The Mechanism Of Dna Polymerase

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DNA Polymerases Use a Single Active Site to Catalyze DNA Synthesis

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 the addition of any of the four deoxynucleosido triphosphates. DNA polymerase accomplishes this catalytic flexibility by exploiting the neatly identical geometry of the A:T and G:C base pairs (remember that the dimensions of the DNA helix are largely independent of the DNA sequence). The DNA polymerase monitors the ability of the incoming nucleotide to form an A:T or G:C base pair rather than detecling the exact nucleotide that enters the active site (Figure 8-3), Only when a correct base pair is formed are the 3'OH of the primer and the «-phosphate of the incoming nucleoside triphosphate in the optimum position for catalysis to occur. Incorrect base-pairing leads to dramatically lower rates of nucleotide addition due to a catalylically unfavorable alignment of these substrates (see Figure 8-3b). This is an example of 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. Indeed, the rate of incorporation of an incorrect nucleotide is as much as 10,000-fold slower than incorporation when base-pairing is correct.

DNA polymerases show an impressive ability to distinguish between ribo- and deoxyribonucieoside triphosphates. Although rNTPs are present at approximately ten-fold higher concentration in the cell, they are incorporated at a rate that is more than 1,000-fold lower than dNTPs. This discrimination is mediated by the steric exclusion of rNTPs from ihe DNA polymerase active site (Figure 8-4). In DNA polymerase, the nucleotide binding pocket is too small to allow the presence of a 2'OH on the incoming nucleotide. This space is occupied by two amine acids that make van rler Waals contacts with the sugar ting. Interestingly, changing these amino acids to others with smaller side chains (for example, by changing a gl utamate to an alanine) results in a DNA polymerase with significantly reduced discrimination between dNTPs and rNTPs.

a correct base pair b incorrect base pair template a correct base pair template

What Are The Dna Pairs

b incorrect base pair

Nucleotide Addition Dna Pol

FIGURE 8-3 Correctly paired bases are required for DNA polymerase catalyzed nucleotide addition, (a) Schematic diagram of the attack of a primer 3'OH end on a correctly base-paired dNTP (b) Schematic diagram of the consequence of incorrect base-paring on catalysis by DNA polymerase. In the example shown, the incorrect AA base par displaces the u-phosphate of the incoming nucleotide. This incorrect ati griment reduces the rate of catalysis dramatically resulting in the DNA polymerase preferential!/ adding correctly base-paired dNTPs {Source Based on Brautigan CA. and Steitz TA 1998. Structural and functional insights provided by crystal structures of DMA polymerase Curt Opin Structural Bblogy 8: SO, fig 4, part d. Copyright © 199S with permission from Elsevier)

a template

primer

DNA polymerase

FIGURE 8-3 Correctly paired bases are required for DNA polymerase catalyzed nucleotide addition, (a) Schematic diagram of the attack of a primer 3'OH end on a correctly base-paired dNTP (b) Schematic diagram of the consequence of incorrect base-paring on catalysis by DNA polymerase. In the example shown, the incorrect AA base par displaces the u-phosphate of the incoming nucleotide. This incorrect ati griment reduces the rate of catalysis dramatically resulting in the DNA polymerase preferential!/ adding correctly base-paired dNTPs {Source Based on Brautigan CA. and Steitz TA 1998. Structural and functional insights provided by crystal structures of DMA polymerase Curt Opin Structural Bblogy 8: SO, fig 4, part d. Copyright © 199S with permission from Elsevier)

FIGURE 8-4 Schematic illustration of the steric constraints preventing catalysis using rNTPs by DNA polymerase, (a) Binding of a correctly base-paired dNTP to the DNA polymerase. Under these conditions, the 3'OH of the primer and the <*-phosphate of the dNTP are m close proximity, (b) Addition of a 2'OH results in a stem: clash with amino acids (the discriminator amino sods) tn the nucleotide binding pocket This results tn tlve ci-phosptiate of the dNTP being displaced and a misalignment with the 3'OH ol the primer, dramatically reducing the rate ot catalysis.

