The Replicon Model of Replication Initiation

The initial replication fork requires the separation of the two strands of the DNA duplex to provide a template for the synthesis of both the RNA primer and new DNA. Specific genomic DNA sequence direct the initiation of DNA Replication. The specific sites at which DNA unwinding and initiation of replication occur are called Origin of Replication.

The Replicon Model of Replication Initiation:

In 963 Frabcois Jacob, Sydney Brenner and Jacques Cuzin proposed a model to explain the events controlling the initiation of replication in bacteria. It proposed two components that control the initiation of replication :

  • Replicator: Replicator is defined as the entire set of cis-acting DNA sequences that is sufficient to direct the initiation of Replication.
  • Initiator: Initiator is the protein which specifically recognizes a 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.

The model proposed that all the DNA replicated from a particular origin as a replicon. In prokaryotes since there is only one origin of replication at the single chromosome, the entire chromosome is a single replicon. In contrast the presence of multiple origins of replication divides each eukaryotic chromosomes into multiple replicons.

Replicator:

The origin of replication is the site on DNA where the DNA is unwound and DNA synthesis initiates. Although the origin of replication is always part of the replicator, sometimes the origin of replication is only a fraction of the replicator.

DNA Sequences of Replicator Share two common features:

  • First, they include a binding site for the initiator protein.
  • They include a stretch of AT-rich DNA that unwinds readily but not spontaneously. Unwinding of DNA at replicators is controlled by the replication Initiation Proteins.

The single replicator required for E-Coli chromosomal replication is called oriC. There are two repeated motif that are critical for oriC function. The 9-mer motif is the binding site for the E-Coli initiator, DnaA, and is repeated four times at oriC. The 13-mer motif, repeated 3 times, is the initial site of ssDNA formation during initiation.

Initiator:

Initiator proteins typically perform 3 different functions:

  1. Binding to the replicator – Bind a specific sequence DNA sequence within the replicator.
  2. Unwind DNA – once bound to the DNA, they frequently distort or unwind a region of DNA adjacent to the site of binding.
  3. Recruiting other replication proteins – Initiator proteins interact with additional factors required for replication initiation, thus recruiting them to the replicator.

In Prokaryotes, for example, the E-coli initiator, DnaA binds the repeated 9-mer elements in oriC and is regulated by ATP. When bound to ATP, DnaA also interacts with DNA in the region of the repeated 13-mer repeats of oriC. These additional interactions result in the separation of the DNA strands over more than 20bp within the 13-mer repeat region. this unwound DNA provides an ssDNA template for additional replication proteins such ad DNA helicase to begin the RNA and DNA synthesis steps of replication.

In Eukaryotes, the initiator is known as the origin recognition complex (ORC). It is a complex formed of 6 proteins. Like DnaA in E-Coli, ORC binds and hydrolyze ATP. ATP is required for sequence-specific DNA binding at the origin. Unlike DNA binding ORC at the replicator does not separate the DNA strands. ORC is , however, required to recruit all the remaining replication proteins to the replicator before it unwinds. Unwinding the strands requires the origin to get activated. Origin activation occurs only when the cell enters S phase of cell cycle.

Unlike prokaryotic initiator, ORC of eukaryotes have two functions:

  1. Binding to the replicator
  2. Recruiting other replication proteins

once the initiator binds to the replicator, the remaining steps in the initiation of replication is largely driven by the protein-protein interactions and protein-DNA interactions that are sequence independent. The end result is the assembly of two replication fork.

