D loops

(mitochondria and chloroplast DNA)

RNA polymerase transcribes a primer, whose 3’ ends are generated by cleavage by an endonuclease at several discrete sites.The endonuclease is specific for the triple structure of DNA-RNA hybrid. The 3’ end is then extended by the DNA polymerase.

D loop expansion continues until it reaches a pointabout 2/3 of the way around the circle. Replication of this region exposes an origin in the displaced L strand. Synthesis of an H strand initiates at this site, which is used by a special primase that synthesizes a short RNA. The RNA is then extended by DNA polymerase, proceeding around the displaced single-stranded L template in the opposite direction from L-strand synthesis.

H strand is used as template DNA

L strand begins to function as template DNA

Rolling circle

Circular duplex DNA (RF)

Initiation occurs on one strand. The A protein, nicks the (+) strand of the duplex DNA at a specific site that defines the origin for replication.

Elongation of growing strand displaces old strand

Continued elongation generates displaced strand of multiple unit lengths

Circular ssDNA is packaged into the phage virion (M13, f1, φX174). Upon infection single-stranded DNA is converted to double-stranded DNA (RF)It may be used as a rolling circle to generate more progeny

Replicative form

(ssDNA)

(dsDNA)

Plasmid ColE1

origin

5’

5’

5’

5’

The sequence of RNA I is complementary to the 5’region of primer RNA

Base pairing with RNA I may change the secondary structure of the primer RNA

RNase H

3’-OH

Proposed model

DNA replication

No DNA replication

No base pairing between RNA I and RNA primer

Rop protein stabilizes RNA structure

The problem of linear replicons

-The DNA may form an unusual structure (hairpin).

- Instead of being precisely determined, the end may be variable (eukaryotic chromosomes).

- The most direct solution is for a protein to intervene to make initiation possible at the actual terminus.

Adenovirus55 K daltons protein

Initiation of DNA replication

In several viruses that use such mechanisms, a protein is found covalently attached to each 5’end. In the case of adenovirus, a terminal protein is linked to the mature viral DNA via a phosphodiester bond to serine.

- The problem may be circumvented by converting the linear replicon into a circular or multimeric molecule (T4 and lambda phages).

Telomeric DNA

Completation of the extension process at the end of a chromosome

Preventing the ends of a linear dsDNA molecule from gradually getting shorter during successive rounds of DNA replication.

Telomerase binding proteins

It is believed that after telomerase has extended the 3’-end a sufficient amount, DNA pol. α primes and DNA pol. δ synthesizes a new Okazaki fragment.

The telomerase is a reverse transcriptase

Telomerase was found in abundance in cancer cells indicating that such enzyme is essential for cancer cells to continue dividing. Most cells in human undergo a limited number of cell divisions and then cease dividing. In these cells telomerase is basically not present.

The telomerase extends the telomeric DNA at the 3’-end

Strategy for the identification of origins of

replication in E. coli

Structure of origins of replication

The DNA sequences of replicatorsshare two common features:-Binding sites (green) for initiation proteins that nucleates the assembly of replication machinery- A stretch of A-T rich DNA that unwinds readily but not spontaneously (blue)

DnaA

T antigen

ORC

(Factor Accumulation model)

v

In addition:* over-expression of DnaA leads to a burst of initiation. * under-expression of DnaA leads to increased initiation mass.

E. coli replication origin oriC

Model for initiation complex at oriC

Base changes in individual DnaA boxes have surprisingly little effects. Any change of distances, however, inactivates oriC.

A compact complex is formed in the presence of HU protein and ATP which contains about 10 monomers of DnaA per oriC. The result of DnaA binding is that double helix melts within the 3 AT-rich, 13-nucleotides repeats.

Mutants with a defect in any one of the genes for the main histone-like proteins (HU, FIS, H-NS, IHF) are viable. Even deficiency in two histone-like proteins is possible.

Mutations in fis or him (IHF) genes result in asynchronous initiations.

Cells depleted of HU, IHF and H-NS simultaneously are not viable.

A model for E. coli initiation of DNA

replication

Replication initiates bidirectionally. Initially only the leading strand is synthesized. After helicase has moved 1000 bases a second primer on each lagging strand is synthesized.

Initiation of lagging strand synthesis.

The primase synthesizes the primer complementary to the leading strand

The DnaA protein binds oriC

oriC

Due modelli a confronto

For bacteria dividing more frequently than every 60 minutes, a cycle of replication must be initiated before the end of the preceding division cycle.

At division (35/0 minutes), the cell receives a partially replicated chromosome.

At 10 minutes, when this "old" replication fork has not yet reached the terminus, initiation occurs at both origins on the partially replicated chromosome. The start of these "new" replication forks creates a multiforkedchromosome.

