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.

DNA and its structural Properties.

DNA is the prime genetic molecule carrying all the hereditary information within chromosomes.

DNA Structure.

The most important feature of DNA is that it is usually composed of two polynucleotide chains twisted around each other in the form of right handed double helix. These two strands are complementary to each other.

However, since each strand has a both a free 5′ hydroxyl group at one end and a free 3′ hydroxyl group at the other, each strands has a polarity or directionality. The polarity of these two strands of macromolecule are in opposite directions, and thus DNA is described as an ‘anti-parallel‘ structure.

These macro molecule structures are composed of regular repeating polymers formed from nucleotides. These are the basic building blocks of nucleic acids. The nucleotides are derived from nucleosides that are composed of two elements; a five-membered pentose sugar, known as 2′-deoxyribose and a nitrogenous base. The sugar is called 2′-deoxyribose because there is no hydroxyl at position 2′. (just two hydrogen). The carbon atom of the sugar are designated ‘prime’ (1′,2′,3′,…5′) to distinguish them from the carbon atoms of nitrogenous bases.

A nucleotide or nucleoside phosphate is fromed by the attachment of a phosphate to the 5′ position of a nucloside by an ester linkage. Such nuclotides can be joined together by the formation of a second ester bond by reaction between the terminal phosphate group of one nucleotide and the 3′ hydroxyl of another, thus generating a 5′ to 3′ phophodiester bond between adjacent sugars; this process can be repeated indefinitely to give long polynucleotide molecule.

The bases in DNA falls in to two classes; purines and pyrimidines. The purines are adenine (A) and Guanine (G) and the pyrimidines are cytosin (C) and thymine (T). The purines are derived from the double-ringed structure but with different groups attached. The pyrimidines are single-ringed structures. The bases are attached to the deoxyribose by Glucosidic linkages at N1 of the pyrimidines or N9 of the purines.

The sequence of bases at one strand is complementary to that in the other. A purine base attached to a sugar residue on one strand is always hydrogen bonded to a sugar residue on the other strand. Adenine always pairs with thymine, via two hydrogen bonds, and guanine always pairs with cytosin by three hydrogen bonds. This is called the specificity of base pairing.

These bases exists in two alternative tautomeric states. The nitrogen atoms attached to the purines and pyrimidine rings are in amino form in the predominant state and only rarely assume the imino configuration. Likewise, the oxygen atoms attached to the guanine and thymine normally have the keto form and only rarely take on the enol configuration.

The high degree of thermodynamic stability of DNA double helices result in part from the large number of hydrogen bonds between base-pairs and in part from the hydrophobic bonding (or “staking force“) between staked base-pairs.

An organic molecule in aqueous solution has all its hydrogen bonding properties satisfied with by water molecules, that comes on and off very rapidly. This means for every hydrogen bond formed requires breaking of H-bond with water molecule that was there before the base pair formed. However, when the polynucleotide chains are seperated, water molecules are lined up on the bases. when strands come together in the double helix, the water molecules are displaced from the bases. This creates disorder and increase entropy, thereby stabilizing the double helix.

DNA double helix is also stabilized by the stacking interactions between the bases. The bases are flat, relatively insoluble molecules, and they stack above each other roughly perpendicular to direction of double helix. Electron cloud interaction (Pi-Pi) between bases in the helical stacks contribute significantly to the stability of the double helix.

Hydrogen bonding is also important for the specificity of the base pairing. Suppose we tried to pair an Adenine with a Cytosine, then they have a hydrogen acceptor (N3 of cytosine) lying opposite a H-Bond acceptor (N1 of Adenine) with no room to put a water molecule in between to satisfy the two acceptors. And like wise two hydrogen bond donor opposite each other. Thus an A:C base pair is unstable in forming H-Bond.