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What initiates primase to add an RNA primer to a DNA strand and what makes it stop?


What initiates primase to add an RNA primer to a DNA strand and what makes it stop adding RNA nucleotides? Is there tags added to the DNA back-bone?


Prokaryotic primases are activated by DNA helicase [1, 2] while the eukaryotic ones are triggered when they form a complex with DNA polymerase alpha and its accessory B subunit [2].

I couldn't find too much information about what exactly triggers activation, but according to De Falco M et al. (2004):

[… ] synthetic function (of the prokaryotic primase) is specifically activated by thymine-containing synthetic bubble structures that mimic early replication intermediates. [3]

It stops when it finishes reading a DNA template:

The Sso DNA primase utilizes poly-pyrimidine single-stranded DNA templates with low efficiency for de novo synthesis of RNA primers [3].


References:

  1. Wikipedia contributors, "Primase," Wikipedia, The Free Encyclopedia, http://en.wikipedia.org/w/index.php?title=Primase&oldid=593928284 (accessed December 1, 2014).
  2. David N. Frick, and Charles C. Richardson. DNA PRIMASES. Annual Review of Biochemistry. Vol. 70: 39-80 (Volume publication date July 2001). DOI: 10.1146/annurev.biochem.70.1.39
  3. De Falco M, Fusco A, De Felice M, Rossi M, Pisani FM. The DNA primase of Sulfolobus solfataricus is activated by substrates containing a thymine-rich bubble and has a 3'-terminal nucleotidyl-transferase activity. Nucleic Acids Res. 2004 Sep 30;32(17):5223-30. Print 2004.

What is the role of the primers in PCR?

Primers are the strands of DNA (or RNA) that serve as this initial foundation for the DNA replication process, and they are used to demarcate the segment of the DNA template to be amplified. In the PCR process, two primers are matched to the segment of DNA.

Beside above, what is the role of the forward and reverse primers used in PCR? Two primers are utilized, one for each of the complementary single strands of DNA released during denaturation. The forward primer attaches to the start codon of the template DNA (the anti-sense strand), while the reverse primer attaches to the stop codon of the complementary strand of DNA (the sense strand).

Also to know, what is the role of a primer?

In living organisms, primers are short strands of RNA. The synthesis of a primer is necessary because the enzymes that synthesize DNA, which are called DNA polymerases, can only attach new DNA nucleotides to an existing strand of nucleotides. The primer therefore serves to prime and lay a foundation for DNA synthesis.

Why does a PCR reaction require a primer?

PCR (Polymerase Chain Reaction) Because DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, it needs a primer to which it can add the first nucleotide. This requirement makes it possible to delineate a specific region of template sequence that the researcher wants to amplify.


Subjective & Short Questions of Molecular Genetics

Ans: The DNA polymerase III can add nucleotides only to a chain of nucleotides that is already paired with the parent strands. Hence DNA polymerase cannot itself initiate synthesis of DNA. Thus another enzyme, primase synthesize an RNA primer.

Ans: Leading strand elongates toward the replication fork. In this

case, the nucleotides are added continuously to its growing 3′ end. Lagging strand elongates away from the replication fork.

Ans: Lagging strand is synthesized discontinuously. A series of short segments are synthesized. These segments are connected later on. These segments are called Okazaki fragments.

Ans: The central dogma is composed of transcription and translation. These two steps of central dogma are also collectively called gene expression.

Ans: The synthesis of mRNA from the DNA is called transcription.

Ans: Transcription starts at the promoter on the DNA template strand. Promoter is the RNA polymerase binding site.

Ans: It is found in ribosome. The rRNA provide

Ans: Transfer RNA molecules read massage (code) on the mRNA and transport the amino acids to the ribosomes.

10-Differentiate between codes and anticodes.

Ans: Codes are present on mRNA and anticodes are present on tRNA.

11-What is the function of mRNA?

Ans: The mRNA is long strands of RNA. It is transcribed from DNA and it travels to the ribosomes and direct precisely which amino acids are assembled into polypeptide.

12-Differentiate between translation and transcription.

Ans: The synthesis of mRNA from DNA is called transcription. The synthesis of protein from mRNA is called translation.

13-Differentiate between template strand or the antisense strand and coding strand or the sense strand.

Ans: Template is the strand which is transcribed during transcription. The opposite strand is called coding or sense strand.

14-What is transcription bulb?