a template primer

DNA polymerase

DNA Polymerases Resemble a Hand that Grips the Primer:Template Junction

A molecular understanding of how the DNA polymerase catalyzes DNA synthesis has emerged from studies of the atomic structure of various DNA polymerases bound to primer:templaie junctions, These structures reveal that the DNA substrate sits in a large cleft that resembles a partially closed right hand {Figure 8-5). Based on the analogy to a hand, Ihe three domains of the polymerase are called the thumb, fingers, and palm.

The palm domain is composed of a p sheet and contains the primary elements of the catalytic site. In particular, this region of DNA polymerase binds two divalent metal ions (typically Mgt:; or Znthat alter the chemical environment around the correctly base-paired dNTP and the 3'QH of the primer (Figure 8-6). One

FIGURE 8-5 The three-dimensional structure of DNA polymerase resembles a right hand, (a) Schematic of DMA polymerase bound to a primertemplate junction. The fingers, thumb, and palm are noted. The recently synthesized DNA is associated with the palm and the site of DNA catalysis ts located in the crevice between the fingers and live thumb. The single-stranded region Of the template Strand is bent sharply and does not pass between the thumb and tile fingers, {b) A similar view of the 17 DNA polymerase bound to DNA. The DNA is shown in a spacefilling manner and the protein is shown as a nbbon diagram Tlte fingers and the thumb are composed of a helices. The palm domain is obscured by the DNA The incoming dlW1 is shown in red (for the base and the deoxyribose) and yellow (for the triphosphate moiety). The template strand of the DNA is shown in dark gray and the primer strand is shown in light gray. (Doubtie S-, Tabor S„ l ong AM, Richardson CC, and Elfenberger T 1998. Nature 391: 251 ) Image prepared with BobScript, MolScnpt, and Raster 3D.

Green Metal Primer
primer
Dna Polymerase Metal Ion

FIGURE 8-6 Two metal ions bound to DNA polymerase catalyze nucleotide addition.

(a) Illustration of the active ate of a DNA polymerase. The two metal ions (shown in green) are held in place by interactions with two highly conserved Aspartate residues Metal ion A primarily interacts with tl^e 3'OH resulting in reduced association between the O and the H, This leaves a nudeophitic 3'0 , Metal ion B interacts with the triphosphates of the incoming dNTP to neutralize their negative charge After catalysis, the pyrophosphate product is stabilised through similar interactions with metal ion B (not shown), (b) Three-dimensional structure of the active site metal ions associated with the DNA polymerase, the 3'OH end of the primer and the incoming nucleotide. The metal ions are shown in green and the remaining elements ate shown m the same colors as in figure 8-5b. The vtew ol the polymerase shown here is roughly equivalent to rotating the image shown in Figure 8-5b -180° around the axis of the DMA helix (Doublie S,r Tabor S., Long AM.. Richardson C.C, and Ellenberger I 1998. Nature 391 251 ) Image prepared with BobScript. MdSaipt, and Raster 3D.

metal ion reduces the affinity of the 3'OH for its hydrogen. This generates a 3fO" that is primed for the nucleophilic attack of the a-phns-phate of the incoming dNTP., The second metal ion coordinates the negative charges of the and -y-phosphates of the dNTP and stabilizes the pyrophosphate producer! by joining the primer and the incoming nucleotide, in addition to its role in catalysis, the palm domain also monitors the accuracy of base-pairing for the most recently added nucleotides. This region of the polymerase makes extensive hydrogen bond contacts with base pairs in the minor groove of the newly synthesized DNA. These contacts are not base-specific but only form if the recently added nucleotides [whichever they may bo) are correctly base-paired. Mismatched DNA in this region dramatically slows catalysis, The combination of the slowed catalysis and reduced affinity for the newly synthesized DNA allows the release of the primer:template from the polymerase active site and binding to a separate proofreading nuclease active site on the polymerase.

