Gentica

Modelos de replicacin del ADN

HealthCare

Replicacin del ADN

El experimento de Meselson y Stahl

Principales elementos implicados

Eucariotas
ADN de doble hebra Topoisomerasas I y II Complejo del Origen de Replicacin (ORC) Complejo del Origen de Replicacin (ORC) Complejo del Origen de Replicacin (ORC) Complejo del Origen de Replicacin (ORC) Antigeno-T (helicasa) Protena de replicacin A (RP-A: replication protein)

Procariotas
ADN de doble hebra ADN Girasa Origen de replicacin (Ori C) Protena Hu ADNA (protenas que reconocen el Ori C ADNC (cargador de la helicasa) Helicasa (ADN-B) Single-strand binding protein (SSB)

ADN polimerasa / ADN polimerasa III (replicacin y correccin de errores) ADN polimerasa- ADN polimerasa I (replicacin y correccin de errores) ADN polimerasa , , , , , , , (repara el ADN daado por ADN polimerasa II, IV, V (repara el ADN daado por agentes agentes fsicos, qumicos, etc.) fsicos, qumicos, etc.) Primasa Primasa (ADN-G) dNTPs: dATP, dTTP, dUTP, dCTP, dGTP dNTPs: dATP, dTTP, dUTP, dCTP, dGTP Magnesio (Mg++) Magnesio (Mg++) Sliding clamp - abrazadera de deslizamiento Sliding clamp - abrazadera de deslizamiento Proliferador del antgeno celular nuclear (PCNA: proliferating cell nuclear antigen) (homotrimer) Sliding clamp loader (cargador): Factor de replicacin C (RFC: replication factor C) (consisting of 5 subunits A, B, C, D and E) ARNase-H FEN-1 (Flap endonuclease 1) Ligasa clamp (homodimer) Sliding clamp loader (cargador): Gamma complex (consisting of 5 subunits) DNA polimerasa I Ligasa

A replicon includes two components, the replicator and the initiator Replicators comprise all DNA sequences necessary to the initiation of DNA replication Replicators include two types of sequences : initiator-binding sites and AT-rich sites where the two strands can be easily unwound. In E. coli, the replicator is called oriC Initiators are proteins which interact with replicator to initiate DNA replication. Initiators have three functions: (1) recognize and bind to a sequence within the replicator (2) unwind the AT-rich region of the replicator, (3) recruit other proteins to efficiently initiate DNA replication. Initiator protein in E. coli is called DnaA. Its homolog in eukaryotes is the Origin Recognition Complex (ORC)

Iniciacin

Replicon - DNA replicated from a single origin

Organi sm # of replicons Average length of replicon 4200 kb 40 kb 40 kb 200 kb 150 kb 300 kb Vel ocity of fork movement 50,000 bp/min 3,600 bp/min 2,600 bp /min 500 bp/min 2,200 bp /min

Procariotas - Ori C

Escherichia coli (bacteria) Saccharomyces cerevisiae (yeast) Drosophila melanogaster (fruit fly) Xenopus laevis (frog) Mus musculus (mouse) Homo sapiens

1 500 3,500 15,000 25,000 10,000 to 100,000

Initiation of replication Ori C in vitro

--Kornberg, 1953
Three 13-mers

+ ATP + Protein Hu on the origin (already bound to the DNA)

helicase loader

GATCTnTTTATTT GATCTnTTnTATT GATCTCTTATTAG

9a-mer

TGTGGATAA
Three 9b-mers

TTATACACA TTTGGATAA TTATCCACA

245 bp minimal region

Ready to bind primase!

How to ensure initiation of replication only once per cycle ?

Regulation for DNA replication In Bacteria, hemimethylated origins are resistant to initiation, delayed methylation leads to delayed initiation at the second phase Dam methylase

Orgenes de replicacin - Eucariotas

Origen De replicacin

The origins of DNA replication on chromosome III of the yeast S. cerevisiae

A close look at an origin of replication in yeast ORC: origin recognition complex B1, B2, B3: other regions binding to required proteins

The replication origins of human genes are more complex Even far distant DNA sequences could be important

Some facts about Replication in eucaryotes

Multiple replication origins occurring in clusters Replication units activated at different times Within replication units, replication origins are separated 40,000-300,000 pairs apart. Replication forks form in pairs and create a replication bubbles moving in opposite directions Different regions on the same chromosome are replicated at distinct times in S phase Condensed Chromatin replicates late, while less condensed regions replicate earlier

Elongacin

Replication in E.coli

RFC

PCNA

Polimerasa-/ RP-A

(Delta/epsilon)

Helicasa (Antgeno T) polimerasa- Topoisomerasa Primasa Iniciador de ARN RP-A Fragmento de Okazaki

Polimerasa-/

(Delta/epsilon)

Topoisomerasas

DNA topoisomerase I

DNA winding

DNA topoisomerase II

DNA Helicase

DNA double helix are tightly coupled. High temperature is needed to break them (95oC)

DNA Binding Protein SSB: Single Strand DNA-binding Proteins, also called helix destabilizing proteins SSB Proteins DNA

DNA Clamping Protein

DNA replication Separation, Base pair

DNA Synthesis by DNA polymerase

The Chemistry of DNA replication

This Doesnt Work!

DNA replication Fork (tenedor)

DNA Primer synthesis On Lagging strand

DNA Ligase

DNA Replication at the Lagging strand

Cycle of DNA Polymerase/Clamping Protein loading and unloading At the lagging strand (how about leading strand?)

Replication in E.coli

Replicacin en eucariotas
RFC PCNA Polimerasa-/ RP-A Helicasa (Antgeno T) polimerasa- Topoisomerasa Primasa Iniciador de ARN RP-A Fragmento de Okazaki (Delta/epsilon)

DNA Proofreading

Polimerasa-/

(Delta/epsilon)

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Structures of DNA polymerase during polymerizing and editing E: exonucleolytic; P: polymerization

Site-directed mismatch repair in eucaryotes In procaryotes, old DNAs are usually methylated on A while newly synthesized ones are not. So Cells can distinguish old and newly synthesized DNAs and mutate mismatches on new ones.

DNA Proofreading RNA usually doesnt have this. Why?

Pairing, correct nucleotide has higher affinity binding to the moving polymerase

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Addition of new histones Chromatin assembly factors (CAFs) help to add and assemble new nucleosomes

Otro modelo
A model for nucleosome replication

Finalizacin

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Bacteria DNAs are circular, not a problem There is a problem for eucaryote DNAs: ??? Hint: Telomere

Telomerase and its function

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