DUPLICACION DEL MATERIAL
GENETICO
The Eukaryotic Cell Cycle
Restriction
Point
DNA
Synthesis
S
Quiescence
G1
S
G1
S
G2
S
M
S
Mitosis
II. Historical Background
A. 1953 Watson and Crick: DNA Structure Predicts a Mechanism of Replication
“It has no escaped our notice that the specific pair we have postulated immediately suggests
a possible copying mechanism for the genetic material.”
B. 1958 Meselson and Stahl: DNA Replication is Conservative
The Meselson-Stahl Experiment
“the most beautiful experiment in biology.”
All
hybrids
1/2 old:
1/2 new
1/4 old:
3/4 new
1/2 hybids:
1/2 new
All
hybrids
Three potential DNA replication models and their predicted outcomes
The actual data!
III. General Features of DNA Replication
DNA Synthesis:
1. requires a DNA template and a primer with a 3’ OH end.
(DNA synthesis cannot initiate de novo)
2. requires dNTPs.
3. occurs in a 5’ to 3’ direction.
Short RNA molecules
act as primersin vivo
DNA replication is extremely accurate
Error rates of ~1 in 109 to 1010 for cellular DNA replication
This would allow approximately 1 human genome to be
replicated with only a few errors!!
How can this happen if the intrinsic error rate of the best
polymerases is only ~1 per 104 to 105 nucleotides?
Proofreading – additional 102 to 103-fold increased fidelity
Uses 3´ to 5´ exonuclease activity
Mismatch repair – final 102 to 103-fold increased fidelity
IV. DNA Polymerases of E. coli
The first DNA polymerase was discovered by Arthur Kornberg in 1957  DNA
Polymerase I
A. E. coli DNA Pol I has 3 enzymatic activit ies:
1) 5’  3’ DNA polymerase
2) 3’  5’ exon cuclease (For proofreading)
Klenow Fragment
3) 5’  3’ DNA exonu clease (To edit out sections of damaged DNA)
1
323
5’ to 3’ Exo.
Klenow Fragment
aa 928
5’ to 3’ Pol & 3’ to 5’ Exo
Hans Klenow showed that limited proteolysis with either subt ilisin or trypsin will cleave
Pol I into two biologically act ive fragments.
Fact s about DNA Synthesis Error Rates:
—DNA polymerase inserts one incorrect nucleot ide for every 105 nucleot ides added.
—Proofreading exonucleases decrease the appearance of an incorrect paired base to one
in every 107 nucleot ides added.
—Actual error rate observed in a typical cell is one mistake in every 1010 nucleot ides
added.
—Error rate for RNA Polymerase is 1/105 nucleot ides.
Model for the Interaction of Klenow Fragment with DNA
How the Proofreading Activity of Klenow Fragment Works
Aplicación de la Polimerización
Traslado del Corte o “Nick Translation”
DNA Polymerase I can Perform “Nick Translation”
They act together to edit out
sections of damaged DNA
The 5’ to 3’ Exonu clease and 5’ to 3 Polymerase of Pol I Result in “ Nick
Translation”:
nick
5’
3’
3’
5’
I
DNA Polyme rase I
Newly synthesized. DNA
nick
5’
3’
5’  3’ exonu clease edits damaged DNA
3’
5’
+
5’-dNMPs
Procesividad de la Duplicación del
DNA
B. Processivi ty
DNA Polymerases Can be Processive or Distributiv e
Processivi ty is continuou s synthesis by polymerase without dissociation
from the template.
A DNA polymerase that is Distributive will di ssociate from the template
after each nu cleotide addi tion.
Processive Polymerization
Used in
DNA
Replication
Distributive Polymerization
1 nucleot ide
Suitable for
DNA Repair
How to Measure Processivity
primer
dATP
dCTP
dGTP
[ P]-dTTP
32
Mg2+
5 min. @ 37oC
M13
DNA Pol
STOP w/ EDTA
Proc.
ssDNA
template
Dist.
Processivi ty experiments
requir e a large excess of
template to Pol to prevent
reassociation to the same
template.
Poly acrylamide Gel
DNA replication is highly processive
Pol III holoenzyme of E. coli can synthesize hundreds of
thousands of nucleotides before falling off the template.
Processivity is effected by the beta subunit of the polymerase,
called the sliding clamp.
Replication in Eukaryotes
Replication in eukaryotes (~50 nucleotides/sec) is much slower
than in prokaryotes (~1,000 nucleotides/sec).
