What is dATP?
Deoxyadenosine triphosphate (dATP) is a nucleotide that is used as a building block in DNA synthesis, during both in vivo replication and transcription and in vitro replication techniques such as PCR. Structurally, dATP is defined as a purine nucleoside triphosphate; a deoxyribose sugar bound to adenine and 3 phosphates (2’-Deoxyadenosine-5′-triphosphate).
The dATP organic compound has a molecular weight of 491.1816 g/mol. It consists of three subunits: a nucleobase, a five-carbon sugar, and a phosphate group. The sugar component is deoxyribose, used in the makeup of DNA. A different sugar component, ribose, is used in RNA.
A nucleobase is a nitrogen-containing compound that is classified into two major forms: purines and pyrimidines. They are involved in the formation of nucleosides and nucleotides.
Nucleotides are the monomers of nucleic acid polymers; nucleosides are a phosphate group. The phosphate group present in the nucleotides connects the sugar rings of two adjacent nucleotide monomers. Both a phosphate group and the sugar moiety act as a backbone of a nucleic acid.
How is dATP synthesized?
dATP is enzymatically synthesized from DNA (the starting material) using deoxyribonuclease (DNase)—a nuclease enzyme that breaks down DNA into smaller fragments: nuclease P1, adenylate kinase, and pyruvate kinase. The general steps of dATP synthesis are as follows:
- The first step in the synthesis is the heat denaturation of DNA, followed by treatment with DNase I to produce oligomers.
- Then, the solution is treated with nuclease P1 to form deoxynucleoside monophosphates.
- A mixture of adenylate kinase and pyruvate kinase is used to selectively convert the deoxyadenosine monophosphate to dATP.
- The last step is to purify the prepared solution.
What does dATP do?
2′-Deoxyadenosine-5′-triphosphate (dATP) is one of the five deoxynucleotides (dNTPs) present in organisms, which include dTTP (deoxythymidine), dCTP (deoxycytidine), dUTP (deoxyuridine), and dGTP (deoxyguanosine). They act as substrates in the process of nucleic acid synthesis.
In DNA synthesis, dATP pairs with dTTP. It also has a role as an Escherichia coli (or E.coli) metabolite and a mouse metabolite.
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How Does dATP Work?
dATP, along with other dNTPs, provide nucleotides to the “unzipped” single-strand template DNA during the process of replication. With the help of DNA polymerase, they expand the growing DNA strand to complete its synthesis.
At the time of DNA synthesis, the DNA polymerase enzyme covalently links the free hydroxyl (-OH) group, on the 3’ carbon of a growing chain of nucleotides, to the α-phosphate on the 5’ carbon of the next dNTP, releasing the β- and γ-phosphate groups as pyrophosphate (PPi). This creates a phosphodiester linkage between dNTPs.
Role of dATP in the inhibition of Ribonucleotide reductases (RNR)
Ribonucleotide reductases is an enzyme that catalyzes the formation of deoxyribonucleotides from ribonucleotides by removing the 2′-hydroxyl group from its ribose ring.
It has two classes: RNR I and RNR II.
The RNR I enzyme is regulated by dATP and ATP and the RNR II enzyme is regulated by GTP and dGTP. ATP and GTP activate the enzyme’s functions and dATP and dGTP act as inhibitors of the enzyme.
dATP regulates the activity of ribonucleotide reductase by binding to its active site. In the class I ribonucleotide reductase of E. coli, the inhibition is due to the increased space between the cysteine and tyrosine radical cofactor at C-site and 𝛃2. This inhibits the formation of the active site that is required for the reduction process.
In human ribonucleotide reductase, dATP induces an α6 structure that inhibits the activity of the ribonucleotide reductase. It facilitates the function by preventing 𝛃 subunits from accessing the active site of the of 𝛂 subunit.
What can you use dATP for?
Commercially available dATP has applications in several in vitro biochem and molecular biology assays to achieve the desired end products or goals. It’s recommended to review a manufacturer’s MSDS and datasheet of the product before purchasing dATP for your lab.
The manufacturer’s documents should highlight these key features of their product:
- Greater than 99% purity (should be confirmed by HPLC)
- Be free of human and E. coli DNA
These guidelines should help you to protect your experiments from getting spoiled because of low-quality reagents.
dATP is used in lab workflows along with other reagents including polymerases, other dNTPs, tris buffer, SDS, and antibodies (based on the type of experiment). Some of the in vitro workflows that involve dATPs are given below:
- 3′-end labeling of RNA with recombinant yeast poly(A) polymerase
- 3′-end-labeling of oligonucleotide primers
- A-tailing at the 3′ end of a double-stranded DNA
- Other molecular biology applications, including PCR amplification, real-time PCR, DNA labeling, DNA sequencing, and whole genome amplification
dATP vs. ATP: What’s the Difference?
The main difference between ATP and dATP is that ATP is a ribonucleotide whereas dATP is a deoxyribonucleotide. And, a functional difference between them is that ATP serves as the energy currency of the cell as well as a building block of RNA and dATP serves as a precursor for DNA synthesis.
The structural differences between ATP and dATP are given below:
|It’s a phosphorylated nucleotide, composed of three phosphate groups and adenosine.||It’s one of the two purine nucleotides that is involved in the process of DNA synthesis.|
|It consists of a ribose sugar, which has a hydroxyl group at the 2’ position of the ring.||It consists of deoxyribose sugar, which has a hydrogen group at the 2’ position of the ring.|
|It’s a ribonucleotide.||It’s a deoxyribonucleotide.|
|Image: The structural representation of ATP.||Image: The structural representation of dATP.|
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dATP is deoxyadenosine triphosphate that acts as a precursor for nucleic acid synthesis in the replication and transcription process. Structurally, it consists of a purine nitrogen base (adenine), deoxyribose sugar, and three phosphate groups.
dATP is different from ATP in both structural and functional aspects. ATP has a 2’ hydroxyl group on the ribose ring and serves as an energy source in organisms, whereas dATP lacks a 2’ hydroxyl group and instead acts as a precursor molecule for DNA and RNA synthesis.
dATP has applications in many biochemistry and molecular biology experiments including PCR, real-time PCR, whole-genome sequencing, and many more.
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