duplicacion del material genetico. the eukaryotic cell cycle dna synthesis restriction point mitosis...
TRANSCRIPT
DUPLICACION DEL MATERIAL GENETICO
The Eukaryotic Cell Cycle
DNASynthesis
RestrictionPoint
Mitosis
Quiescence
SS
SG1
SM
SG2SG1
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.”
Three potential DNA replication models and their predicted outcomes The actual data!
1/4 old:3/4 new
1/2 hybids:1/2 new
Allhybrids
1/2 old:1/2 new
Allhybrids
III. General Features of DNA Replication
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.
DNA Synthesis:
Short RNA moleculesact 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 bereplicated with only a few errors!!
How can this happen if the intrinsic error rate of the bestpolymerases 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 DNAPolymerase I
A. E. coli DNA Pol I has 3 enzymatic activities:
1) 5’ 3’ DNA polymeraseKlenow Fragment
2) 3’ 5’ exoncuclease (For proofreading)
3) 5’ 3’ DNA exonuclease (To edit out sections of damaged DNA)
1 323
Hans Klenow showed that limited proteolysis with either subtilisin or trypsin will cleavePol I into two biologically active fragments.
Facts about DNA Synthesis Error Rates:—DNA polymerase inserts one incorrect nucleotide for every 105 nucleotides added.—Proofreading exonucleases decrease the appearance of an incorrect paired base to onein every 107 nucleotides added.—Actual error rate observed in a typical cell is one mistake in every 1010 nucleotidesadded.—Error rate for RNA Polymerase is 1/105 nucleotides.
aa 928Klenow Fragment
5’ to 3’ Exo.5’ to 3’ Pol & 3’ to 5’ Exo
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’ Exonuclease and 5’ to 3 Polymerase of Pol I Result in “NickTranslation”:
5’ 3’ 3’ 5’
I
5’ 3’ 3’ 5’
5’ 3’ exonuclease edits damaged DNA
Newly synthesized. DNA
DNA Polymerase I
+
5’-dNMPs
nick
nick
Procesividad de la Duplicación del DNA
B. Processivity
DNA Polymerases Can be Processive or Distributive
Processivity is continuous synthesis by polymerase without dissociationfrom the template.
A DNA polymerase that is Distributive will dissociate from the templateafter each nucleotide addition.
Processive Polymerization
Distributive Polymerization
1 nucleotide
Used inDNAReplication
Suitable forDNA Repair
Proc. Dist.
How to Measure Processivity
dATPdCTP
dGTP[32P]-dTTP
ssDNAtemplate
M13Mg2+
5 min. @ 37oC
STOP w/ EDTA
DNA Pol
Polyacrylamide Gel
Processivity experimentsrequire a large excess oftemplate to Pol to preventreassociation to the sametemplate.
primer
DNA replication is highly processive
Pol III holoenzyme of E. coli can synthesize hundreds ofthousands 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 theautoimmune 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´ endof 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
DNA Pol I
RNA
Okazaki fragment
>10 kb
1 kb
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)
RNA primer replaced withDNA by Pol I’s nick translatiton activity
Okazaki fragment
DNA Pol III is highly “processive” DNA Pol I is” distributive”
Pol I & II – main DNA repair enzymePol III – main DNA replication enzyme
Dirección de la Replicación
Initiation of replication
Prokaryotic and eukaryoticcellular 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. 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.
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
starts.
The lagging L strand replication gives 2 type of molecules: and is gapped
on L strand.
L strand finishes replicating, and then both and are converted to supercoiled forms.
En la replicación del DNA participan otras enzimas además de las DNA
polimerasas
DNA replication is semi-discontinuous
Lagging strand synthesis MUST besemi-discontinuous
Functional aspects of DNA replication
Function ProteinsUnwind helix DNA helicasesRelieve torsional stress TopoisomeraseDNA polymerization DNA polymerasePrimer (RNA) synthesis PrimaseElimination of RNA primers 5´-3´ exonucleaseProofreading 3´-5´ exonucleaseJoining DNA strands following DNA ligase
primer eliminationProtect local single-strand regions Single-strand binding
proteins
Replication of the E. coli Chromosome is Semidiscontinuous
Replicates continuously
DNA synthesis is going in same direction as replication fork
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.
Replicates discontinuously
DNA synthesis is going in opposite direction as replication fork
However all known DNA polymerases synthesize DNA in the 5’ to 3’ direction only.
