DNA structure and mechanism of replication

Introduction

DNA is a helically twisted double-chain poly deoxyribonucleotide macromolecule that constitutes the genetic material of all the organisms with the exception of ribovirus. In prokaryotes, it occurs in nucleoid and plasmid. The DNA is usually circular. Unlike, in eukaryotes, it occurs in chromatin of the nucleus and this is linear. Single-stranded DNA occurs as genetic material in some viruses. In addition, smaller quantities of DNA are also present in mitochondria and plastids. In this case, it can either be circular or linear.

History

DNA was discovered in 1869 by J. Friedrich Miescher, a Swiss researcher. He named the phosphorus-containing substance as “nuclein”. Erwin Chargoff and his colleagues (in the late 1940s) found that the four nucleotide bases of the DNA occur in different ratios in different organisms. Furthermore, the demonstration that DNA contains genetic information made in 1944 by Avery, Macleod, and MacCary.

Structure of DNA

It is the largest macromolecule that has a diameter of 2nm or 20Å. An interesting thing to know is that human DNA has 3 billion bases and more than 99 percent of those bases are the same in all people. A DNA molecule has two unbranched complementary strands. They are spirally coiled and collectively known as DNA duplex. The duplex has two types of alternate grooves, major and minor. Basically, one turn of the spiral has about 10-10.5 nucleotides. Altogether, it occupies a distance of 34Å. Moreover, the two adjacent nucleotides or bases are separated by a space of less than 3.4Å. Each base is also attached to a sugar molecule and a phosphate molecule.

DNA structure
Deoxyribonucleic Acid

A DNA is formed by cross-linking of three components.

  1. a phosphate group,
  2. a pentose sugar, and
  3. nitrogenous bases (purines and pyrimidines)

Nitrogenous bases

The nitrogenous bases are derivatives of two parent compounds, purines and pyrimidine.

  • Purine
  • Pyrimidine

Purines

Purines are 9-membered double rings with nitrogen at 1, 3, 7, and 9 positions. Altogether, they contain four nitrogen atoms and two carbon rings. They are larger molecules. Basically, purines are of two types, adenine (A) and guanine(G). In adenine, we have an amino group (NH2) at 6 carbon atom. Whereas in guanine, an amino group is present at the second and C=O at sixth carbon atom.

purine structure
structure of a purine

Pyrimidine

It is a heterocyclic aromatic organic compound and is 6-membered rings with nitrogen at 1 and 3 positions. Altogether, they contain two nitrogen atom and only one carbon atom. On the contrary, they are smaller. Pyrimidines are also of two types and these are cytosine(C) and thymine(T). The DNA & RNA contains the same purines namely adenine(A) and guanine(G). In a similar fashion, pyrimidine cytosine(C) is present in both DNA & RNA. But, the second major pyrimidine (thymine) is not the same In both DNA and RNA. Instead of thymine(T), Uracil(U) is present in RNA. On rare occasion, thymine occurs in RNA and uracil occur in DNA. In pyrimidine, we have an amino group (NH2) at the fourth carbon atom and C=O at the second carbon atom in cytosine.

pyrimidine structure
structure of a pyrimidine

DNA double helix

The double helical structure was proposed by James Watson and Francis Crick in 1953. Later, they receive a nobel prize for the same in 1962.

It consists of two helical DNA strands wound around the same axis to form a right-handed double helix. Although the two strands are antiparallel i.e., they run parallel to each other but In opposite directions. In one chain, the direction is 5′ to 3′ and in another, it is 3′ to 5′.

The two strands are held together by H-bonds formed by complementary base pair. Here, complementary base pairing means a purine of one chain lies exactly opposite to pyrimidine of another chain. Adenine binds to thymine and Cytosine bonds to Guanine. A-T base pair has two H-bonds whereas G-C has three H-bonds. This allows a sort of lock and key arrangement between large size purines and small size pyrimidine.

Why purine binds to pyrimidine?

Do you wonder why a purine never binds to a purine and a pyrimidine to a pyrimidine? Want to know the answer? Then, stay connected…

Here is the answer

The H-bonds are formed between a purine and a pyrimidine only. Because if two purines face together, they won’t fit into the allowable space(because of their large size). Likewise, two pyrimidines won’t bind because they would be too far to form H-bonds(because of their smaller size). Therefore, the only base pair arrangement possible in the structure from spatial consideration is A-T, T-A, G-C, and C-G. Thus, the complementary base-pairing in DNA helix proves the Chargaff’s rule.

According to Chargaff’s rule, the number of adenosine residues is equal to the number of thymidine residue and the no. of guanosine residue is equal to the no. of cytidine residue. From this relationship, we can say that the sum of purine residue is equal to the sum of pyrimidine residue. I.e.