Function
E. coli
Human
Genomic replication
pol III
pol delta
Primer synthesis (RNA/DNA)
Primase
pol alpha
Sliding clamp
beta-subunit of pol III
proliferating cell nuclear
antigen (PCNA)
PCNA originally discovered in sera of patients with the
autoimmune disorder, SLE (systemic lupus erythematosis).
It is a highly-regulated marker of cell proliferation.
The problem of replication of the ends
of linear chromosomes
3´
5´
3´
DNA replication cannot complete the 3´ end
of linear chromosomes
The cell addresses this issue by generating hundreds
to thousands of simple repeats (5´TTAGGG)n at the ends
of chromosomes of all vertebrates - telomeres
The enzyme, telomerase, is an RNA-directed DNA polymerase.
DNA Pol I y DNA Pol III trabajan
juntas
Roles of DNA Pol III and Pol I in E. coli
Pol III—main DNA replication enzyme. It exists as a dimer to coordinate the synthesis
of both the leading and lagging strands at the replication fork.
Pol I—repair enzyme to remove RNA primers that initiate DNA synthesis on both
strands. It is need predominantly for maturation of Okazaki fragments.
1) Removes RNA primers (5’3’ Exo)
2) Replaces the RNA primers with DNA (5’3’ Pol & 3’5’ Exo proofreading)
>10 kb
DNA Pol I
1 kb
RNA primer replaced with
DNA by Pol I’s nick translatiton
activity
RNA
Okazaki fragment
Okazaki fragment
DNA Pol III is highly “processive”
Pol I & II – main DNA repair enzyme
Pol III – main DNA replication enzyme
DNA Pol I is” distributiv e”
Dirección de la Replicación
Initiation of replication
Prokaryotic and eukaryotic
cellular replication
Some viruses
In higher eukaryotes, number and characteristics of origins are not well
defined.
Origin activation is extremely complex, and involves both sequence (cis)
elements and protein (trans) elements.
Replication of the E. coli Chromosome is Bidirectional
DNA mitocondrial
Un ejemplo de replicación alternativa
Mammalian Mitochondrial DNA
(MtDNA)
Multi-copy, circular molecule of ~16,000 bp.
2.
3.
Encodes genes for respiration (13
proteins) and translation (22 tRNAs, 2
rRNAs).
2 promoters (1 on each strand); the
STOP codons for the protein genes,
UAA, created post-transcriptionally by
Mammalian Mt DNA
Mt DNA replication
Mammalian (mouse) mtDNA
Replication
Two origins of replication: H (for heavy
strand) and L (for light strand) that are used
sequentially for
unidirectional replication.
Persistent D-loop at H ori, which is extended
to start
replication of the H strand.
Once ~2/3 of H strand is replicated, L ori is
exposed and
replication of L strand
En la replicación del DNA participan
otras enzimas además de las DNA
polimerasas
DNA replication is semi-discontinuous
Lagging strand synthesis MUST be
semi-discontinuous
Functional aspects of DNA replication
Function
Proteins
Unwind helix
Relieve torsional stress
DNA polymerization
Primer (RNA) synthesis
Elimination of RNA primers
Proofreading
Joining DNA strands following
primer elimination
Protect local single-strand regions
DNA helicases
Topoisomerase
DNA polymerase
Primase
5´-3´ exonuclease
3´-5´ exonuclease
DNA ligase
Single-strand binding
proteins
Replication of the E. coli Chromosome is Semidiscontinuous
Replicates continuously
DNA synthesis is going in same direction as replication fork
Replicates discontinuously
DNA synthesis is going in
opposite direction as
replication fork
Joined by DNA ligase
Because of the anti-parallel structure of the DNA duplex, new DNA must be synthesized in the
direction of fork movement in both the 5’ to 3’ and 3’ to 5’ directions overall.
However all known DNA polymerases synthesize DNA in the 5’ to 3’ direction only.
The solution is semidiscontinuous DNA replication.
Review of DNA synthesis – E. coli as paradigm
At Each Replication Fork is A Replisome
LAS TOPOISOMERASAS
Additional Terms Used To Describe Topology
The Linking Number Difference = DL = L – L0
The difference between the linking number of a DNA molecule (L)
and the linking number of its relaxed form (L0)
It is a measure of the number of writhes
For a relaxed molecule: DL = 0
The superhelical density (s )= DL – L0
It is a measure of supercoiling that is independent of length.
For a relaxed molecule: s = 0
DNA in cells has a s of –0.06
What Topoisomerases Do
1. Change the linking number of a DNA molecule by:
A) Breaking one or both strands then
B) Winding them tighter or looser, and rejoining the ends.
2. Usually relax supercoiled DNA
Type I Topoisomerases
They relax DNA by nicking then closing one strand of duplex DNA. They cut one strand of the
double helix, pass the other strand through, then rejoin the cut ends. They change the linking
number by increments of +1 or –1.
