nt/s
compared to 800-1000
bp/s from bacteria, but
there are many origins
of replication (>25,000)
spaced 30-300Kb apart,
+ pol IV and pol V for repair
In eukariotes replicators are inactivated by DNA
replication (no replicator can initiate after it has been
replicated)

DNA replication once and only once per

cell cycle
 PROTEINS WITH SIMILAR FUNCTION IN DNA
REPLICATION IN PROKARIOTES AND EUKARYOTES

Protein Prokariotes Eukaryotes

Helicase DnaB MCM2

Helicase loader DnaC ORC, Cdc6, Cdt1
Primase DnaG DNA pol 
Polimerase lagging strand Pol III DNA pol 
Polimerase leading strand Pol III DNA 
Protein stabilizing SS-DNA SSB RPA
Clamp loader  Complex of DNA pol III RF-C
DNA clamp  clamp of DNA pol III PCNA
In eukaryotes elicases (MCM2-7) are loaded (in G1
phase) by ORC, Cdc6 and Cdt1

ATP hydrolysis
is required

Two (MCM2-7) are

loaded head to tail
but they are inactive !!!
Inactive pre-RC
08_Figure33.jpg

loading activation
In eukariotes elicases (MCM2) are loaded in G1 phase but
they stay inactive till activation in S phase by CCKs

DDK phosphorilates MCM2-7 (helicase)

CCK phosphorilates other auxiliary proteins
which activate MCM2-7 by binding to it

Activated MCM2 (helicase) is called CGM complex

08_Figure32.jpg
08_Figure33.jpg
08_Figure34.jpg
 The end replication problem

As the lagging-strand replication

machinery reaches the 3’ end of the
chromosome, primase no longer
have sufficient space to synthetize a
new RNA primer: this results in a
shorter chormosome
 Linear chromosomes (eukaryotic) cannot easily replicate
the ends of chromosomes

No place for
a primer
5′

3′
3′

5′

DNA polymerase cannot link

Brooker, fig 13.21
these two nucleotides together
without a primer.
Linear chromosomes (eukaryotic) must fill in gap left by RNA
primer

Telomeric repeat sequences

3′
T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG T T A GGG
AA AA AA AA AA AA AA AA
TCCC TCCC TCCC TCCC TCCC TCCC TCCC T
5′
Overhang

In humans and most complex organisms, telomerase is only used in

continuously dividing stem cells (e.g. spermatogonia stem cells)  most cells
get shorter telomeres over time (age).
What happened to Dolly, the cloned sheep? (she was generated from a skin
cell with shorter telomeres, and she aged early)
Brooker, fig 13.20
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
 TELOMERES
•Head to tail repeat of TG sequence positioned at the 3’
end of linear eukariotic chromosomes (overhang).
• In Humans is 5’-TTAGGG-3’

• These repeats consitute a 3’ overhang (single strand)

DNA that acts as point of replication.

•TELOMERASE is a DNA polimerase RNA dependent,

and uses its own RNA (TERT) as a template to extend
the 3’ of the linear chromosomes.

•By providing an extended 3’end, telomerases provides

additional template for the lagging-strand replication
machinery
 Telomere
How telomerase
5′ 3′
3′ 5′ “finishes” the replication
of linear chromosomes
Eukaryotic
chromosome

Repeat unit

T T A G G GT T A GG G T T A G GG T TA G G G 3
The binding- CA AU C

polymerization- A A T C C CAA T
RNA
Step 1 Binding
translocation cycle Telomerase synthesizes
3′ 5′

can occurs many a 6-nucleotide repeat.

Telomerase
times G
G

T T A G G GT T A GG G T T A G GG T T A G G GT T A G
CAAU C Step 2 Polymerization
A A T C C CAA T

Telomerase moves 6
This greatly nucleotides to the right and
begins to make another repeat.
lengthens one of T
T
Step 3 Translocation
the strands T T A G G GT T A GG G T T A G GG T T A G G GT T A G G G
CA A U C
A A T C C CAA T

The complementary
strand is made by primase,
DNA polymerase, and ligase.

T T A G G G T T A G G G T T A G G G T T A G G G T T A G G G T T A G G G 3′
The end is now
A A T C C C A A T C C C A A T C C C A A T C C C A A U C C C A A U 5’ lengthened
RNA primer

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
08_Figure37.jpg

repeat
08_Figure38.jpg
 Telomers isolated from young people is longer
than that isolated from older people

 Perhaps the lenght of telomeric DNA limits the

number of time a cell could divide

 Normal cell have limited telomerase activity

 Stem cell and tumor cell have higher level of

telomerase activity
 TELOMERES PROTECTION
yeast
Telomeres binding proteins
regulate telomerase activity
by inhibiting it

In Humans, telomeres binding

protein complex «Shelterin»,
protects telomeres from the
human
action of DNA repairing
Pot1 binds single enzymes which would start
strand telomer and repair by recombination
inhibits telomerase causing chromosome breaks.
Telomer length regulation by telomer binding protein (in
yeast)
08_Figure41.jpg

TELOMERES PROTECTION: T-loop

Model where the 3’ overhang end is attached to the rest of

the chromosome by intramolecular base pairing:
displacement loop

TRF-2 may promote the formation

of the loop
 Anticancer and Antiviral Agents Target DNA
Replication
5-fluorouracil (5-FU) and 6-mercaptopurine (6-MP)
analogs of nucleotide precursors that inhibit the synthesis of
pyrimidine and purine nucleotides, respectively.
5-FU is used in the treatment of colorectal cancer and also of
stomach, pancreatic, and advanced breast cancer.

6-MP is used to treat patients with acute leukemia (blood cell

cancers).
 Anticancer and Antiviral Agents Target DNA
Replication
Cytosine arabinoside (AraC) is a deoxycytidine analog that
once incorporated causes termination of DNA synthesys.
Like 6-MP, AraC is primarily used in the treatment of
acute leukemia.

Cisplatin and bischloroethyl Cisplatin is for metastatic

testicular cancer, and BCNU is
nitrosourea (BCNU) block DNA
used for brain tumors and
replication causing crosslinks leukemias.
between nucleotides
Anticancer and Antiviral Agents Target DNA
Replication

Camptothecin and etoposide are inhibitors of

topoisomerases that block the ability of these proteins to
re-form a phosphodiester bond after cleaving the DNA
backbone
Antibiotics
Block
bacterial
gyrase

Block human
topoisomerase I
Anticancer drugs

Camptothecin
Block human
topoisomerase II
Anticancer
drugs
 SIDE EFFECTS OF CHEMIOTHERAPY

Not surprisingly, these DNA replication inhibitors are also toxic

toward rapidly growing host cells such as red and white blood
cells, hair cells, and gastrointestinal mucosal cells. Inhibiting
the
growth of these cells leads to the now familiar side effects of
many chemotherapies, including immunosuppression (due to
loss of white blood cells), anemia (due to loss of red blood
cells), diarrhea (due to gastrointestinal defects), and hair loss.
 Anticancer and Antiviral Agents Target DNA
Replication
Replication inhibitors have also been used as antiviral
agents.
In acyclovir, the ribose of a normal nucleoside is replaced with an open-
chain structure that resembles the part of ribose closest to the base.
Once incorporated into DNA act as chain terminators because of their
Lack of a ribose group and therefore the3’-OH requiredfor further
nucleotide addition.

(f ) azidothymidine (AZT), and (g) acyclovir.

08_Table01.jpg