DUPLICACION DEL MATERIAL GENETICO

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DUPLICACION DEL MATERIAL GENETICO
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The Eukaryotic Cell Cycle
DNA Synthesis
Restriction Point
Quiescence
Mitosis
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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
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The Meselson-Stahl Experiment the most beautiful
experiment in biology.
All hybrids
1/2 old 1/2 new
1/2 hybids 1/2 new
1/4 old 3/4 new
All hybrids
Three potential DNA replication models and their
predicted outcomes
The actual data!
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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)
Short RNA molecules act as primersin vivo
2. requires dNTPs.
3. occurs in a 5 to 3 direction.
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Model for the Interaction of Klenow Fragment with
DNA
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How the Proofreading Activity of Klenow Fragment
Works
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Aplicación de la Polimerización

• Traslado del Corte o Nick Translation

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DNA Polymerase I can Perform Nick Translation
They act together to edit out sections of damaged
DNA
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Procesividad de la Duplicación del DNA
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The problem of replication of the ends
of linear chromosomes
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DNA replication cannot complete the 3 end of
linear chromosomes
The cell addresses this issue by generating
hundreds to thousands of simple repeats
(5TTAGGG)n at the ends of chromosomes of all
vertebrates - telomeres
The enzyme, telomerase, is an RNA-directed DNA
polymerase.
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DNA Pol I y DNA Pol III trabajan juntas
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Roles of DNA Pol III and Pol I in E. coli
Pol IIImain DNA replication enzyme. It exists
as a dimer to coordinate the synthesis
of both the leading and lagging strands at
the replication fork.
Pol Irepair 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)
gt10 kb
DNA Pol I
1 kb
RNA primer replaced with DNA by Pol Is nick
translatiton activity
Okazaki fragment
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Dirección de la Replicación
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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.
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Replication of the E. coli Chromosome is
Bidirectional
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DNA mitocondrial

• Un ejemplo de replicación alternativa

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Mammalian Mitochondrial DNA (MtDNA)

• Multi-copy, circular molecule of 16,000 bp.
• 2. Encodes genes for respiration (13 proteins)
  and translation (22 tRNAs, 2 rRNAs).
• 3. 2 promoters (1 on each strand) the STOP
  codons for the protein genes, UAA, created
  post-transcriptionally by polyadenylation
• 4. Some genetic diseases caused by mutations in
  mtDNA. MtDNA mutations accumulate during aging.
• 5. MtDNA used to define phylogenetic
  relationships between species, subspecies, etc.,
  or define breeding populations.

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Mammalian Mt DNA
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Mt DNA replication
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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 starts.
• The lagging L strand replication gives 2 type of
  molecules a and b. b is gapped on L strand.
• b L strand finishes replicating, and then both a
  and b are converted to supercoiled forms.

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En la replicación del DNA participan otras
enzimas además de las DNA polimerasas
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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.
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At Each Replication Fork is A Replisome
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LAS TOPOISOMERASAS
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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
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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
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Type I Topoisomerases
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
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Relaxation of SV40 DNA by Topo I
Maximum supercoiled
3 min. Topo I
25 min. Topo I
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Type II Topoisomerases
All Type II Topoisomerases Can Catenate and
Decatenate cccDNA molecules
Circular DNA molecules that use type II
topoisomerases E. coli Eukaryotes -plasmids
-mitochondrial DNA -E. coli chromosome -circular
dsDNA viruses (SV40)
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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
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DNA Gyrase Adds Negative Supercoils to DNA
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Topo II from Eukaryotes 1) Relaxes only
negatively supercoiled DNA 2) Increases the
linking number by increments of 2 3) Requires ATP
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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
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Example 2 Replicated circular DNA molecules are
separated by type II topoisomerase
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A Review of the Different Topoisomerases
Cleaves 1 strand (nicks)
1 or 1
supercoils
Cleaves 2 strands (ds cut)
Can catenate and decatenate DNA
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How Does Eukaryotic DNA Become Neg. Supecoiled?
Plectonemic
Toroidal (Solenoidal)
Q What happens when you remove the histone core?
A The negative supercoil adopts a plectonemic
conformation
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Aplicación del conocimiento de las
Topoisomerasas
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At Each Replication Fork is A Replisome
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Targeting DNA Replication Topoisomerase
Inhibitors
different agents used in Bacterial infection or
cancer chemotherapy
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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
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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
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Early Quinolones Used for UTI
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• 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.