Replication in Eukaryotes

Eukaryotic replication occurs during s-phase of cell cycle.  Replication usually occurs only one time in a cell.  Replication in eukaryotes occur in five stages namely,

  1. Pre-initiation
  2. Initiation
  3. Elongation
  4. Termination
  5. Telomerase function
  1. Pre-initiation:

Actually during pre-initiation stage, replicator selection occurs.  Replicator selection is the process of identifying the sequences that will direct the initiation of replication and occur in G1 phase. and occurs in Gl (prior to S phase). This process leads to the assembly of a multiprotein complex at each replicator in the genome. Origin activation only occurs after cells enter S phase and triggers the Replicator – associated protein complex to initiate DNA unwinding and DNA polymerase recruitment.  Replicator selection is mediated by the formation of pre-replicative complexes (pre-RCs). The first step in the formation of the pre-RC is the recognition of the replicator by the eukaryotic initiator, ORC (Origin recognition Complex). Once ORC is bound, it recruits two helicase loading proteins (Cdc6 and Cdtl). Together, ORC and the loading proteins recruit a protein that is thought to be the eukaryotic replication fork helicase (the Mem 2-7 complex). Formation of the pre-RC does not lead to the immediate unwinding of origin DNA or the recruitment of DNA polymerases. Instead the pre-RCs that are formed during Gl are only activated to initiate replication after cells pass from the Gl to the S phase of the cell cycle.

rep3.14

 

  1. Initiation:

Pre-RCs are activated to initiate replication by two protein  kinases namely Cdk (Cyclin Dependant Kinase) and Ddk (Ddt4 Dependant Kinase). Kinases are proteins that  covalently attach phosphate groups to target proteins. Each of these kinases is inactive in Gl and is activated only when cells enter S phase. Once activated, these kinases target the pre-RC and other replication proteins. Phosphorylation of these pro-proteins results in the assembly of additional replication proteins at the origin and the initiation of replication.

 

These new proteins include the three eukaryotic DNA polymerases and a number of other proteins required for their recruitment. Interestingly, the polymerases assemble at the origin in a particular order. DNA Pol d and e associate first, followed by DNA Pol a/primase. This order ensures that all three DNA polymerases are present at the origin prior to the synthesis of the first RNA primer (by DNA Pol a/primase).   Once present at the origin, DNA Pol a/primase synthesizes an RNA primer and briefly extends it. Thus initiation of replication started.

rep3.15

 

  1. Elongation:

 

The resulting primer-template junction is recognized by the eukaryotic sliding clamp loader (RF-C), which assembles a sliding clamp (PCNA) at these sites. Either DNA Pol d or e recognizes this primer and begins leading strand synthesis. After a period of DNA unwinding, DNA Pol a/primase synthesizes additional primers, which allow the initiation of lagging strand DNA synthesis by either DNA Pol d or e. In the diagram, Pol d was used for leading strand and Pol e was used for lagging strand synthesis.  DNA Pol e possess activity to remove primer and fills the gap with DNA like DNA Pol I in prokaryotes.  SSB like activity was played by replication protein A (RP A) which is denoted as accessory factors during replication.

 

  1. Termination:

 

When the replication forks meet each other, then termination occurs.  It will result in the formation of two duplex DNA.  Eventhough replication terminated, 5’ end of telomeric part of the newly synthesized DNA found to have shorter DNA strand than the template parent strand.  This shortage corrected by the action of telomerase enzyme and then only the actual replication completed.

  1. Terlomerase Function:

In Linear eukaryotic chromosome, once the first primer on each strand is remove, then it appears that there is no way to fill in the gaps, since DNA cannot be extended in the 3′–>5′ direction and there is no 3′ end upstream available as there would be in a circle DNA.  If this were actually the situation, the DNA strand would get shorter every time they replicated and genes would be lost forever.

Elizabeth Blackburn and her colleagues have provided the answer to fill up the gaps with the help of enzyme telomerase.  So, that the genes at the ends, are conserved.  Telomerase is a ribonucleoprotein (RNP) i.e. it has RNA with repetitive sequence.  Repetitive sequence varies depending upon the species example Tetrahymena thermophilia RNA has AACCCC sequence and in Oxytrica it has AAAACCCC.  Telomerase otherwise known as modified Reverse Transcriptase.  In human, the RNA template contains AAUCCC repeats.  This enzyme was also known as telomere terminal transferase.