What are the roles of the fingers am"! the thumb? The fingers are also important for catalysis. Several residues located within the fingers bind to the incoming dNTP. More importantly, once a correct base pair is formed between the incoming dNTP and the template, the finger domain moves to enclose the dNTP (Figure 8-?}. This closed form of the polymerase hand stimulates catalysis by moving the incoming nucleotide in close contact with the catalytic metal ions.

The finger domain also associates with the template region, leading to a nearly 90° turn of the phosphodiester backbone of the template immediately after the active site. This bend serves to expose only the first template base after the primer at the catalytic site. This conformation of the template avoids any confusion concerning which template base is ready to pair with the next nucleotide to be added (Figure 8-8).

In contrast to the fingers and I he palm, the thumb domain is not intimately involved in catalysis. Instead, the thumb interacts with the DNA that has been most recently synthesized (see Figure 8-9). This serves two purposes. First, it maintains the correct position of the primer and the active site. Second, the thumb helps to maintain a strong association between the DNA polymerase and its substrate. This association contributes to the ability of the DNA polymerase to add many dNTPs each time il binds a primer:templatr junction (see below).

To summarize, an ordered series of events occurs each time the DNA polymerase adds a nucleotide lo the growing DNA chain. The incoming nucleotide base-pairs with the next available template base. This interaction causes the "fingers" of the polymerase to close around the base-paired dNTP. This conformation of the enzyme places the critical catalytic metal ions in a position to catalyze formation of the next phosphodiester bond. Attachment of the base-paired nucleotide to the primer leads lo the re-opening of the fingers and the movement of the primer [template junction by one base pair. The polymerase is then ready for the next cycle of addition. Importantly, each of these events is strongly stimulated by correct base-pairing between the incoming dNTP and the template.

DNA Polymerases Are Proccssive Enzymes

Catalysis by DNA polymerase is rapid. DNA polymerases are capable of adding as many as 1,000 nucleotides per second to a primer strand.

Dntp Strand

jvr incoming j)? dNTP

rotation

DNA polymerase

(open) ARG

Lys jvr incoming j)? dNTP

5' primer

template

O-helix (closed)

rotation

Polymerase Enzyme Mechanism
of O-helix

O-helix (closed)

Dna Base Pairs Interacting With Arg

FIGURE 8-7 DMA polymerase "grips" the template and the incoming nucleotide when a correct base pair is made, (a) An illustration of the changes m DNA polymerase structure after the incoming nudeotide base-pairs correctly to the template DNA. The primary change is a 40" rotation of one of the helices in the ftnger domain called the O-hetix. In the open conformation this helix is distant from the incoming nudeobde When the polymerase is rn the dosed conformation, this helix moves and makes several important interactions with the incoming dNTP. A tyrosine makes stacking interactions with the bast ot the dNTP and two charged residues assoaate with the triphosphate The combination of these interactions positions the dNTP for catalysis mediated by the two metal ions bound to the DNA polymerase, (b) The structure of T7 DNA polymerase bound to its substrates in the dosed conformation The O-helix is shown in purple and the rest of the protein struc cure is shown as transparent for clarity. Ihe critical tyrosine, lysine, and arginine can be seen behind the O-heiix in pink. The base and the deoxyribose of the incoming dNTP are shown in red, the primer is shown in light gray, and the template strand is shown in dark gray. The two catalytic metal ions are shown in green, and the phosphates are shown in yetbw. (Doublie S., tabor S., Long A M., Richardson C.C., and Elenberger T. 5998. Nature 39}: 251.) Image prepared with BobScript, MolScript, and Raster 3D

The speed of DNA synthesis is largely due to the processive nature of DNA polymerase. Processivity is a characteristic of enzymes thai operate on polymeric substrates. In the case of DNA polymerases, the degree of processivity is defined as the average number of nucleotides added each time the enzyme binds a pnnier.fempJate junction. Each DNA polymerase has a characteristic processivity that can range from only a few nucleotides to more than 50,000 bases added per binding event (Figure 8-9).