Ans: The DNA strands open tip at the place where enzyme is attached with the template strand. It forms transcription bubble.

15-What is cap and tail of mRNA?

Ans: Cap is in the form of 7 methyl GTP. It is linked to 5′ end with the first nucleotide of mRNA. Tail is in the form of poly A tail. It is linked to 3′ end of the RNA.

16-What is genetic code?

Ans: Genetic code is a combination of 3 nucleotides, which specify a particular amino acid.

17-Why are three nucleotides taken as most appropriate Mr genetic codes?

Ans: Three Nucleotides can form 43 or 64 different combinations. It is more than enough to code for the 20 amino acids. Thus genetic code is a triplet code and the reading occurs continuously.

18-What are terminations or nonsense codes?

Ans: These are three codons UAA, UAG and UGA. They do not code for any amino acid. So they are known as nonsense codon.

19-What is initiation codon?

Ans: It is present at the start of every gene. This initiation codon
AUG. It encodes the amino acid methionine.

20-Are genetic codes universal?

Ans: No, genetic codes are not universal. The study of genetic

code of mitochondrial DNA showed that genetic codes are not so universal. For example, UGA codon is normally a stop codon but in mitochondria it codes for tryptophan amino acid.

21-What is translation?

Ans: The synthesis of protein from mRNA is called translation.

22-What is anticodon?

Ans: The tRNA molecules possess complementary three nucleotide sequence. It is called anticodon.

23-What is the function of P site (peptidyl site) of ribosome?

Ans: It is the site of the ribosome where peptide bonds are formed.

24-What is the function of A site (aminoacyl site)?

Ans: This site contains successive (next) amino acid with its

25-What is initiation factor?

Ans: A proteins initiation factor control the attachment of the

tRNA on the ribosomal surface at the P site.

26-What is translocation?

Ans: During Translocation, the ribosome now moves (translocates) to next code along the mRNA molecule in the


What initiates primase to add an RNA primer to a DNA strand and what makes it stop? - Biology

By the end of this section, you will be able to do the following:

  • Explain the process of DNA replication in prokaryotes
  • Discuss the role of different enzymes and proteins in supporting this process

DNA replication has been well studied in prokaryotes primarily because of the small size of the genome and because of the large variety of mutants that are available. E. coli has 4.6 million base pairs in a single circular chromosome and all of it gets replicated in approximately 42 minutes, starting from a single site along the chromosome and proceeding around the circle in both directions. This means that approximately 1000 nucleotides are added per second. Thus, the process is quite rapid and occurs without many mistakes.

DNA replication employs a large number of structural proteins and enzymes, each of which plays a critical role during the process. One of the key players is the enzyme DNA polymerase, also known as DNA pol, which adds nucleotides one-by-one to the growing DNA chain that is complementary to the template strand. The addition of nucleotides requires energy this energy is obtained from the nucleoside triphosphates ATP, GTP, TTP and CTP. Like ATP, the other NTPs (nucleoside triphosphates) are high-energy molecules that can serve both as the source of DNA nucleotides and the source of energy to drive the polymerization. When the bond between the phosphates is “broken,” the energy released is used to form the phosphodiester bond between the incoming nucleotide and the growing chain. In prokaryotes, three main types of polymerases are known: DNA pol I, DNA pol II, and DNA pol III. It is now known that DNA pol III is the enzyme required for DNA synthesis DNA pol I is an important accessory enzyme in DNA replication, and along with DNA pol II, is primarily required for repair.

How does the replication machinery know where to begin? It turns out that there are specific nucleotide sequences called origins of replication where replication begins. In E. coli, which has a single origin of replication on its one chromosome (as do most prokaryotes), this origin of replication is approximately 245 base pairs long and is rich in AT sequences. The origin of replication is recognized by certain proteins that bind to this site. An enzyme called helicase unwinds the DNA by breaking the hydrogen bonds between the nitrogenous base pairs. ATP hydrolysis is required for this process. As the DNA opens up, Y-shaped structures called replication forks are formed. Two replication forks are formed at the origin of replication and these get extended bi-directionally as replication proceeds. Single-strand binding proteins coat the single strands of DNA near the replication fork to prevent the single-stranded DNA from winding back into a double helix.

