DNA replication

Semiconservative DNA replication(Meselson-Stahl experiment)

Replication of DNA

Hartl

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3’5’

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3’- 5’

New nucleotides are added to DNA only during replication in the5’-3’ direction

DNA synthesis takes place

simultaneouslybut in

opposite directionson the two DNA templates

Direction of synthesis: 5´-3’

ss

ss

DNA synthesis iscontinuous (leading strand) on one template of DNA

anddiscontinuous (lagging strand) on the other

Lagging strand replication requiresthe formation of Okazaki fragments

Priming of DNA synthesis with a RNA segment(RNA primer)

Primase synthesizes short streches of RNA nucleotides, provinding a 3’-OH groupto which DNA polymerase can add DNA nucleotides

10-12 nts long

(3’-OH)

On the leading strand, where replication is continuous,a primeris required

only at the 5’ endof the newly

synthesized strand

On the lagging strand, with discontinuous replication, anew primer

must be generated at the begining ofeach Okazaki

fragment

Helicase, Primase, SSB and DNA polimerase

DNA helicase unwinds DNA by binding to the lagging-strand template ateach replication fork and moving in the 5’-3’ direction along the strandby breaking hydrogen bonds

Primase forms a complex with helicase (primosome )

SSB (single-stranded binding) proteins stabilize the exposed single-stranded DNA

How double helix unwind

DNA polimerase IIIDNA polimerase I

DNA polimerasesin E. coli

DNA polymerase III

5’-3’ polymerase activity

Proofreading role of DNA polymerase III

3’-5’ exonuclease activity

Hydrolysis due to 3’-5’ exonucleolytic activity

DNA polymerase III- a large multiprotein complex

αααα- actividade polimerase 5’-3’

εεεε- “ exonuclease 3’-5’

ββββ- aumenta processividade da enzima

θθθθ- necessária ao assembly de α ε τα ε τα ε τα ε τ

ττττ- mantém estrutura do dímero e contacta com DnaB (heli case)

Modelo assimétrico da DNA polimerase III apoia o mod elo da replicação simultânea das duas cadeias

A ββββ subunit tethers the core of E. coli DNA polymerase III to DNA thereby increasing its processivity

DNA polymerase I

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5’-3’ exonuclease activity

5’-3’ polymerase activity

DNA ligase

dNTP

Nick

DNA polymerase I also have 3’-5’ exonuclease activity

1st ribonucleotide of RNA primer is trifosphatated

DNA polymerase IDNA polymerase III

DNA polymerase IDNA polymerase III

DNA polymerase I

Synthesis

Removes incorrect nucleotide

Displaces incorporatednucleotides

ACTIVITIES

Characteristics of DNA polymerases in E. coli

Klenow fragment(large DNA polymerase I fragment)

DNA polymerase I(103 kDa)

5’-3’ polymerase activity

5’-3’ exonuclease activity

3’-5’ exonuclease activity

Klenow fragment(large DNA polymerase I fragment)

(68 kDa)

5’-3’ polymerase activity

5’-3’ exonuclease activity

3’-5’ exonuclease activity

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Small fragment (35 kDa)- 5’-3’ exonuclease activity

DNA polymerase I has the unique ability to start replication in vitro at a nick in DNA

Topoisomerase I

Replisome

Relationship between E. coli replication proteins at a growing fork

In this model, DNA must form a loop so that both strands can replicate simultaneously

Model of DNA replication in E. coli, where two units of DNA polymerse III are connected

The lagging strand loops around

so that 5’-3’ synthesis can take place

on both antiparallel strands

Cooper 4.19

Origin of replication

Model of initiation of replication at E. coli oriC(Konberg and collab.)

245 bp

Typical 13-mer GATCTATTTATTT

Typical 9-mer TTATCCACA

DNA forced to unwind in 13-mers

Ligation of DnaA (initiator proteins )occurs when DNA is negatively supercoiled

Activates DnaG (primase )

Unwinding allows helicase and other SSBproteins to attach to single-stranded DNA

Unidirectional vs bidirectional replication

Circular DNA molecule

Linear DNA molecule

Modes of Replication

ThetaRolling circle

Linear

Theta replication is a type of common in E. coli andother organisms possessing circular DNA

Double-stranded DNA unwinds at thereplication of origin

Producing single-stranded templatesfor the synthesis of new DNA. A replication buble forms, usualllyhaving a replication fork at each end(bidirectional replication)

