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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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DNA polymerases catalyze the development of polynucleotide chains through the addition of successive nucleotides obtained from deoxynucleoside triphosphates. The polymerase reaction takes place only in the existence of an proper DNA template. Each incoming nucleoside triphosphate an initial forms an ideal base pair through a base in this template. Only then go the DNA polymerase connect the just arrive base v the predecessor in the chain. Thus, DNA polymerases space template-directed enzymes.

DNA polymerases add nucleotides to the 3′ end of a polynucleotide chain. The polymerase catalyzes the nucleophilic assault of the 3′-hydroxyl group terminus of the polynucleotide chain on the α-phosphate team of the nucleoside tree phosphate to be added (see figure 5.22). Come initiate this reaction, DNA polymerases call for a primer v a complimentary 3′-hydroxyl group currently base-paired come the template. They cannot start from scratch by adding nucleotides come a free single-stranded DNA template. RNA polymerase, in contrast, can initiate RNA synthesis there is no a primer (Section 28.1.4).

27.2.1. All DNA Polymerases have Structural attributes in Common

The three-dimensional structures of a number of DNA polymerase enzymes room known. The very first such structure to be figured out was the of the so-called Klenow fragment the DNA polymerase ns from E. Coli (Figure 27.11). This fragment comprises two key parts of the full enzyme, including the polymerase unit. This unit almost right the shape of a appropriate hand with domains that are referred to as the fingers, the thumb, and also the palm. In addition to the polymerase, the Klenow fragment includes a domain v 3′ → 5′ exonuclease task that participates in proofreading and also correcting the polynucleotide product (Section 27.2.4).


Figure 27.11

DNA Polymerase Structure. The very first DNA polymerase structure determined was that of a fragment that E. Coli DNA polymerase I referred to as the Klenow fragment. Like various other DNA polymerases, the polymerase unit resembles a right hand v fingers (blue), palm (yellow), (more...)

DNA polymerases room remarkably similar in all at once shape, although they differ substantially in detail. In ~ least 5 structural classes have been identified; some of them are clearly homologous, whereas rather are probably the products of convergent evolution. In all cases, the finger and also thumb domains wrap about DNA and also hold it across the enzyme"s active site, i beg your pardon comprises residues mostly from the palm domain. Furthermore, all the polymerases catalyze the exact same polymerase reaction, i m sorry is dependency on two steel ions.

27.2.2. Two Bound steel Ions take part in the Polymerase Reaction

Like all enzymes through nucleoside triphosphate substrates, DNA polymerases require metal ions for activity. Check of the frameworks of DNA polymerases v bound substrates and also substrate analogs expose the presence of two metal ions in the energetic site. One steel ion binding both the deoxynucleoside triphosphate (dNTP) and also the 3′-hydroxyl group of the primer, conversely, the other interacts only with the 3′-hydroxyl group (Figure 27.12). The two metal ions are linked by the carboxylate groups of two aspartate residual water in the palm domain the the polymerase. These side chains hold the metal ions in the ideal position and orientation. The steel ion bound to the primer activates the 3′-hydroxyl group of the primer, facilitating its strike on the α-phosphate group of the dNTP substrate in the active site. The two steel ions together aid stabilize the an unfavorable charge that accumulates top top the pentacoordinate shift state. The metal ion originally bound come dNTP stabilizes the negative charge ~ above the pyrophosphate product.


Figure 27.12

DNA Polymerase Mechanism. Two metal ions (typically, Mg2+) participate in the DNA polymerase reaction. One steel ion coordinates the 3′-hydroxyl group of the primer, whereas the phosphate team of the nucleoside triphosphate bridges between the (more...)