DNA polymerase has two important restrictions: it is able to add nucleotides only in the 5′ to 3′ direction (a new DNA strand can be only extended in this direction). It also requires a free 3′-OH group to which it can add nucleotides by forming a phosphodiester bond between the 3′-OH end and the 5′ phosphate of the next nucleotide. This essentially means that it cannot add nucleotides if a free 3′-OH group is not available. Then how does it add the first nucleotide? The problem is solved with the help of a primer that provides the free 3′-OH end. Another enzyme, RNA primase, synthesizes an RNA segment that is about five to ten nucleotides long and complementary to the template DNA. Because this sequence primes the DNA synthesis, it is appropriately called the primer. DNA polymerase can now extend this RNA primer, adding nucleotides one-by-one that are complementary to the template strand ((Figure)).

Art Connection

Figure 1. A replication fork is formed when helicase separates the DNA strands at the origin of replication. The DNA tends to become more highly coiled ahead of the replication fork. Topoisomerase breaks and reforms DNA’s phosphate backbone ahead of the replication fork, thereby relieving the pressure that results from this “supercoiling.” Single-strand binding proteins bind to the single-stranded DNA to prevent the helix from re-forming. Primase synthesizes an RNA primer. DNA polymerase III uses this primer to synthesize the daughter DNA strand. On the leading strand, DNA is synthesized continuously, whereas on the lagging strand, DNA is synthesized in short stretches called Okazaki fragments. DNA polymerase I replaces the RNA primer with DNA. DNA ligase seals the gaps between the Okazaki fragments, joining the fragments into a single DNA molecule. (credit: modification of work by Mariana Ruiz Villareal)

Question: You isolate a cell strain in which the joining of Okazaki fragments is impaired and suspect that a mutation has occurred in an enzyme found at the replication fork. Which enzyme is most likely to be mutated?

The replication fork moves at the rate of 1000 nucleotides per second. Topoisomerase prevents the over-winding of the DNA double helix ahead of the replication fork as the DNA is opening up it does so by causing temporary nicks in the DNA helix and then resealing it. Because DNA polymerase can only extend in the 5′ to 3′ direction, and because the DNA double helix is antiparallel, there is a slight problem at the replication fork. The two template DNA strands have opposing orientations: one strand is in the 5′ to 3′ direction and the other is oriented in the 3′ to 5′ direction. Only one new DNA strand, the one that is complementary to the 3′ to 5′ parental DNA strand, can be synthesized continuously towards the replication fork. This continuously synthesized strand is known as the leading strand. The other strand, complementary to the 5′ to 3′ parental DNA, is extended away from the replication fork, in small fragments known as Okazaki fragments, each requiring a primer to start the synthesis. New primer segments are laid down in the direction of the replication fork, but each pointing away from it. (Okazaki fragments are named after the Japanese scientist who first discovered them. The strand with the Okazaki fragments is known as the lagging strand.)

The leading strand can be extended from a single primer, whereas the lagging strand needs a new primer for each of the short Okazaki fragments. The overall direction of the lagging strand will be 3′ to 5′, and that of the leading strand 5′ to 3′. A protein called the sliding clamp holds the DNA polymerase in place as it continues to add nucleotides. The sliding clamp is a ring-shaped protein that binds to the DNA and holds the polymerase in place. As synthesis proceeds, the RNA primers are replaced by DNA. The primers are removed by the exonuclease activity of DNA pol I, which uses DNA behind the RNA as its own primer and fills in the gaps left by removal of the RNA nucleotides by the addition of DNA nucleotides. The nicks that remain between the newly synthesized DNA (that replaced the RNA primer) and the previously synthesized DNA are sealed by the enzyme DNA ligase, which catalyzes the formation of phosphodiester linkages between the 3′-OH end of one nucleotide and the 5′ phosphate end of the other fragment.

Once the chromosome has been completely replicated, the two DNA copies move into two different cells during cell division.

The process of DNA replication can be summarized as follows:

  1. DNA unwinds at the origin of replication.
  2. Helicase opens up the DNA-forming replication forks these are extended bidirectionally.
  3. Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA.
  4. Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling.
  5. Primase synthesizes RNA primers complementary to the DNA strand.
  6. DNA polymerase III starts adding nucleotides to the 3′-OH end of the primer.
  7. Elongation of both the lagging and the leading strand continues.
  8. RNA primers are removed by exonuclease activity.
  9. Gaps are filled by DNA pol I by adding dNTPs.
  10. The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds.

(Figure) summarizes the enzymes involved in prokaryotic DNA replication and the functions of each.

