Initiation of DNA replication (In E.coli)

Initiation of DNA replication (In E.coli)

Replication does not initiate randomly at any place in DNA. There is a definite region in E. coli DNA where the replication originates. Such regions are termed as origin of replication.

At least nine different enzymes or proteins participate in the initiation phase of replication.

 Two Major components that control the initiation of replication:

1) Replicator: Replicator is defined as the entire set of cis-acting DNA sequences that is sufficient to direct the initiation of Replication. They include a binding site for the initiator protein.

2) Initiator: Initiator is the protein which specifically recognizes DNA elements in the replicator and activates the initiation of replication. The initiator protein is the only sequence specific DNA-binding protein involved in the initiation of replication.

Example: DnaA protein

Origin of replication in E. coli

  • The circular chromosome of E.coli has a single replication origin (oriC).

The E. coli oriC consists of 245 bp and several critical sites (DNA sequence elements that are highly conserved among bacterial replication origins).

Figure 1: Arrangement of sequences in the E. coli replication origin, oriC.

Two series of highly conserved short repeats in oriC:

 1) Three repeats of a 13 bp sequence (AT-rich segments) – locate at the left boundary of OriC

Consensus sequence: 5’- GATCTNTTNTTTT-3’

Where N marks nonspecific positions that are known as DNA unwinding elements (DUEs).

2) Four repeats of a 9 bp sequence (DnaA boxes)

Consensus sequence: 5’-TTATCCACA-3’

They are specifically bound by DnaA Protein (Initiator protein)

  • DNA synthesis requires a single stranded template. So double-stranded DNA must be unwound before DNA synthesis can take place.
  • The cell relies on several proteins and enzymes to accomplish the unwinding.
  • Helicases cannot initiate the unwinding of double-stranded DNA.
  • The crucial component in the initiation process is the DnaA protein.
  • The initiator proteins first separate DNA strands at the origin, providing a short stretch of single-stranded DNA to which a helicase binds.
  • Further unwinding occurs through the activity of the DnaB protein (helicase).

The initial unwinding by DnaA and helicase loading occurs via three steps.

Step 1: OriC binding of DnaA protein (DnaA protein in ATP bound state)

  • Initially DnaA tetramers recognize and bind to the four 9 bp repeats (DnaA boxes) in the OriC.
  • Additional molecules of DnaA (20 – 40 monomers) bind cooperatively and form a closed complex in which negatively supercoiled OriC DNA wrapped around the DnaA protein complex (forms a protein core).
  • This process requires the presence of the histone like HU or 1 HC proteins to facilitate DNA bending.
Figure 2: OriC binding of DnaA protein

Step 2: OriC melting (open complex formation)

  • The three A=T-rich 13 bp repeats of the OriC are denatured sequentially by DnaA protein complex.

(In prokaryotes, DnaA hydrolyzes ATP in order to unwind DNA at the oriC. This denatured region is accessible to the DnaB helicase and DnaC helicase loader).

Figure 3: OriC melting and open complex formation

Step 3: Helicase loading

  • This process requires ATP and the bacterial histone like protein HU
  • The DnaA protein then guides a DnaB – DnaC complex into the melted region to form a so called pre priming complex. 
Figure 4: Helicase loading by DnaC protein

DnaC: Helicase loader (an ATPase)

Function: Facilitate the loading of the DnaB hexamers onto the unwound region of DNA.

  • Two ring shaped hexamers of DnaB are loaded onto DNA (One DnaB loaded onto each DNA strand)
  • After loading DnaB onto DNA, the ATP bound to DnaC is hydrolyzed and DnaC subsequently released.

DnaB: Act as helicases

Function:

  • Unwinding of the open DNA complex bidirectionally to form the pre priming complex. It will lead to the creation of two potential replication forks.
  • DNA helicases break the hydrogen bonds that exist between the bases of the two nucleotide strands of a DNA molecule.

These enzymes use energy from ATP hydrolysis to unwind short stretches of helix just ahead of the replication fork.

DnaB migrates along the single-stranded DNA in the 5’→3′ direction (DnaB is a 5’→3′ helicase) and causes unwinding of the DNA as it travels. The DnaB helicases loaded onto the two DNA strands thus travel in opposite directions, creating two potential replication forks. All other proteins at the replication fork are linked directly or indirectly to DnaB.