The solution is semidiscontinuous DNA replication.
Joined by DNA ligase
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 = L = L – L0
It is a measure of the number of writhes
For a relaxed molecule: L = 0
The difference between the linking number of a DNA molecule (L)and the linking number of its relaxed form (L0)
The superhelical density ( )= L – L0
It is a measure of supercoiling that is independent of length.
For a relaxed molecule: = 0
DNA in cells has a of –0.06
What Topoisomerases Do
1. Change the linking number of a DNA molecule by:A) Breaking one or both strands thenB) Winding them tighter or looser, and rejoining the ends.
2. Usually relax supercoiled DNA
Type I TopoisomerasesThey relax DNA by nicking then closing one strand of duplex DNA. They cut one strand of thedouble helix, pass the other strand through, then rejoin the cut ends. They change the linkingnumber by increments of +1 or –1.
Topo I from E. coli 1) acts to relax only negative supercoils2) increases linking number by +1 increments
Topo I from eukaryotes 1) acts to relax positive or negative supercoils2) changes linking number by –1 or +1 increments
Maximumsupercoiled
3 min.Topo I
25 min. Topo I
Relaxation of SV40 DNA by Topo I
Type II Topoisomerases
They relax or underwind DNA by cutting then closing both strands. They change the linkingnumber 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 Eukaryotes-plasmids -mitochondrial DNA-E. coli chromosome -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 DNA2) Increases the # of neg. supercoils by increments of 23) Requires ATP
DNA Gyrase Adds Negative Supercoils to DNA
Topo II from Eukaryotes1) Relaxes only negatively supercoiled DNA2) Increases the linking number by increments of +23) Requires ATP
The Role of Topoisomerases in DNA Replication
DNA gyrase
Example 1: DNA gyrase (a type II topo of E. coli removes positive supercoilsthat normally form ahead of the growing replication fork
Example 2: Replicated circular DNA molecules are separated by type II topoisomerase
A Review of the Different TopoisomerasesType E. coli Eukaryotic
I Topo I Topo I
cleaves Relaxes only - supercoils Relaxes – and +supercoils1 strand(nicks) Changes linking # by +1 Changes linking # by +1or -1
Requires no cofactors Requires no cofactors
II DNA Gyrase Topo II
cleaves Acts on – or + supercoils Relaxes only -supercoils2 strands(ds cut) Changes linking # in steps of –2 Changes linking # by +2
Introduces net neg. supercoils Requires ATP
Requires ATP
Needed to introduce neg. supercoils near the OriC sitebecause DnaA can initiate replication only on a negativelysupercoiled template
Can catenate and decatenate DNA
If eukaryotic topoisomerasescannot introduce netsupercoils, how caneukaryotic DNA becomenegatively supercoiled?
+1 or –1
supercoils
Cleaves1 strand(nicks)
Cleaves2 strands(ds cut)
Can catenate and decatenate DNA
How Does Eukaryotic DNA Become Neg. Supecoiled?
PlectonemicToroidal (Solenoidal)
Q: What happens when you remove the histone core? A: The negative supercoil adopts a plectonemic conformation
Aplicación del conocimiento de las Topoisomerasas
At Each Replication Fork is A Replisome
different agents used in Bacterial infection or cancer chemotherapy
Targeting DNA Replication: Topoisomerase Inhibitors
nick DNA, pass other strand through nickATP-independent; change linking number in steps of 1
Inhibitors (e.g., camptothecin)can freeze enzyme-DNA covalent complex
Type I Topoisomerase
break DS DNA, pass DS DNA through enzyme-bound nickrequire ATP; change linking number in steps of 2
bacterial DNA gyrase uses ATP to increase linking number
Type II Topoisomerases
N N
O
COOH
CH3
CH2CH3
Nalidixic acid
12
364
7
5
• 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.
N
O
COOH
CH2CH3
O
O
Cinoxacin
• Resistance developed due to gyrase mutations.
• 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.
Early Quinolones Used for UTI
• 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
• 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.
N
O
COOHF
N
NH CH2CH3
CH3
F
lomefloxacin
N
O
COOHF
N
NH
ciprofloxacin
N
O
COOHF
N
NH CH2CH3
norfloxacin
N
O
COOHF
N
NHCH3
OCH3
ofloxacin
• 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.