Prokaryotic DNA Replication: Enzymes and Their Function
Enzyme/protein Specific Function
DNA pol I Removes RNA primer and replaces it with newly synthesized DNA
DNA pol III Main enzyme that adds nucleotides in the 5′-3′ direction
Helicase Opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases
Ligase Seals the gaps between the Okazaki fragments to create one continuous DNA strand
Primase Synthesizes RNA primers needed to start replication
Sliding Clamp Helps to hold the DNA polymerase in place when nucleotides are being added
Topoisomerase Helps relieve the strain on DNA when unwinding by causing breaks, and then resealing the DNA
Single-strand binding proteins (SSB) Binds to single-stranded DNA to prevent DNA from rewinding back.

Link to Learning

Review the full process of DNA replication here.

Section Summary

Replication in prokaryotes starts from a sequence found on the chromosome called the origin of replication—the point at which the DNA opens up. Helicase opens up the DNA double helix, resulting in the formation of the replication fork. Single-strand binding proteins bind to the single-stranded DNA near the replication fork to keep the fork open. Primase synthesizes an RNA primer to initiate synthesis by DNA polymerase, which can add nucleotides only to the 3′ end of a previously synthesized primer strand. Both new DNA strands grow according to their respective 5′-3′ directions. One strand is synthesized continuously in the direction of the replication fork this is called the leading strand. The other strand is synthesized in a direction away from the replication fork, in short stretches of DNA known as Okazaki fragments. This strand is known as the lagging strand. Once replication is completed, the RNA primers are replaced by DNA nucleotides and the DNA is sealed with DNA ligase, which creates phosphodiester bonds between the 3′-OH of one end and the 5′ phosphate of the other strand.

Art Connections

(Figure) You isolate a cell strain in which the joining of Okazaki fragments is impaired and suspect that a mutation has occurred in an enzyme found at the replication fork. Which enzyme is most likely to be mutated?


3 Main Enzymes of DNA Replications | Cell Biology

A primase is an enzyme which makes the RNA primers required for initiation of Okazaki pieces on the lagging strand. Primase activity needs the formation of a complex of primase and at least six other proteins. This complex is called the primo-some.

The primo-some contains pre-priming proteins—arbitrarily called proteins i, n, n’ and n”—as well as the product of genes dna B and dna C. The primo-some carries out the initial priming activity for leading strand wherein the synthesis takes place continuously in the overall 5′ to 3′ direction.

It also carries out the repeating priming of the synthesis of Okazaki fragments for the lagging strand where the synthesis occurs discontinuously in the overall 3′ to 5′ direction.

The primase shows a very strong preference to initiate with adenosine followed by guanosine and this suggests that initiation of Okazaki fragments may occur at particular sites on the lagging strand. However, the small phage P4, which needs only about 20 Okazaki fragments per round of replication, shows no preferential initiation sites.

The primase that is tightly as­sociated with the eukaryotic DNA polymerase Q is made of two sub-units and shows no stringent sequence requirements. But it does not act at random.

Enzyme # 2. DNA Polymerase:

DNA polymerase is an enzyme that makes a new DNA on a template strand. Both prokaryotic and eukaryotic cells contain more than one species of DNA polymerase enzymes. Only some of these enzymes actually carry out replication and sometimes they are designated as DNA replicases. The others are involved in subsidiary roles in replication and/or par­ticipate repair synthesis of DNA to replace damaged sequences.

DNA polymerase catalyses the formation of a phosphodiester bond between the 3′ hydroxyl group at the growing end of a DNA chain (the primer) and the 5′ phosphate group of the incoming deoxyribonucleoside triphosphate (Fig. 20.8).

Growth is in the 5’→3′ direc­tion and the order in which the deoxyribonucleotides are added is dictated by base pairing to a template DNA chain. Thus, besides four types of deoxyribonucleotides and Mg++ ions, the enzyme requires both primer and template DNA (Figs. 20.9 and 20.10). No DNA polymerase has been found which is able to initiate DNA chains.

DNA polymerases isolated from prokaryotes and eukaryotes differ from each other in several aspects a brief account of these enzyme is given below:

(i) Prokaryotic DNA Polymerase:

There are three different types of prokaryotic DNA polymerases which are called DNA poly­merase I, II and III. These enzymes have been isolated from prokaryotes. DNA polymerase I or Romberg enzyme was first to be isolated from E.coli by Arthur Kornberg etal and was used for DNA synthesis in 1956. Kornberg received (jointly with Severo Ochoa) the Nobel Prize for this work in 1959.