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Fluoroquinolones

• 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.

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• 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.

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Patología por falla de Helicasa
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Sindrome de Werner
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Genes implicated in progerias

• Werners
• found gene implicated in Werners
• Werners gene appears to be responsible for
  making a protein

• The genetic sequence of Werners gene closely
  resembles
• a sequence of genes that code for helicases in
  normal cells

• helicase is responsible for unwinding dsDNA

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DNA Replication

• Mutations of helicases
• may affect unwinding
• of DNA
• Could affect following
• - DNA repair
• - DNA replication
• - gene expression
• - chromosome
• recombination

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• Aging Hypothesis
• With ? age there are a of defects in genes
  that code for
• helicases in the cell
• This produces abnormal proteins that cant
  unwind ds DNA
• Result in a ? in the efficiency of above
  cellular functions
• Ultimately leads to a ? in functional capacity.

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Quimioterapia Anti-viral basado en el
conocimiento de la replicación
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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)

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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

1990s Protease inhibitors
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Anti-Viral Chemotherapy
Nucleic Acid Synthesis Polymerases are often
virally encoded Other enzymes in nucleic acid
synthesis e.g. THYMIDINE KINASE in Herpes Simplex
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Anti-Viral Chemotherapy
Thymidine Kinase Deoxy-thymidine
Deoxy-thymidine triphosphate
Intracellular viral or cellular thymidine kinase
adds first phosphate
PO4
PO4
PO4
Cellular kinases add two more phosphates to form
TTP
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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 Deoxynucleoside kinase
NON-SPECIFIC
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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
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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

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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

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Anti-Viral Chemotherapy

• Guanine analogs
• Acyclovir acycloguanosine Zovirax
• Ganciclovir Cytovene
• Activated by viral TK
• Activated ACV is better (10x) inhibitor of viral
  DNA polymerase than inhibitor of cell DNA
  polymerase

Acyclovir Ganciclovir
Excellent anti-herpes drug
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Anti-Viral Chemotherapy

• Acyclovir
• Chain terminator
• Good anti-herpes drug

Normal DNA synthesis
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Anti-Viral Chemotherapy

• Acyclovir
• Chain terminator

Termination

• Selective
• Virus phosphorylates drug
• Polymerase more sensitive

• Also inhibits
• Epstein Barr
• Cytomegalovirus

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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
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Anti-Viral Chemotherapy

• Adenine arabinoside (Ara-A)
• Problems Severe side effects
• Resistant mutants (altered polymerase)
• Chromosome breaks (mutagenic)
• Tumorigenic in rats
• Teratogenic in rabbits
• Insoluble
• Use topical applications in ocular herpes simplex

Competitive inhibitor of virus DNA polymerase
which is much more sensitive than host polymerase
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Anti-Viral Chemotherapy

• Adenine arabinoside
• HSV encephalitis
• Neonatal herpes
• Disseminated herpes zoster
• Hepatitis B
• Poor in vivo efficacy
• DEAMINATION

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Anti-Viral Chemotherapy
Other sugar modifications
AZTazidothymidine
DDCdideoxycytidine
DDIdideoxyinosine
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Anti-Viral Chemotherapy
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Anti-Viral Chemotherapy
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Prodrugse.g. Famciclovir
Penciclovir Available as topical cream
P
P
P
Taken orally
Converted by patients metabolism
HSV thymidine kinase
Host kinase
Glaxo-SmithKlein
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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
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Anti-Viral Chemotherapy
DuPont
Sustiva
(S) -6- chloro-4-(cyclopropylethynyl)-1,4-dihydro-
4-(trifluoromethyl)-2H-3, 1-benzoxazin-2-one.
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Anti-Viral Chemotherapy

• Nevirapine Approved for AIDS patientsGood
  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

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Anti-Viral Chemotherapy
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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

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