  • Another protein essential for the unwinding process is the enzyme DNA gyrase, a topoisomerase.
  • Topoisomerases control the supercoiling of DNA.
  • In replication, DNA gyrase reduces torsional strain (torque) that builds up ahead of the replication fork as a result of DNA unwinding by DnaB helicase.
Figure 5: DNA helicase unwinds DNA duplex, SSB stabilize the single stranded DNA and topoisomerase II relieves the topological stress generated as a result of unwinding

Stabilization of single stranded DNA long enough for replication

  • After DNA has been unwound by helicase, the single stranded nucleotide chains have a tendency to form hydrogen bonds and reanneal (stick back together). Secondary structures, such as hairpins also may form between complementary nucleotides on the same strand.
  • Many molecules of single-strand-binding (SSB) proteins attach tightly to the exposed single-stranded DNA and stabilize them.
  • Unlike many DNA-binding proteins, SSBs are indifferent to base sequence—they will bind to any single-stranded DNA.
  • Single-strand-binding proteins form tetramers (groups of four) that together cover from 35 to 65 nucleotides.

Pre priming complex:

  • A pre-replication complex (pre-RC) is a protein complex that forms at the origin of replication during the initiation step of DNA replication.
  • The main component of the pre-RC is DnaA. The pre-RC is complete when DnaA occupies all of its binding sites within the bacterial origin of replication (oriC).
Figure 6: Formation of pre – replication complex during initiation stage of DNA replication (detailed picture)

Once pre priming complex formed it will recruit DnaG enzyme which is a primase and form primosome

 Primosome

  • Protein complex capable of duplex DNA unwinding and RNA primer synthesis at the replication fork DNA during DNA replication.
  • The primosome consists of seven proteins:

               Core unit: DnaG primase and DnaB helicase

               Associated proteins: DnaC helicase assistant, DnaT, PriA, Pri B, and PriC.

(Associated proteins contribute to the processes of replication initiation, lagging strand elongation, and replication restart).

Primase enzyme (DnaG)

(Primase is an RNA polymerase. It does not require an existing 3’-OH group to which nucleotides can be added.)

  • Enzyme that synthesizes a short RNA primer (usually around 10 nucleotides long, complementary to the DNA) that provides a 3’-OH group to which DNA polymerase can attach DNA nucleotides..
  • The free 3’ end of the primer is called the primer terminus.
  • On the leading strand, where DNA synthesis is continuous, a primer is required only at the 5’ end of the newly synthesized strand.
  • On the lagging strand, where replication is discontinuous, a new primer must be generated at the beginning of each Okazaki fragment
  • Primase forms a complex with helicase at the replication fork and moves along the template of the lagging strand.
Figure 7: Primase synthesize RNA primer providing a 3′ OH group to which DNA polymerase can add DNA nucleotides

Primosome recruits the replicative DNA polymerase III, and replication begins.

To know how DNA polymerases are loaded onto the DNA template and how they act, Refer my lecture notes on DNA polymerase in prokaryotes.

Link:http://easylifescienceworld.com/dna-dependent-dna-polymerase-dnap-of-prokaryotes/

Figure 8: Assembly of primosome and replisome

How nucleotides are added onto the 3’ end of the RNA primer?

  • RNA primer have free 3’ OH group with a lone pair of electron.
  • This will catalyse the nucleophillic attack on the α phosphate of the first entering deoxynucleoside triphosphate (dNTP) with the splitting off of pyrophosphate.
  • The 3′-hydroxyl group of the recently attached deoxyribonucleoside monophosphate is then free to carry out a nucleophilic attack on the next entering deoxyribonucleoside triphosphate again at its α phosphate moiety, with the splitting off of pyrophosphate.
  • Selection of the proper deoxyribonucleotide whose terminal 3′-hydroxyl group is to be attacked is dependent upon proper base pairing with the other strand of the DNA molecule according to the rules proposed originally by Watson and Crick

Example: When an adenine deoxyribonucleoside monophosphoryl (dAMP)moiety is in the template position, a thymidine triphosphate will enter and its α phosphate will be attacked by the 3′-hydroxyl group of the deoxyribonucleoside monophosphoryl most recently added to the polymer.

By this stepwise process, the template dictates which deoxyribonucleoside triphosphate is complementary and by hydrogen bonding holds it in place while the 3′-hydroxyl group of the growing strand attacks and incorporates the new nucleotide into the polymer.

Figure 9: The initiation of DNA synthesis upon a primer of RNA and the subsequent attachment of the second deoxyribonucleoside triphosphate

Replisome (DNA replicase system)

The replisome is a large protein complex that assembles at the replicating fork to undertake synthesis of DNA.  It contains DNA polymerase and other enzymes.

Replisome assembly involves three main stages:

1) Formation of pre-replication complex.

2) Formation of pre-initiation complex.

(SSB binds to the single strand and then gamma (clamp loading factor) binds to SSB).

3) Formation of initiation complex.

(Clamp loader deposits the sliding clamp (beta) and attracts DNA polymerase III).

Figure 10: Components of replisome

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