DNA polymerase is a protein of Mr109, 000 in the form of a single polypeptide chain. It contains only one sulphydryl group and one disulphide’ group—the residue at the N- terminus is methionine.

Most of the prokaryotic DNA polymerase I exhibits the following activates:

ii. 3′ → 5′ exonuclease activity.

iii. 5′ → 3′ exonuclease activity.

iv. Excision of the RNA primers used in the initiation DNA synthesis.

DNA polymerase I is mainly responsible for the synthesis of new strand of DNA. This is the polymerase activity. The direction of synthesis of the new strand’ is always 5′ → 3′. But it is estimated that DNA polymerase incorporates wrong bases during DNA replication with a frequency of 10-5. This is not desirable.

Hence DNA polymerase has also 3′ 5′ exonuclease activity (Fig. 20.11) which enables it to proof­read or edit the newly synthesised DNA strand and, thereby, correct the errors made during DNA replication. An exonuclease is an enzyme that degrades nucleic acids from the free ends.

Therefore, whenever the DNA chain being syn­thesised has a terminal mismatch, i.e., insertion of a wrong base in the new chain, the 3’→ 5′ exonuclease activity of DNA polymerase I in reverse direction clips off the wrong base and immediately the same enzyme, i.e., DNA polymerase I, reinitiates the synthesis of correct base in the growing new chain.

Therefore, due to this dual activity of DNA polymerase I, the chance of errors in DNA replication is reduced.

The 5′ → 3′ exonuclease activity of DNA polymerase I is also very important. It func­tions in the removal of the DNA segment damaged by the irradiation of ultraviolet ray and other agents. An endonuclease (degrades nucleic acid by making internal cut) must cleave the DNA strand close to the site of damage before 5′ → 3′ exonuclease action of the DNA polymerase I may take place.

The 5′ → 3′ exonuclease activity of DNA polymerase I also functions in the removal of RNA primers from DNA. The ribonucleotides are Immediately replaced by deoxyribonucleotides due to the 5′ → 3′ polymerase activity of the enzyme.

The prokaryotic DNA polymerase II was discovered in pol A – mutant of E.coli. Pol A is a gene responsible for the synthesis of polymerase I. Therefore, the mutant of pol A – are deficient in DNA polymerase I or Kornberg enzyme. But, in absence of DNA polymerase I, replication of DNA also takes place in such mutant type.

Therefore, it is obvious that DNA poly­merase II plays a role in DNA replication of such mutant. DNA polymerase II has 5’→ 3′ polymerase activity but it uses gapped DNA template. This enzyme also has the 3′ → 5′ but not the 5′ → 3′ exonuclease activity. The function of E.coli DNA polymerase II in vivo is unknown.

Prokaryotic DNA polymerase III was also discovered in pol A – mutant. There is a strong evidence that unlike DNA polymerase I and II, polymerase III is essential for DNA synthe­sis. The best template for DNA polymerase III is double-stranded DNA with very small gaps containing 3′-OH priming ends. In the DNA polymerase II, the core enzyme is tightly associated with two small sub-units.

The core enzyme has both 3′ → 5′ exonuclease (which could be involved in proof-reading) and 5’→ 3′ exonuclease activities, although the latter is only manifest in vitro on duplex DNA with a single-stranded 5′ tail.

This enzyme has a higher affinity for nucleotide triphosphate than DNA polymerase I and II and catalyses the synthesis of DNA chains at very high rates, i.e., 10-15 times the rate of polymerase I. The major properties of the three DNA polymerases are summarised in Table 22.3.

A DNA polymerase molecule has four func­tional sites which are involved in polymerase activity.

These sites are:

(iii) Primer terminus site, and

The template site binds to the DNA strand functioning as template during DNA replica­tion and holds it in the correct orientation. The primer site is the site where the primer chains to which the nucleotides will be added are attached.

The primer terminus site ensures that the primer binding to the primer site has a free 3′- OH. A primer without a free 3′-OH is not able to bind to this site.

The triphosphate site is the site for bind­ing deoxyribonucleotide 5′-triphosphate that is complementary to the corresponding nucleotide of the template and catalyses the formation of phosphodiester bond between the 5′ phosphate of this nucleotide and the 3′-OH of the terminal primer nucleotide. In addition, there is a 3’→ 5′ exonuclease site and a 5′-3′ exonuclease site of DNA poly­merase I.