The 3′-end of the lagging strand template basepairs with a unique region of the telomerase associated RNA.  Hybridization facilitated by the match between the sequence at the 3′-end of telomere and the sequence at the 3′-end of the RNA.  The telomerase catalytic site then adds deoxy ribonucleotides using RNA molecule as a template, this reverse transcription proceeds to position 35 of the RNA template.  Telomerase then translocates to the new 3′-end by pairing with RNA template and it continues reverse transcription.  When the G-rich strand sufficiently long, Primase can make an RNA primer, complementary to the 3′-end of the telomere’s G-rich strand.   DNA polymerase uses the newly made primer to prime synthesis of DNA to fill in the remaining gap on the progeny DNA.  The primer is removed and the nick between fragments sealed by DNA ligase.

rep3.17

Mechanism of DNA Polymerase

The synthesis of DNA is catalyzed by an enzyme called DNA Polymerase.  Unlike most enzymes, which have an active site dedicated to a single reaction, DNA polymerase uses a single active site  to catalyze addition of any of the four deoxynucleotide triphosphates. Polymerase shows kinetic selectivity, in which an enzyme favors catalysis using one of several possible substrates by dramatically increasing the rate of bond formation only when the correct substrate is present. Only when the correct pair is formed the catalysis occurs. Incorrect base pairing leads to dramatically lower rate of nucleotide addition due to the catalytically unfavorable alignment of these substrates. DNA polymerase show an impressive ability to discriminate between ribo- and deoxyribonucleoside phosphates. This ability is mediated by steric exclusion of rNTPs from the DNA polymerase active site, because in DNA polymerase the nucleotide binding pocket is too small to allow the presence of a 2’OH on the incoming nucleotide.

DNA Polymerase Structure

DNA Polymerase resemble a right hand that partially grips the primer: Template junction. Based on anatomy of the hand, there are three domains in the polymerase:

  • Palm: The palm domain is composed of a B Sheet and is the active site of DNA Polymerase. This region of DNA polymerase binds two divalent metal ions (Mg2+ or Zn2+) that alter the chemical environment around the correctly base-paired dNTP and the 3’OH of the primer. One metal ion reduces the affinity of the 3’OH for its hydrogen. This generates a 3’O- that is primed for the nucleophilic attack of the alpha-phosphate of the incoming dNTP. The secong metal ion coordinates the negative charges of the Beta and Gamma phosphates of the dNTP and stabilizes the pyrophosphate produced by joining the primer and the incoming nucleotide. The palm domain also monitors the accuracy of base-pairing for the most recently added dNTP.
  • Fingers: once the correct base-pair is formed, the finger domain moves to enclose the dNTP. this close form of the polymerase hand stimulates catalysis by moving the incoming nucleotide in close contact with the catalytic metal ions.
  • Thumb: Thumb interacts with the DNA that has been most recently synthesized. this serves two purpose: (1) It maintains the correct position of the primer and the active site, (2) helps to maintain a strong association between the DNA polymerase and its substrate, which contribute to the processive nature of DNA polymerase.

Features of DNA Polymerase:

  • High Processivity: Catalysis by DNA polymerase is rapid, are capable of adding as many as 1000 nucleotide per second to a primer strand. In case of DNA polymerase, the degree of processivity is defined as the number of nucleotides added each time the enzyme binds a primer:template junction.
  • Multiple substrates: In processive enzyme each time the enzyme binds a primer:template junction multiple substrates, dNTP is added to the primer. In contrast with no processive enzyme adds a single dNTP to primer and then is released from the primer:template junction.

Increased processivity is facilitated by the ability of DNA polymerase to slide along the DNA Template. DNA polymerase interacts tightly with much of the double-stranded portion of the DNA in a sequence non specific manner. These interactions include electrostatic interactions between the phosphate backbone and the thumb domain, and interaction between the minor groove of the DNA and the palm domain. Further increase in processivity are achived through the interactions between the DNA polymerase and the “Sliding clamp” protein.