Fluoroquinolones
•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’sWerner’s::
found gene implicated in Werner’s
Werner’s gene appears to be responsible for making a protein
• helicase is responsible for unwinding dsDNAhelicase is responsible for unwinding dsDNA
• The genetic sequence of Werner’s gene closely resembles a sequence of genes that code for helicaseshelicases in normal cells
DNA ReplicationDNA Replication
Mutations of helicasesmay affect unwindingof DNA
Could affect following:- DNA repair- DNA repair- DNA replication- DNA replication- gene expression- gene expression- chromosome- chromosome recombinationrecombination
Helicase enzyme isresponsible forunwinding the DNAstrand
Aging Hypothesis:
With age there are a # of defects# of defects in genes that code for helicases in the cell This produces abnormal proteinsabnormal 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
Viral enzymesNucleic 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 ChemotherapyAnti-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 cells1990’s Protease inhibitors
Anti-Viral ChemotherapyAnti-Viral Chemotherapy
Binding
FusionReverse transcription
Nuclear localization
Uncoating
Integration
Transcription
SplicingRNA export
Genomic RNA
mRNA
Translation
ModificationBuddingAssembly
Maturation
Endocytosis
Lysosome
Nucleic Acid Synthesis
Polymerases are often virally encoded
Other enzymes in nucleic acid synthesis
e.g. THYMIDINE KINASE in Herpes Simplex
Anti-Viral ChemotherapyAnti-Viral Chemotherapy
Thymidine Kinase
Deoxy-thymidine
Deoxy-thymidine triphosphate
Intracellular viral or cellular thymidine kinase adds first phosphate
PO4PO4PO4Cellular kinases add two more phosphates to form TTP
Anti-Viral ChemotherapyAnti-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 kinaseNON-SPECIFIC
Anti-Viral ChemotherapyAnti-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
ACG P P P
Anti-Viral ChemotherapyAnti-Viral Chemotherapy
Cellular TK (where expressed) does not phosphorylate (activate) the drug
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 ChemotherapyAnti-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 ChemotherapyAnti-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
Excellent anti-herpes drug
Acyclovir Ganciclovir
Anti-Viral ChemotherapyAnti-Viral Chemotherapy
Acyclovir:
• Chain terminator
Good anti-herpes drug
T P
PG
PC
PA
Normal DNA synthesis
Anti-Viral ChemotherapyAnti-Viral Chemotherapy
T P
PG
PC
PA
PA
ACGP-P-P
Termination
Also inhibits:
• Epstein Barr
• Cytomegalovirus
Acyclovir:
• Chain terminator
Selective:
• Virus phosphorylates drug
• Polymerase more sensitive
Anti-Viral ChemotherapyAnti-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 ChemotherapyAnti-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
Anti-Viral ChemotherapyAnti-Viral Chemotherapy
Adenine arabinoside• HSV encephalitis
• Neonatal herpes
• Disseminated herpes zoster
• Hepatitis B
Poor in vivo efficacy:
DEAMINATION
Anti-Viral ChemotherapyAnti-Viral Chemotherapy
Other sugar modifications:
AZTazidothymidin
e
DDIdideoxyinosin
e
DDCdideoxycytidin
e
Anti-Viral ChemotherapyAnti-Viral Chemotherapy
Base change analogs
Altered base pairing
Mutant DNA
Resistant mutants
TrifluorouridineViroptic
anti-HSV
Idoxuridine
Anti-Viral ChemotherapyAnti-Viral Chemotherapy
Fluoroiodo aracytosine has both a base and a sugar alteration
OHOCH2
O
NH2
I
F
Anti-Viral ChemotherapyAnti-Viral Chemotherapy
Prodrugse.g. Famciclovir
Taken orally Converted bypatient’s metabolism
HSV thymidine kinase
P
Host kinase
PP
Penciclovir: Available as topical cream
Glaxo-SmithKlein
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 ChemotherapyAnti-Viral Chemotherapy
Sustiva(S) -6- chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3, 1-benzoxazin-2-one.
DuPont
Anti-Viral ChemotherapyAnti-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
Anti-Viral ChemotherapyAnti-Viral Chemotherapy
Phosphono acetic acid (PAA)
Phosphono formic acid
O O
HO P 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 ChemotherapyAnti-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
Anti-Viral ChemotherapyAnti-Viral Chemotherapy