(ii) Eukaryotic DNA Polymerase:

In higher eukaryotes, there are at least four DNA polymerases known as α, β,y and δ and a fifth (ɛ) has recently been described. In yeast DNA, polymerase I corresponds to DNA polymerase a, polymerase II to e, polymerase III to 6 and polymerase m to S and they have renamed accordingly.

Polymerase α is present in the nuclei of the cell. DNA polymerase a shows optimal activity with a gapped DNA template but shows a remarkable ability to use single-stranded DNA by forming transient hairpins. It will not bind to duplex DNA.

The native, undegraded enzyme consists of a 180 K Da polymerase together with three sub- units—the 60 and 50 K Da sub-units of about 70, 60 and 50 K Da. Association of the 180 K Da polymerase with the 70KDa protein makes the 3’→ 5′ exonuclease activity of the larger sub-units comprise a primase activity which allows the enzyme to initiate replication on unprimed single-stranded cyclic DNAs.

There­fore, polymerase a have dual activity, i.e., both the polymerase and primase activity. The association of primase with DNA polymerase α is restricted to the DNA synthetic phase.

Polymerase β is also present in the nuclei. It shows optimal activity with native DNA acti­vated by limited treatment with native DNA-ase I to make single-stranded nicks and short gaps bearing 3′-OH priming termini and also shows negligible activity with denatured DNA. DNA polymerase β is believed to play a role in repair of DNA.

Polymerase δ is present in the dividing cell and have got similar properties polymerase a, but having 3′ → 5′ exonuclease activity. The activity of polymerase δ is dependent on activity on two auxiliary proteins: cyclin and activator I.

Due to presence of approximately equal activities of DNA polymerase α and δ it has been proposed that they act as a dimer at the replication fork with the highly processive polymerase δ acting on the leading strand and the primease-associated polymerase a acting on the lagging strand.

Cyclin or PCNA (proliferating cell nuclear antigen) independent form of DNA polymerase 6 is known as polymerase e which has two ac­tive polymerase sub-units of 220 and 145 K Da. DNA polymerase e is also probably involved in replication and it has been proposed that it takes over from DNA polymerase a in the synthesis of Okazaki fragments.

Polymerase y is found in small amount in animal cells. It is also found in mitochon­dria and chloroplasts and is believed to be responsible for replication of the chromosome of these organelles. DNA polymerase 7 isolated from chick embryos is a tetramer having four identical sub-units. It has also a proof-reading exonuclease activity.

Enzyme # 3. DNA Ligases:

DNA ligase is an important enzyme involved in DNA replication. DNA ligases catalyse the formation of a phosphodiester bond between the free 5′ phosphate end of an oligo or polynu­cleotide and the 3′-OH group of a second oligo or polynucleotide next to it.

A ligase-AMP complex seems to be an obligatory intermediate and is formed by reaction with NAD in case of E.coli and B. subtilis and with ATP in mam­malian and phage-infected cells.

The adenyl group is then transferred from the enzyme to the 5′ phosphoryl terminus of the DNA. The activated phosphoryl group is then attached by the 3′-hydroxyl terminus of the DNA to form a phosphodiester bond. DNA ligases join successive Okazaki fragments produced during discontinuous DNA replication and seal the nicks left behind by DNA polymerase.

Reverse Transcriptase:

The enzymes so far discussed are required for the synthesis of DNA on parental tem­plate strand of DNA. But in certain RNA virus or retrovirus, there is an enzyme—called RNA-dependent DNA polymerase or reverse transcriptase—which uses parental RNA strand as a template for the synthesis of DNA.

The immediate product of this enzyme activity is the formation of double-stranded RNA-DNA hybrid which is the result of the synthesis of a complementary strand of DNA using single- stranded viral RNA as template. This enzyme uses viral RNA as template.