  • Exonuclease Activity: Occasional flickering of the bases into the ‘wrong’ tautomeric form. This alternate form of bases allow incorrect base pairs to be correctly positioned for catalysis. Exonucleases proofread newly synthesized DNA. These type of exonuclease is called proofreading exonuclease. Theses exonucleases are capable of degrading DNA starting from the 3′ DNA end, that is the from the growing end of the new DNA strand and remove the incorrect nucleotide.

Mispaired DNA alters the geometry of the 3′ OH and the incoming nucleotide due to poor interactions with the palm region. This altered geometry reduces the rate of nucleotide addition and increase the rate of proofreading exonuclease activity. proofreading can occur without releasing the DNA from the polymerase. When a mismatched pair is detected, the primer:template junction slides away from the DNA polymerase active site and into the exonuclease site. after the incorrect nucleotide is removed the correctly paired primer:template junction slides back ingto the active site of polymerase and DNA synthesis continues.

The DNA polymerase performing synthesis and editing

  • Nucleotides are added in to both templates in replication fork at the same time.
  • Primer is required. DNA polymerase require primer to initiate DNA Polymerase.

Specialization of DNA Polymerase

DNA polymerases are specialized for different roles in the cell. The central role of DNA polymerases in the efficient and accurate replication of the genome requires that cell have multiple specialized DNA polymerases.

DNA polymerases in Prokaryotes:

1. DNA polymerase II (pol II) is a minor DNA polymerase in E. соli.

  • Function. Pol II may be involved in some DNA repair processes, but E соli mutants lacking this enzyme show no replication or growth deficiencies.
  • Structure. Pol II is a single polypeptide.
  • Other enzymatic activities. Pol II has proofreading (3′ to 5′ exonuclease) activity but lacks excision-repair (5′ to 3′ exonuclease) activity.

2. DNA polymerase III (pol III) is the primary DNA polymerase involved in cellular replication.

  • Function. Pol III catalyzes leading and lagging strand synthesis.
  • Structure. Pol III is structurally complex. The pol III core enzyme, made of three different polypeptides, was first isolated. Gentle purification schemes and careful reconstitution showed that the active cellular form of pol HI, called pol III holoenzyme, is made of 10 subunit polypeptides. The subunits are organized into an asymmetric dimeric structure with two catalytic centers. This is important in understanding how it catalyzes both leading and lagging strand syntheses at the replication fork.
  • Other enzymatic activities. Pol III holoenzyme has proofreading (3′ to 5′ exonuclease) activity but no excision-repair (5′ to 3′ exonuclease) activity. The two α subunits both contain the DNA polymerase activity, and the two ε subunits both contain the 3′ to 5′ exonuclease activity. The two subunits form a dimer that circles around the DNA. This β2 “DNA clamp” enables the pol III holoenzyme to stay bound to the DNA and facilitates its high rate of replication.

3. DNA polymerase I (pol I) was the first DNA polymerase discovered.

  • Function. Pol I functions in the replication of DNA and in the repair of damaged DNA.
  • Structure. Pol I is a single polypeptide.
  • Other enzymatic activities. Pol I has two enzymatic activities—besides DNA polymerase activity—that are important to its cellular function.
    • (1) Proofreading. Pol I does not typically add a nucleotide to the growing DNA chain that cannot properly base pair with the template strand. If a mismatched nucleotide is added, the enzyme halts polymerization. A 3′- to 5′-exonuclease activity removes the mismatched nucleotide, and polymerization resumes. This activity is called proofreading, and it assures the high-level fidelity of replication that is a desirable trait of genetic material.
    •  (2) Excision-repair. Pol I has a 5′ to 3′ exonuclease activity, called excision-repair activity, that can hydrolytically remove a segment of DNA from the 5′ end of a strand of duplex DNA.
      • (a) From 1-10 nucleotide segments of DNA can be removed simultaneously.
      • (b) This activity is essential for the removal of primers in DNA replication.
      • (c) This activity is essential for the repair of damaged DNA.
    • (3) Nick-translation. A nick is a break in a phosphodiester bond of one strand of DNA in a double helix. A nick leaves a free 3′-hydroxyl and a free 5′-phosphate. Pol I can function at nicks as an exonuclease and a polymerase at the same time. As the 5′-phosphate nucleotide is removed or displaced by pol I, it is replaced with the polymerase activity of pol I. Pol I cannot reseal the nick; therefore, the nick is moved, or translated, along the DNA in the direction of synthesis.