Topo I from E. coli
1) acts to relax only negative supercoils
2) increases linking number by +1
increments
Topo I from eukaryotes
1) acts to relax positive or negative supercoils
2) changes linking number by –1 or +1
increments
Relaxation of SV40 DNA by Topo I
Maximum
supercoiled
3 min.
Topo I
25 min.
Topo I
Type II Topoisomerases
They relax or unde rwind DNA by cutting then closing bo th strands . They change the linking
number by increments of +2 or –2.
All Type II Topoisomerases Can Catenate and Decatenate cccDNA molecules
Circular DNA molecules that use type II topoisomerases:
E. coli
-plasmids
-E. coli chromosome
Eukaryotes
-mitochondrial DNA
-circular dsDNA viruses (SV40)
An E. coli Type II Topoisomerase: DNA Gyrase
Topo II (DNA Gyrase) from E. coli
1) Acts on both neg. and pos. supercoiled DNA
2) Increases the # of neg. supercoils by increments of 2
3) Requires ATP
DNA Gyrase Adds Negative Supercoils to DNA
Topo II from Eukaryotes
1) Relaxes only negatively
supercoiled DNA
2) Increases the linking number by
increments of +2
3) Requires ATP
The Role of Topoisomerases in DNA Replication
Example 1: DNA gyrase (a type II topo of E. coli removes positive supercoils
that normally form ahead of the growing replication fork
DNA gyrase
Example 2: Replicated circular DNA molecules are separated by type II topoisomerase
A Review of the Different Topoisomerases
Type
E. coli
Eukaryotic
I
Topo I
Topo I
cleaves
Cleaves
1 strand
supercoils
1(nicks)
strand
(nicks)
or -1
Relaxes only
- supercoils
Relaxes – and +
Chang es link ing # by +1
Chang es link ing # by +1
+1 or –1
Requires no cofactors
Requires no cofactors
II
DNA Gyrase
Topo II
cleaves
Cleaves
2supercoils
strands
(ds
cut)
2 strands
Acts on – or + supercoils
Relaxes only
(ds cut)
Chang es link ing # in steps of –2
Chang es link ing # by +2
Introduces net neg. supercoils
Requires ATP
- supercoils
Requires ATP
Needed to introduce neg.
supercoils near the OriC site
because DnaA can initi ate
replication only on a negat ively
supercoiled t emplate
Can catenate and decatenate DNA
If eukaryotic topoisomerases
canno t int roduc e net
supercoils, how can
eukaryotic DNA become
negatively supercoiled?
Can catenate and decatenate DNA
How Does Eukaryotic DNA Become Neg. Supecoiled?
Plectonemic
Q: What happens when you remove the histone core?
Toroidal (Solenoidal)
A: The negative supercoil adopts a plectonemic conformation
Aplicación del conocimiento de
las Topoisomerasas
At Each Replication Fork is A Replisome
Targeting DNA Replication: Topoisomerase
Inhibitors
different agents used in
Bacterial infection or cancer chemotherapy
Type I Topoisomerase
nick DNA, pass other strand through nick
ATP-independent; change linking number in steps of 1
Inhibitors (e.g., camptothecin)
can freeze enzyme-DNA
covalent complex
Type II Topoisomerases
break DS DNA, pass DS DNA through enzyme-bound nick
require ATP; change linking number in steps of 2
bacterial DNA gyrase uses ATP to increase linking number
Early Quinolones Used for UTI
CH2CH3
CH2CH3
CH3
N
7
6
5
N
1
O
2
N
3
4
COOH
O
Nalidixic acid
O
COOH
CinoxacinO
• Quinolones and fluoroquinolones bind to two enzymes needed for bacterial
replication, DNA gyrase (A subunit mainly) and topoisomerase IV, causing inhibition
of DNA replication and cell death. Mammalian homologues show 100-1000 times less
affinity for these drugs.
• Nalidixic acid and cinoxacin are well absorbed from GI tract and rapidly metabolized
in the liver (one metabolite, OH-nalidixic acid is active). They only reach effective
concentration in urine.
• Resistance developed due to gyrase mutations.
Fluoroquinolones
CH3
NH
NH
NH
N
F
N
N
F
CH2CH3
O
N
CH3
NH
N
CH3
O
N
N
N
F
COOH
ciprofloxacin
CH2CH3
F
lomefloxacin O
COOH
COOH
norfloxacin O
F
COOH
ofloxacinO
• Rapidly and incompletely absorbed from the GI tract.