The fork proceedsaround the circle

The products of theta replication are two circular DNA mo lecules

Two DNA moleculesare produced

(vector)

Rolling-circle replication(circular DNA)

Nick

3’-OH at the nick is the growing point where DNA synthesis begins. The inner strand is used as a template

The 3’ end grows around the circle giving rise to the name ro lling-circle model

Displaced strand5’

Cleavage in one of the nucleotide strands

Takes place in some virus and in the F factor of E. coli

5’ 3’

The cycle may be repeated

Two copies (or more)of same sequence oflinear DNA

The linear molecule circularizes

- after serving as a template for the synthesis of a complementa ry strand

- or either before serving as a template

1st revolution

2nd revolution

New synthesized DNA(discontinuous replication-lagging strand)

Bidirectional model of linear eukaryotic replication

- D-loop

- One or several linear molecules

Termination of DNA replication in E. coli:the role of terminator sequences ter during DNA replication in

E. coli

Replication termini in E. coli are located beyondthe point at which the replication forks actually meet

Eucaryotic replication

Replication bubbles fusewhere they meet

Replication beginsand is bidireccional

Synthesis starts at all theorigins of replication

Linear DNA replication takes place in eukaryoticchromosomes at several origins of replication

Structure of yeast origin of replication

< 200 bpARS- autonomously replicating sequence, thatacts as an origin of replication in S. cerevisae

A, B1, B2 and B3- functional sequences

Melting of the helix occurs within the subdomain B2, induced by the attachment ofARS binding protein (ABFI) to subdomain B3.

The proteins of the origin of replication complex (ORC) are permanentlyattached to subdomains A and BI

Number and lenght of replicons

Organism

Number ofreplicationorigins

Average lenghtof replicon (bp)

Escherichia coli 1 4 200 000(bacterium)

Saccharomyces 500 40 000cerevisae (yeast)

Drosophila 3 500 40 000melanogaster(fruit fly)

Xenopus laevis 15 000 200 000

Mus musculus 25 000 150 000(mouse)

Events in DNA replication in E. coli and eukaryotes

Polimerase δδδδ on leading and maybe lagging strand ;

Polimerase εεεε maybe on lagging strand ;

Polimerase αααα initiates both the leading and lagging strands (contains primase activity);

Polimerase γγγγ in mithocondrialDNA replication.

Priming of DNA synthesis

In eukaryotes the primase forms a complex with DNA polymer ase αααα, which is shownsynthesizing the RNA primer followed by the first few nucle otides of DNA

(complexed with primase activity)

Removal of the RNA primer from each Okazakifragment in eukaryotes, by FEN I endonuclease

The “flap endonuclease” FEN I cannot initiate primer deg radation because its activity isblocked by the triphosphate group present at the 5’ end of th e primer

There appears to be no DNA polymerase with

5’-3’ exonuclease activity in eukaryotes

Two models for completion of lagging strandreplication in eukaryotes

RNaseH- degrades the RNAstrand of RNA-DNA hybrids

Telomeres replication

Telomeres:

- have simple repeating sequences

- seal the chromosome ends

- are synthesized by a ribonucleoprotein enzyme

- are essential for survival

Como se resolve o problema da extremidade 3’ projec tada originada durante o processo de replicação do DNA?

The telomere has a protruding endwith a G-rich repeated sequence

Sequências de DNA nas extremidades dos Sequências de DNA nas extremidades dos cromossomascromossomas

The mechanism of restoring the ends of a DNA molecule in a chromosome relies on an enzyme called TELOMERASE

3’

5’

5’3’

Telomerase elongatesthe template DNA strand at the 3’ end

The telomerase contains nainternal RNA with a sequencecomplementary to the telomererepeat

Mechanism of action of telomerase

G-quartet structure formed by hydrogen bonding between fou r guaninebases present in a single DNA strand folded back upon itse lf

Models of telomere structure inOxytricha and Tetrahymena

Replication slippage

A trinucleotide repeat in the act of replication

The 3’ end of the growing strand momentrily detachesfrom the template and reanneals to the template at a point upstream from its original location

Continued replication duplicates the region betweenthe points of detachment and reannealing

Mismatch repair of the shorter strand createsa duplex with a trinucleotide expansion

Doenças neurodegenerativas (doenças de expansão de r epetições de trinucleotídeos)

Fragile-X syndrome: CGG repeat present in the FMR1 gene