INTRODUCTION

Mycobacterium tuberculosis is a deadly pathogen that claims nearly 2 million lives annually and infects an estimated 2 billion people, who serve as a reservoir of latently infected individuals (1). Most tuberculosis (TB) cases are not the result of new infections but are caused by the reactivation of dormant M. tuberculosis (2). TB caused by drug-sensitive strains is fully treatable, but patients must take three or four drugs for approximately 𢙖 months. Premature termination of drug therapy results in the emergence of resistant strains. The World Health Organization estimates that 50 million individuals harbor multidrug-resistant (MDR) M. tuberculosis, which is resistant to at least rifampin and isoniazid. Treating these MDR strains requires second-line drugs, which are expensive, have side effects, and take longer to work (up to 2 years). More disturbing is that strains of untreatable extensively drug-resistant (XDR) TB, which are additionally resistant to any fluoroquinolone and at least one of three injectable second-line drugs (capreomycin, kanamycin, or amikacin), have already been identified in 58 countries. This XDR form, together with totally drug-resistant (TDR) TB, seems to represent the greatest health threat (3). The options for treating MDR/XDR/TDR TB infections are becoming seriously limited, threatening to return TB control to the preantibiotic era (4, 5). The first-line drugs for treating TB are restricted to a few sensitive targets, including inhA (NADH-dependent enoyl-[acyl carrier protein] reductase) and kasA (3-oxoacyl-[acyl carrier protein] synthase 1) for isoniazid, rpoB (DNA-directed RNA polymerase subunit beta) for rifampin, and the embCAB operon for ethambutol. Also in this category are enzymes required for the intracellular activation of currently used drugs, such as katG (catalase peroxidase peroxynitritase T) for isoniazid, pncA (pyrazinamidase/nicotinamidase) for pyrazinamide, and etaA (monooxygenase) for ethionamide (6). The identification of new drugs and sensitive targets would appear to be indispensable for the control of drug-resistant forms of TB. One requirement for a promising antibacterial enzyme target is that it be essential for the organism and that it not be present in the host. Such candidates might be found among basic essential metabolism pathways, including DNA replication processes.

Bacterial DNA replication is performed by PolIII, which is unable to synthesize DNA de novo and therefore requires a primer to allow the initiation of DNA synthesis. The replication of leading strands requires at least a single primer to initiate the process, but replication of the lagging strand requires an individual starter for each Okazaki fragment. In Escherichia coli, the enzyme that synthesizes such primers is the RNA polymerase, DnaG. Eukaryotes also possess a distinct primase responsible for the synthesis of RNA primers. DNA primase is a single-strand DNA (ssDNA)-dependent RNA polymerase that plays a key role in DNA synthesis (7). The DNA primases of bacteria and bacteriophages are classified into one group, and the primases of eukaryotes and archaea belong to a second group. All primases share many catalytic properties, but the proteins in the two classes differ both in structure and in their relationship with other proteins in the replication complex (8, 9). The prokaryotic primase associates with the replicative DNA helicase. DnaG primase contains three distinct domains, an N-terminal zinc-binding region, a middle RNA polymerase domain, and a C-terminal domain containing either a DNA helicase (phage) or a region for interaction with the DNA helicase (bacteria) (10). In contrast to DnaG primase, which is monomeric, eukaryotic primase is a heterodimeric complex of DNA polymerase α and an accessory β subunit. The small primase subunit (PriS) belongs to the archaeo-eukaryotic primase (AEP) superfamily (11). The PriS complex contains an active site for RNA primer synthesis and the large primase accessory subunit (PriL), which may coordinate primase and polymerase action and is required for the initiation of primer synthesis (12). Previous studies have demonstrated that AEPs are also present in diverse bacteria (13, 14).

An AEP domain constitutes one of three domains in ATP-dependent ligase (LigD), which is a key protein in the nonhomologous end-joining (NHEJ) DNA repair system (11, 15, 16, 17, 18). The primase domain has terminal transferase, DNA-dependent RNA primase, and DNA-dependent DNA/RNA gap-filling polymerase activities (15, 16, 18, 19, 20). In mycobacteria, both DnaG and AEPs have been reported. The replicative DnaG primase is encoded by the dnaG gene, which is located in the dnaG operon (21).

The viability of DnaG primases as antibiotic targets rests on the presumption that these enzymes are essential for all bacteria because they are required for initiating DNA replication. However, it is difficult to definitively establish this indispensability, which is a fundamental prerequisite if these enzymes are to be considered potential antibiotic targets. In this report, we undertook a series of experiments that unequivocally demonstrate that dnaG is essential in Mycobacterium smegmatis, even in AEP-overexpressing backgrounds. We also characterized the enzymatic activities of M. smegmatis and M. tuberculosis DnaG proteins. A detailed analysis of the amount of DnaG in various strains revealed that the level of protein can vary by 𢏆-fold without producing a major effect on growth under standard laboratory conditions. Strains engineered during this study will be useful in any future detailed evaluation of antibiotics targeting DnaG.


Watch the video: How DNA Polymerase Works (January 2022).