Widely distributed to body fluids but concentrations in CSF are low.
Plasma lifetime varies from 4-11 hours.
• Fluoroquinolones are active against most urinary tract pathogens: E. coli and
Klebsiella. Also most bacteria that cause enteritis: Salmonella, Shigella, E. coli.
Inactive against anaerobes: Clostridium difficile
• Ciprofloxacin reaches high concentration in respiratory, urinary and GI tract, bones,
joints, skin, and soft tissues. It is eliminated mostly by renal clearance.
• Newer derivatives Grepafloxacin, Levofloxacin, Gatifloxacin, Clinafloxacin
Moxifloxacin, Trovafloxacin can have increased activity against gram (+) and
anaerobic bacteria, but are not generally first line drugs for these organisms.
•Fluoroquinolone resistance mutations:
DNA gyrase is the primary target in E. coli and other gram-negative organisms
topoisomerase IV is primary target for S. aureus and other gram-positive bacteria.
Patología por falla de Helicasa
Sindrome de Werner
Genes implicated in progerias:
Werner’s:
 found gene implicated in Werner’s
 Werner’s gene appears to be responsible for making a protein
• The genetic sequence of Werner’s gene closely resembles
a sequence of genes that code for helicases in normal cells
• helicase is responsible for unwinding dsDNA
DNA Replication
Helicase enzyme is
responsible for
unwinding the DNA
strand
Mutations of helicases
may affect unwinding
of DNA
Could affect following:
- DNA repair
- DNA replication
- gene expression
- chromosome
recombination
Aging Hypothesis:
 With  age there are a # of defects in genes that code for
helicases in the cell
 This produces abnormal proteins that can’t unwind ds DNA
 Result in a  in the efficiency of above cellular functions
 Ultimately leads to a  in functional capacity.
Quimioterapia Anti-viral basado en
el conocimiento de la replicación
Anti-Viral Chemotherapy
Viral enzymes
Nucleic acid polymerases
• DNA-dependent DNA polymerase - DNA viruses
• RNA-dependent RNA polymerase - RNA viruses
• RNA dependent DNA polymerase (RT) - Retroviruses
• Protease (retrovirus)
• Integrase (retrovirus)
• Neuraminidase (orthomyxovirus)
Anti-Viral Chemotherapy
1962 Idoxuridine
• Pyrimidine analog
• Toxic
• Topical - Epithelial herpetic keratitis
1983 Acyclovir
• Purine analog
• Sugar modification
• Chain terminator
• Anti-herpes
• Selective to virus-infected
cells
1990’s Protease inhibitors
Binding
Reverse transcription
Fusion
Integration
Transcription
Endocytosi
s
Nuclear localization
Uncoating
Splicing
Lysosome
RNA export
Maturation
Genomic RNA
Modification
mRNA
Translation
Assembly
Budding
Anti-Viral Chemotherapy
Nucleic Acid Synthesis
Polymerases are often virally encoded
Other enzymes in nucleic acid synthesis
e.g. THYMIDINE KINASE in Herpes Simplex
Anti-Viral Chemotherapy
Thymidine Kinase
Intracellular viral or
cellular thymidine
kinase adds first
phosphate
Deoxy-thymidine
Deoxy-thymidine
triphosphate
Cellular kinases add
two more
phosphates to form
TTP
PO4 PO4 PO4
Anti-Viral Chemotherapy
Why does Herpes simplex code for its own
thymidine kinase?