27.2.3. The Specificity that Replication Is Dictated through Hydrogen Bonding and also the Complementarity that Shape in between Bases

DNA should be replicated through high fidelity. Every base added to the cultivation chain have to with high probability it is in the Watson-Crick complement of the basic in the corresponding position in the theme strand. The binding that the NTP containing the proper base is favored by the development of a base pair, i m sorry is stabilized by details hydrogen bonds. The binding the a noncomplementary basic is unlikely, due to the fact that the interactions are unfavorable. The hydrogen bond linking two complementary bases do a significant contribution come the fidelity that DNA replication. However, DNA polymerases replicate DNA more faithfully 보다 these interaction alone deserve to account for.

The check of the crystal structures of various DNA polymerases suggested several extr mechanisms whereby replication fidelity is improved. First, residual water of the enzyme form hydrogen bonds v the minor-groove side of the basic pair in the energetic site (Figure 27.13). In the young groove, hydrogen-bond acceptors are current in the very same positions for every Watson-Crick base pairs. This interactions act as a “ruler” that procedures whether a effectively spaced base pair has formed in the energetic site. Second, DNA polymerases nearby down around the incoming NTP (Figure 27.14). The binding of a nucleoside triphosphate right into the energetic site that a DNA polymerase root cause a conformational change: the finger domain rotates to type a chop pocket into which only a effectively shaped base pair will conveniently fit. The mutation the a conserved tyrosine residue at the height of the pocket results in a polymerase that is approximately 40 times together error prone as the parental polymerase.


Figure 27.13

Minor-Groove Interactions. DNA polymerases donate 2 hydrogen binding to base pairs in the boy groove. Hydrogen-bond acceptors are current in these two positions for every Watson-Crick basic pairs including the A-T basic pair shown.


Figure 27.14

Shape Selectivity. The binding the a nucleoside triphosphate (NTP) come DNA polymerase root cause a conformational change, generating a chop pocket because that the base pair consists of the NTP and its companion on the template strand. Together a conformational readjust (more...)

27.2.4. Numerous Polymerases Proofread the Newly included Bases and also Excise Errors

Many polymerases additional enhance the fidelity of replication by the usage of proofreading mechanisms. As already noted, the Klenow fragment that E. Coli DNA polymerase I includes an exonuclease domain the does not get involved in the polymerization reaction itself. Instead, this domain clears mismatched nucleotides indigenous the 3′ end of DNA by hydrolysis. The exonuclease energetic site is 35 Å from the polymerase energetic site, however it deserve to be reached by the freshly synthesized polynucleotide chain under suitable conditions. The proofreading device relies top top the raised probability that the finish of a cultivation strand through an incorrectly included nucleotide will leave the polymerase site and also transiently move to the exonuclease site (Figure 27.15).

Figure 27.15

Proofreading. The cultivation polynucleotide chain sometimes leaves the polymerase site of DNA polymerase I and migrates to the exonuclease site. There, the last nucleotide added is gotten rid of by hydrolysis. Because mismatched bases are more likely to leave (more...)

How walk the enzyme sense whether a newly added base is correct? First, an not correct base will certainly not pair correctly with the theme strand. Its better structural fluctuation, permitted by the weaker hydrogen bonding, will frequently carry the recently synthesized strand come the exonuclease site. Second, after the addition of a new nucleotide, the DNA translocates through one base pair into the enzyme. The newly created base pair need to be of the proper dimensions to fit right into a chop binding site and also participate in hydrogen-bonding interaction in the young groove similar to those in the polymerization website itself (see number 27.13). Indeed, the duplex DNA in ~ the enzyme adopts an A-form structure, enabling clear access to the minor groove. If an incorrect base is incorporated, the enzyme stalls, and also the stop provides added time because that the strand to move to the exonuclease site. Over there is a cost to this editing and enhancing function, however: DNA polymerase ns removes roughly 1 correct nucleotide in 20 by hydrolysis. Although the removal of correct nucleotides is contempt wasteful energetically, proofreading rises the accuracy the replication by a element of about 1000.

