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DNA replication
Semiconservative DNA replication(Meselson-Stahl experiment)
Replication of DNA
Hartl
5’
3’5’
3’
5’
3’
3’
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
5’
5’
5’
5’
5’
5’
5’
5’
3’
3’
3’
3’
3’
3’
3’
3’
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
N COOH
synt
hesi
s
proo
frea
ding
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
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