Cells growing more rapidly are larger and possess a greater number of origins and there are correspondingly more chromosomes in the individual bacterium.

Una cellula viene esposta per un breve tempo a [3H] timina(fase di pulse), poi viene somministrato un eccesso di timina fredda (fase di chase).

Replication of eukaryotic DNA

DNA polymerases δ δ δ δ and ε ε ε ε are the main replicative enzymes

Model for DNA replication

The combined DNA polymerase and primase activities of the DNA polymerase α may constitute an evidence that this enzyme synthesizes the Okazaky fragments(??) but the DNA polymerase α has not the 3’→5’ exonuclease activity.

None of eukaryotic DNA pol. have the 5’→ 3’ exonuclease activity like pol. I in E. coli.

Switching delle Polimerasi

Il prodotto del complesso primasi/DNA polimerasi èdi circa 50-100 nucleotidi.

Le DNA polimerasi δ and � sintetizzano rispettivamente il filamento guida e quello copia.

Isolating the origins of yeast replicons (ARSs)

S. cerevisiae mutants can be "transformed" to the wild phenotype by addition of DNA vectors that carries a wild-type copy of the gene (selection). Some yeast DNA fragments (when circularized) are able to transform defective cells very efficiently. These fragments can survive in the cell in the unintegrated (autonomous) state, that is, as self-replicating plasmids. A high-frequency transforming fragment possesses a sequence that confers the ability to replicate efficiently in yeast. This segment is called an ARS (autonomously replicating sequence).

Priming in eukaryotes

5’3’

↓↓↓↓

→→→→DNA pol. αααα DNA pol. δδδδ

RNA

DNA (50-100 bases)

In eukaryotes the Okazaki fragments are much shorter (200 bp) than in bacteria (about 4000 fragments of 1-2 Kb) and priming is a highly repetitive event. This indicates that each round of synthesis replicates the DNA associates with a single nucleosome (140-150 bp wound around the core particle plus 50-70 bp of linker).

RPA (replication protein A) prevents duplex formation.

In eukaryotes the primase is an integral part of DNA pol. α.

200 bp

origin recognition complex

ARS binding factor 1

The subdomain B2appears to correspond with the 13-nucleotide repeat array of E.coli.

The subdomain B3 is the DNA binding site for ABF1 that by introducing a torsional stress, favours DNA melting at B2.

ORCs appear to remain attached to the yeast origins throughout the cell cycle (it is not the initiator proteins) and may have a key role in regulation of DNA replication.

Yeast origin of replication

Possibly DNA synthesis (leading and lagging strands) involves the combined action of both DNA polymerasesδ andε

10 bases

core region (40 bp)

Formation of pre-replicative complex

Cdc6 ↑ multiple genome replication in absence of mitosis

Cdc6 ↓ absence of pre-replication complexes

Cdc6 protein was originally identified in yeast and subsequently shown to have homologs in higher eukaryotes

Helicase loading proteins

Helicase

ORC binds the origin

Cdc6 is a highly unstable protein (half-life <5 minutes). Its rapid degradation means that no protein is available later in the cycle.

The presence of Cdc6 in turn allows Mcm proteins to bind to the complex.

Mutations in the Mcm factors could prevent initiation of replication.

Origin Recognition Complex

Pre-Replicative Complex

pre-RC is formed in G1.

Kinases Cdk and Ddk are inactive in G1 and are activated when cells enter S phase.Phosphorylation of Cdc6 and Cdt1.

Cdc6 and Cdt1 are displaced. Because these proteins are rapidly degraded during S phase, they are not available to support reloading of Mcm complex and so the origin cannot be used for a second cycle of initiation during the S phase.

Synthesis of primer by Primase and positioning of the sliding clamp by PCNA protein.

DNA Pol δ recognizes primer and begins leading strand synthesis.

DNA Pol δ on the leading strand and DNA Polε on the lagging strand.

Activation of the pre-replicative complex

Centinaia o addirittura migliaia di origini devono essere attivate una sola volta in ciascun ciclo.

Le Cdk sono richieste per attivare il pre-RC. Una elevata attività Cdkpromuove l’inizio della replicazione del DNA.

L’attività delle Cdkinibisce la formazione di nuovi complessi pre-RC

I livelli di Cdk variano durante il ciclo cellulare

Gli elevati livelli di Cdk determinano l’associazione con l’origine di replicazione del solo ORC

Gli elevati livelli di Cdk determinano la fosforilazione e conseguente dissociazione dall’origine di replicazione delle proteine Cdc6 e Cdt1

Completation of lagging strand synthesis in eukaryotes

The flap model The RNase H model

None of the eukaryotic polymerase have a 5’→→→→3’exonuclease activity.

FEN1 endonuclease cannot initiate primer degradation because its activity is blocked by the triphosphate group present at the 5’-end of the primer.

DNA-RNA junction