27.2.5. The Separation the DNA Strands Requires particular Helicases and ATP Hydrolysis

For a double-stranded DNA molecule come replicate, the 2 strands the the double helix must be be separate from every other, at least locally. This separation permits each strand to act together a theme on i m sorry a brand-new polynucleotide chain have the right to be assembled. For long double-stranded DNA molecules, the price of voluntary strand separation is negligibly short under physiological conditions. Particular enzymes, termed helicases, utilize the power of ATP hydrolysis to strength strand separation.

The comprehensive mechanisms of helicases space still under energetic investigation. However, the decision of the three-dimensional frameworks of number of helicases has been a resource of insight. Because that example, a bacter helicase dubbed PcrA comprises four domains, hereafter described as domains A1, A2, B1, and also B2 (Figure 27.16). Domain A1 includes a P-loop NTPase fold, as was expected from amino mountain sequence analysis. This domain participates in ATP binding and also hydrolysis. Domain B1 is homologous to domain A1 however lacks a P-loop. Domains A2 and B2 have unique structures.

Figure 27.16

Helicase Structure. The bacterial helicase PcrA comprises 4 domains: A1, A2, B1, and B2. The A1 domain has a P-loop NTPase fold, vice versa, the B1 domain has actually a similar overall structure yet lacks a P-loop and also does not bind nucleotides. Single-stranded (more...)

From an analysis of a set of helicase crystal frameworks bound come nucleotide analogs and appropriate double- and also single-stranded DNA molecules, a device for the activity of these enzymes was proposed (Figure 27.17). Domains A1 and B1 are qualified of binding single-stranded DNA. In the absence of bound ATP, both domain names are bound to DNA. The binding that ATP cause conformational alters in the P-loop and nearby regions that result in the closure the the cleft between these two domains. To achieve this movement, domain A1 publication the DNA and slides follow me the DNA strand, relocating closer to domain B1. The enzyme then catalyzes the hydrolysis of ATP to kind ADP and also orthophosphate. Top top product release, the cleft in between domains A and also B springs open. In this state, however, domain A1 has actually a tighter tight on the DNA than does domain B1, so the DNA is pulled throughout domain B1 toward domain A1. The result is the translocation the the enzyme along the DNA strand in a manner comparable to the way in i beg your pardon an inchworm moves. In regard come PcrA, the enzyme translocates in the 3′ → 5′ direction. When the helicase to meet a an ar of double-stranded DNA, it proceeds to move along one strand and displaces the opposite DNA strand as it progresses. Interactions with certain pockets ~ above the helicase assist destabilize the DNA duplex, aided by ATP-induced conformational changes.

Figure 27.17

Helicase Mechanism. Initially, both domain names A1 and also B1 of PcrA tie single-stranded DNA. ~ above binding the ATP, the cleft between these domains closes and domain A1 slides along the DNA. Top top ATP hydrolysis, the cleft opens up, pulling the DNA native domain B1 (more...)

Helicases constitute a huge and diverse class of enzymes. Few of these enzymes relocate in a 5′ → 3′ direction, whereas rather unwind RNA quite than DNA and participate in processes such as RNA splicing and the initiation that mRNA translation. A to compare of the amino acid sequences of hundreds of these enzyme reveals seven regions of striking preservation (Figure 27.18). Mapping these regions onto the PcrA structure shows that they heat the ATP-binding site and the cleft in between the 2 domains, regular with the concept that various other helicases experience conformational alters analogous to those uncovered in PcrA. However, whereas PcrA shows up to role as a monomer, other members that the helicase class duty as oligomers. The hexameric structures of one vital group are comparable to that of the F1 component of ATP synthase (Section 18.4.1), suggesting potential mechanistic similarities.

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Figure 27.18

Conserved Residues among Helicases. A compare of the amino acid sequences of numerous helicases revealed seven regions of strong sequence preservation (shown in color). When mapped onto the structure of PcrA, this conserved areas lie along the (more...)

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