TK- virus cannot grow in neural cells because they
are not proliferating (not making DNA)
Although purine/pyrimidines are present, levels of
phosphorylated nucleosides are low
Allows virus to grow in cells that are not making
DNA
“Thymidine kinase” is a misnomer
NON-SPECIFIC
Deoxynucleoside kinase
Anti-Viral Chemotherapy
Herpes thymidine kinase will phosphorylate any
deoxynucleoside including drugs – as a result of its
necessary non-specificity
Nucleoside analog may be given in non-phosphorylated form
• Gets drugs across membrane
• Allows selectivity as only infected cell has enzyme to
phosphorylate the drug
Cellular TK (where
expressed) does not
phosphorylate
(activate) the drug
ACG P P P
Anti-Viral Chemotherapy
Need for activation restricts drug to:
• Viruses such as HSV that code for own thymidine
kinase
• Virus such as cytomegalovirus and Epstein-Barr
virus that induce cells to overproduce their own
thymidine kinase
• In either case it is the VIRUS-INFECTED cell
that activates the drug
Anti-Viral Chemotherapy
Thymidine kinase activates drug but phosphorylated drug
inhibits the polymerase
Nucleotide analogs
Sugar modifications
Base modifications
Selectivity
• Viral thymidine kinase better activator
• Cellular enzyme may not be present in non-proliferating cells
• Activated drug is more active against viral DNA polymerase
that against cell polymerase
Anti-Viral Chemotherapy
Guanine analogs
Acyclovir = acycloguanosine
= Zovirax
Ganciclovir = Cytovene
• Activated by viral TK
Acyclovir
Ganciclovir
Excellent anti-herpes drug
• Activated ACV is better
(10x) inhibitor of viral
DNA polymerase than
inhibitor of cell DNA
polymerase
Anti-Viral Chemotherapy
Acyclovir:
• Chain terminator
Good anti-herpes drug
P
P
P
P
Normal DNA synthesis
Anti-Viral Chemotherapy
Acyclovir:
P
• Chain terminator
P
Terminatio
n
P
Selective:
• Virus phosphorylates
drug
• Polymerase more
Also
inhibits:
sensitive
• Epstein Barr
• Cytomegalovirus
P
ACG
P-P-P
P
Anti-Viral Chemotherapy
Acyclovir very effective against:
• Herpes simplex keratitis (topical)
• Latent HSV (iv)
• Fever blisters – Herpes labialis (topical)
• Genital herpes (topical, oral, iv)
Resistant mutants in thymidine kinase or DNA polymerase
Appears not to be teratogenic or carcinogenic
Ganciclovir very effective against cytomegalovirus – viral DNA
polymerase is very sensitive to drug activated by cell TK
Anti-Viral Chemotherapy
Adenine arabinoside (Ara-A)
Problems : Severe side effects
• Resistant mutants (altered polymerase)
Competitive
• Chromosome breaks (mutagenic)
inhibitor of
virus DNA
• Tumorigenic in rats
polymerase
• Teratogenic in rabbits
which is
much more
• Insoluble
sensitive
Use: topical applications in ocular herpes simplex
than host
polymerase
Anti-Viral Chemotherapy
Adenine arabinoside
• HSV encephalitis
• Neonatal herpes
• Disseminated herpes zoster
• Hepatitis B
Poor in vivo efficacy:
DEAMINATION
Anti-Viral Chemotherapy
Other sugar modifications:
AZT
azidothymidin
e
DDI
dideoxyinosi
ne
DDC
dideoxycytidi
ne
Anti-Viral Chemotherapy
Base change analogs
Trifluorouridine
Viroptic
anti-HSV
Altered base pairing
Mutant DNA
Resistant mutants
Idoxuridine
Anti-Viral Chemotherapy
Fluoroiodo aracytosine has both a
base and a sugar alteration
NH2
I
O
HOCH2
O
F
Prodrugs
e.g. Famciclovir
P
P
P
Taken orally
Glaxo-SmithKlein
Converted by
patient’s
metabolism
Penciclovir:
Available as
topical
cream
HSV thymidine kinase
Host kinase
Anti-Viral Chemotherapy
Non-nucleoside Non-competitive RT inhibitors
Combination therapy with AZT
Resistance mutations will be at different sites
The most potent and selective RT inhibitors
Nanomolar range
Minimal toxicity (T.I. 10,000-100,000)
Synergistic with nucleoside analogs (AZT)
Good bio-availability
Resistant mutants - little use in monotherapy
Anti-Viral Chemotherapy
DuPont
Sustiva
(S) -6- chloro-4(cyclopropylethynyl)-1,4-dihydro-4(trifluoromethyl)-2H-3, 1benzoxazin-2-one.
Anti-Viral Chemotherapy
Nevirapine: Approved for AIDS patients
Good blocker of mother to child transmission
peri-natal - breast feeding
• Single dose at delivery reduced HIV transmission
by 50%
• Single dose to baby by 72 hours
Efavirenz (Sustiva, DMP266)
In combination therapy will suppress viral load as
well as HAART and may be better – Approved for
AIDS patients
Anti-Viral Chemotherapy
Phosphono acetic acid (PAA)
Phosphono formic acid
O
HO P
O
C
OH
Binds pyrophosphate site of polymerase
Competitive inhibitor
10 -100x greater inhibition of herpes polymerase
Toxic: accumulates in bones, nephrotoxicity
Rapid resistance
Clinical trial: CMV in AIDS patients
Anti-Viral Chemotherapy
Ribavirin
• Guanosine analog
• Non-competitive inhibitor of
RNA polymerase in vitro
• Little effect on ‘flu in vitro
• Often good in animals but
poor in humans
• Aerosol use: respiratory
syncytial virus
• i.v./oral: reduces mortality in
Lassa fever, Korean and
Argentine hemorrhagic fever