DNA polymerases in Eukaryotes:

1. DNA polymerase alpha /primase

  • Function. DNA polymerase alpha is almost exclusively required for the initiation of DNA replication and the priming of Okazaki fragments during elongation. In most organisms no specific repair role beyond that of checkpoint control has been assigned to this enzyme.
  • Structure. Pol alpha contain 4 subunit. (two subunit DNA Pol alpha and a two-subunit primase)

2. DNA polymerase beta

  • Function. Base excision repair. The role of DNA polymerase beta in base-excision repair is well established for mammalian systems, but in yeast, DNA polymerase delta appears to fulfill that function.
  • Structure. Pol beta contain 1 subunit.

3. DNA polymerase gamma

  • Function. Mitrochondrial DNA replication and repair.
  • Structure. contain 3 subunit.

4. DNA polymerase Delta

  • Function. DNA polymerase delta functions as a dimer and, therefore, may be responsible for both leading and lagging strand DNA replication. In addition, this enzyme is required for mismatch repair and, together with DNA polymerase zeta, for mutagenesis.
  • Structure. contain 2-3 subunits.

5. DNA polymerase Epsilon

  • Function. The function of DNA polymerase epsilon in DNA replication may be restricted to that of Okazaki fragment maturation. In contrast, either polymerase delta or epsilon suffices for the repair of UV-induced damage.
  • Structure. contain 4 subunits.

During Replication, due to relatively low processivity , DNA pol alpha/primase is replaced by the highly processive DNA polymerase Delta and Epsilon by the process called polymerase switching. The majority of other DNA polymerase in Eukaryotes are involved in DNA repair.

DNA Replication: Introduction

DNA replication is the process of producing two identical replicas from one original DNA molecule. This biological process occurs in all living organisms and is the basis for biological inheritance. DNA is made up of two strands and each strand of the original DNA molecule serves as template for the production of the complementary strand, a process referred to as semiconservative replication. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication.

For the DNA synthesis two key substrate is required.

Substrate: The four deoxynucleoside triphosphates (dNTPs)—deoxyadenosine tri- phosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphos-phate (dCTP), and deoxythymidine triphosphate (dTTP)—are needed as substrates for DNA synthesis.

Primer:template junction: second substrate for the DNA Synthesis is a particular arrangement of ssDNA and dsDNA called primer:template junction.

  • Template. DNA replication cannot occur without a template. A template is required to direct the addition of the appropriate complementary deoxynucleotide to the newly synthesized DNA strand. In semiconservative replication, each strand of parental DNA serves as a template. Then, each template strand and its newly synthesized complementary strand serve as the DNA in daughter cells.
  • Primer. DNA synthesis cannot start without a primer, which prepares the template strand for the addition of nucleotides. The primer must also have an exposed 3’OH because new nucleotides are added to the 3′ end of a primer that is properly base paired to the template strand of DNA, new synthesis is said to occur in a 5′ to 3′ direction.

The DNA synthesis that occurs during the process of replication is catalyzed by enzymes called DNA-dependent DNA polymerases. These enzymes depend on DNA to the extent that they require a DNA template. They are more commonly called DNA polymerases.