The rate of DNA synthesis is dramatically increased by adding multiple nucleotides per binding event. It is the initial binding of

FIGURE 8-8 Illustration of the path of the template DNA through the DNA polymerase. The recently replicated DMA is associated with the palm region of the DNA polymerase. At the active site, the first base of the Single stranded region of the template is in a position expected ioi double-stranded DNA. As one follows the template strand toward its 5' end, the phosphodester backbone abruptly bends 90° fhis results in the second and all subsequent single-stranded bases being placed in a position that prevents any possibility of base pairing with a dNTP bound at the active site.

polymerase lo the primerjtemplate junction that is the rate-limiting step. In a typical DNA polymerase reaction, it takes approximately one second for the DNA polymerase to locate and bind a primer; template junction. Once bound, addition of a nucleotide is very fast tin the millisecond range). Thus, a completely nonprocessive DNA polymerase would add approximately 1 base pair per second. In contrast, the fastest DNA polymerases add as many as 1,000 nucleotides per second by remaining associated with the template for multiple rounds of dNTP addition. Consequently, a highly pro-cessive polymerase increases the overall rate of DNA synthesis by

FIGURE 8-9 DNA polymerases synthesize DNA in a processive manner.

This illustration shows the difference between a processive and a nonprocesave DMA polymerase, finth DNA polymerases bind the primer:template junction Upon binding, the nonprocessive enzyme adds a single dNTP to tlie 3* end of the primer and then is released from the new primentemplate junction. In contrast, a processive DNA polymerase adds many dNTPs each time it binds to the template

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Responses

  • Samuel
    What acid is nudeophite?
    4 years ago
  • Rhoda
    How dna pol. distinguish betn rntp and dntp?
    4 years ago
  • demsas
    How dna polymerase differentiate between dntp and rntp?
    4 years ago
  • carl
    Can dna pol discriminate between dntps and rntps.?
    4 years ago
  • arnaldo
    Is dna polymerase discriminate between rntps nd dntp?
    4 years ago
  • claudia
    How dna polymerase discriminates between dntp and rntp?
    4 years ago
  • Denise
    Can Dna polymerase recognize rNTPs because of the absence of 2'OH pocket in it?
    4 years ago
  • Susanne
    Which arediscriminator amino acids recognized 2'oH in dna polymerase?
    4 years ago
  • adamo
    How dna ploymerase differentiate between dNTP and rNTP?
    4 years ago
  • Polo
    How many active site have polymerase to catalyze the addition of nucleoside?
    3 years ago
  • ROSE
    Why does phosphodiester bond bend in dna polymerase?
    3 years ago
  • gertude
    How two divalent metal ions at palm domain helps in catalysis?
    3 years ago
  • myles
    How DNA polymerases diffferentiate between dNTPs and rNTPs?
    2 years ago
  • Michael
    How many active site dose DNA pol have?
    2 years ago
  • dominik
    What type of amino acid present in dna polymerase pocket?
    2 years ago
  • MAYA
    How DNA polymerase distinguish between rNTPs and dNTPs?
    2 years ago
  • stefan
    Why rNTP present about 10 fold higher than dNTP but added less by DNA polymerase?
    2 years ago
  • ATTE
    Why DNA polymarase requires 2 metal ions during catalysis?
    2 years ago
  • ilse
    How does a polymerase base pair?
    1 year ago
  • manlio
    Why Dna polymerase exclude rntp?
    1 year ago
  • julia
    What polymerase corrects incorrect base pairs?
    1 year ago
  • tricia rudisill
    How many amino acids make up DNA polymerase?
    8 months ago
  • giuseppa
    How many base pairs can a dna polymerase copy in a second?
    3 months ago
  • GIOELE
    How polymerases add basses?
    1 day ago

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