DNA replication

Semiconservative DNA replication

Meselson and Stahl

Replication of DNA

Hartl

New nucleotides are added to DNA only during replication in the 5-3 direction

How double helix unwind

DNA synthesis takes place simultaneously but in opposite directions on the two DNA templates

ss

ss Direction of synthesis: 5-3

DNA synthesis is continuous on one template of DNA and discontinuous on the other

Lagging strand replication requires the formation of Okazaki fragments

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

10-12 nts long

(3-OH)

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

On the leading strand, where replication is continuous, a primer is required only at the 5 end of the newly synthesized strand

On the lagging strand, with discontinuous replication, a new primer must be generated at the begining of each Okazaki fragment

Helicase, Primase, SSB and DNA polimerase

DNA helicase unwinds DNA by binding to the lagging-strand template at each replication fork and moving in the 5-3 direction along the strand by breaking hydrogen bonds Primase forms a complex with helicase (primosome) SSB (single-stranded binding) proteins stabilize the exposed single-stranded DNA

DNA polimerases in E. coli

DNA polymerase III

5-3 polymerase activity

Proofreading role of DNA polymerase III

Hydrolysis due to 3-5 exonucleolytic activity

3-5 exonuclease activity

DNA polymerase III- a large multiprotein complex

- actividade polimerase 5-3 exonuclease 3-5 - aumenta processividade da enzima - necessria ao assembly de - mantm estrutura do dmero e contacta com DnaB (primase)

Modelo assimtrico da DNA polimerase III apoia o modelo da replicao simultnea das duas cadeias

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

DNA polymerase I
5 3 5-3 exonuclease activity 5 5-3 polymerase activity 3 3 5 3 5
1st ribonucleotide of RNA primer is trifosphatated

dNTP
5 3 3 5

Nick

DNA ligase

5 3

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

ACTIVITIES

DNA polymerase I DNA polymerase III

Synthesis

DNA polymerase I DNA polymerase III

Removes incorrect nucleotide

DNA polymerase I

Displaces incorporated nucleotides

Characteristics of DNA polymerases in E. coli

Klenow fragment
(large DNA polymerase I fragment)

5-3 polymerase activity

DNA polymerase I (103 kDa)

5-3 exonuclease activity 3-5 exonuclease activity

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

5-3 polymerase activity

Klenow fragment
(large DNA polymerase I fragment)

5-3 exonuclease activity 3-5 exonuclease activity

proofreading

COOH
synthesis

(68 kDa)

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

Topoisomerase I

Replisome

Origin of replication

Cooper 4.19

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

245 bp

DNA forced to unwind in 13-mers

Activates DnaG (primase)

Typical 13-mer Typical 9-mer

GATCTATTTATTT TTATCCACA
Unwinding allows helicase and other SSB proteins to attach to single-stranded DNA

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

DNA polymerase III- a large multiprotein complex

- actividade polimerase 5-3 exonuclease 3-5 - aumenta processividade da enzima - necessria ao assembly de - mantm estrutura do dmero e contacta com DnaB (primase)

Modelo assimtrico da DNA polimerase III apoia o modelo da replicao simultnea das duas cadeias

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

Unidirectional vs bidirectional replication

Circular DNA molecule Linear DNA molecule

Modes of Replication
Theta Rolling circle Linear

Theta replication is a type of common in E. coli and other organisms possessing circular DNA
Producing single-stranded templates for the synthesis of new DNA. A replication buble forms, usuallly having a replication fork at each end (bidirectional replication)

The fork proceeds around the circle Double-stranded DNA unwinds at the replication of origin

Two DNA molecules are produced

The products of theta replication are two circular DNA molecules

Rolling-circle replication
(circular DNA)

Nick
Displaced strand

Cleavage in one of the nucleotide strands

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

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

The 3 end grows around the circle giving rise to the name rollingcircle model

Cleavage may release a single-stranded linear DNA and a double-stranded circular DNA

or
The cycle may be repeated

The cycle may be repeated

Two copies (or more) of same sequence of linear DNA

The linear molecule circularizes after serving as a template for the synthesis of a complementary strand

2nd revolution New synthesized DNA (discontinuous replicationlagging strand)

1st revolution

Or either before serving as a template

- D-loop - One or several linear molecules

Termination of DNA replication in E. coli

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

One of the replication forks has proceeded some distance past the halfway point. This does not happen during DNA replication in E.coli because of the action of the Tus proteins

The role of terminator sequences during DNA replication in E. coli

-strands facing the fork

Eucaryotic replication

Linear DNA replication takes place in eukaryotic chromosomes

Replication begins and is bidireccional

Synthesis starts at all the origins of replication

Replication bubles fuse where they meet

Structure of yeast origin of replication

ARS- autonomously replicating sequence, that acts as na origin of replication in S. cerevisae A, B1, B2 and B3- functional sequences

< 200 bp

Melting of the helix occurs within the subdomain B2, induced by the attachment of ARS binding protein (ABFI) to subdomain B3. The proteins of the origin of replication complex (ORC) are permanently attached to subdomains A and BI

Number and lenght of replicons

Organism

Average lenght of replicon (bp)

Escherichia coli (bacterium) Saccharomyces cerevisae (yeast) Drosophila melanogaster (fruit fly) Xenopus laevis Mus musculus (mouse)

500

40 000

3 500

40 000

15 000 25 000

200 000 150 000

Events in DNA replication in E. coli and eukaryotes

Priming of DNA synthesis

In eukaryotes the primase forms a complex with DNA polymerase , which is shown synthesizing the RNA primer followed by the first few nucleotides of DNA

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

There appears to be no DNA polymerase with 5-3 exonuclease activity in eukaryotes

The flap endonuclease FEN I cannot initiate primer degradation because its activity is blocked by the triphosphate group present at the 5 end of the primer

Two models for completion of lagging strand replication in eukaryotes

Telomer replication

Sequncias de DNA nas extremidades dos cromossomas

The telomere has a protruding end with a G-rich repeated sequence

Como se resolve o problema da extremidade 3 projectada originada durante o processo de replicao do DNA?

The mechanism of restoring the ends of a DNA molecule in a chromosome relies on an enzyme called TELOMERASE
3 5

Telomerase elongates the template DNA strand at the 3 end

3 5

The telomerase contains na internal RNA with a sequence complementary to the telomere repeat

Mechanism of action of telomerase

G-quartet structure formed by hydrogen bonding between four guanine bases present in a single DNA strand folded back upon itself

Models of telomere structure in Oxytricha and Tetrahymena

Replication slippage
A trinucleotide repeat in the act of replication

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

Continued replication duplicates the region between the points of detachment and reannealing

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

Doenas neurodegenerativas (doenas de expanso de repeties de trinucleotdeos)