DUPLICACION DEL MATERIAL GENETICO. The Eukaryotic Cell Cycle DNA Synthesis Restriction Point Mitosis...

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DUPLICACION DEL MATERIAL GENETICO

The Eukaryotic Cell Cycle

DNASynthesis

RestrictionPoint

Mitosis

Quiescence

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)

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’

5’ 3’ exonuclease edits damaged DNA

Newly synthesized. DNA

DNA Polymerase I

5’-dNMPs

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´

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

Okazaki fragment

>10 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

CH2CH3

Nalidixic acid

• 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.

CH2CH3

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.

NH CH2CH3

lomefloxacin

ciprofloxacin

NH CH2CH3

norfloxacin

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

Binding

FusionReverse transcription

Nuclear localization

Uncoating

Integration

Transcription

SplicingRNA export

Genomic RNA

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

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

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

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

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

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

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

Acyclovir:

Good anti-herpes drug

Normal DNA synthesis

ACGP-P-P

Termination

Also inhibits:

• Epstein Barr

• Cytomegalovirus

Acyclovir:

Selective:

• Virus phosphorylates drug

• Polymerase more sensitive

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

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

Adenine arabinoside• HSV encephalitis

• Neonatal herpes

• Disseminated herpes zoster

• Hepatitis B

Poor in vivo efficacy:

DEAMINATION

Other sugar modifications:

AZTazidothymidin

DDIdideoxyinosin

DDCdideoxycytidin

Base change analogs

Altered base pairing

Mutant DNA

Resistant mutants

TrifluorouridineViroptic

anti-HSV

Idoxuridine

Fluoroiodo aracytosine has both a base and a sugar alteration

OHOCH2

Prodrugse.g. Famciclovir

Taken orally Converted bypatient’s metabolism

HSV thymidine kinase

Host kinase

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

Sustiva(S) -6- chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3, 1-benzoxazin-2-one.

DuPont

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

Phosphono acetic acid (PAA)

Phosphono formic acid

HO P C

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

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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