A+G=T+C

Mechanism of DNA replication

Replication occurs in the following steps:

  1. Activation of deoxyribonucleotides
  2. Exposure of parent DNA bases
  3. Formation of RNA primer
  4. Base pairing
  5. Conversion of deoxyribonucleoside monophosphate
  6. Formation of new DNA chains
  7. Editing (proof-reading) and DNA repair.

Activation of deoxyribonucleotide

Depending upon the nitrogen base, it has four kinds of deoxyribonucleotides namely,

  1. first, deoxy adenine monophosphate (de AMP)
  2. second, deoxy guanine monophosphate (de GMP)
  3. third, deoxy cytosine monophosphate (de CMP)
  4. last but not least, deoxy thymine monophosphate (de TMP)

The raw material required in DNA synthesis are four deoxyribonucleotides that float freely in the nuclear sap. Chiefly, these are de AMP, de CMP, de TMP, and de GMP.

Here, please note that the above mentioned four molecules are nucleotides and not the nucleosides. The difference between a nucleotide and a nucleoside is that the nucleoside is without a sugar molecule. A nucleotide is composed of a nitrogenous base, a sugar, and a phosphate group whereas a nucleoside comprises a nitrogenous base and a phosphate group. Please read carefully where I write a nucleoside and where a nucleotide

Now, come to the topic…

Further, for incorporation into Deoxyribonucleic acid, these nucleotides are activated by union with ATP molecule. This reaction is called phosphorylation. It is catalyzed by phosphorylase. Meanwhile, it produces deoxyribonucleotides de ATP, de GTP, de CTP, and de TTP.

Exposure of parent DNA bases

Parent double-strand splits and uncoil into single strands by the breakdown of H-bonds. In fact, unwinding is not an easy job. For this purpose, enzyme helicases help in unwinding the helicase by using ATP hydrolysis as a source of energy. Enzyme helicases are also known as unwindases.

After that, the helix stabilizing protein comes into play. In this case, a non-enzymic single-stranded DNA-binding protein stabilizes the chain in a single-stranded form and reduces the energy required in unwinding. Due to unwinding, a coiling tension builds in both strands. Moreover, other enzymes called topoisomerase, may cut and rejoin a strand of Deoxyribonucleic acid to facilitate uncoiling. In case of prokaryotes, enzyme gyrase does the job of helicases and topoisomerase.

The entire Deoxyribonucleic acid strand does not split into one go. Instead, it starts separating at a specific point. This point is called the origin of replication or ‘Ori’. In other words, DNA separation starts progressing at the origin of replication. Eukaryotic DNA may split at the various origin of replication whereas prokaryotic DNA generally forms a single origin of replication.

replication of DNA helix

Formation of RNA primer

Generally, a short chain of RNA is formed on the DNA template at the 5′ end. This is called an RNA primer. The enzyme polymerase catalyzes the polymerization of RNA building blocks (A.U.G, and C) into the primer. Okazaki fragments will form when the RNA primer binds to the DNA strand. These are short and discontinuous stretches of nucleotides, usually 150-200 base pair long. Later, they will linked together by the enzyme DNA ligase.

Since the DNA polymerase cannot initiate the synthesis of a newly formed DNA strand, this RNA primer is formed. Although, DNA polymerase can catalyze the growth of the strand.

After that, the primer is removed and the gaps so left, are fitted with deoxyribonucleotides to make th chain continuous.

Base pairing

According to base-pairing rule, deoxyribonucleoside triphosphate joined together by H-bonds. As I have said above, base pairing occurs according to Chargaff’s rule i.e., Adenine binds to Thymine and Cytosine bonds to Guanine.

Conversion of deoxyribonucleoside triphosphate to monophosphate

Deoxyribonucleoside triphosphate is attached to each single DNA strand. In order to proceed with the process, they break off their inner high energy bonds and pyrophosphate Deoxyribonucleic acid molecules sat free. In this case, it changes deoxyribonucleoside triphosphate to deoxyribonucleoside monophosphate. These are the normal component of DNA molecules. Thereafter, pyrophosphate undergoes hydrolysis by an enzyme pyrophosphatase and releases energy. As a result, the inorganic phosphate group sat free.

The energy released in the above step used to derive the polymerization of nucleoside to form DNA

Formation of new DNA chain

At this point, the adjacent monophosphate joined to each single DNA strand linked together to form a new chain. Certainly, the process is catalyze by the enzyme polymerase α and δ. Eventually, it produces two double chains that are identical to each other as well as to their mother strand.

Editing (proofreading) and DNA repairs.

Lastly, proofreading and DNA repair occur. It is a very crucial step as it ensures the conservation of genetic material and also prevents genetic mutations and defects.

The specificity of base pairs ensures accurate replications. However, some wrong bases do get in. These wrong bases are observed by DNA polymerase. Moreover, the abnormal regions resulting from mutation are cleaves by enzyme nuclease. Meanwhile, the DNA polymerase resynthesizes the missing segments of DNA.

Lastly, the DNA ligase join the new and old DNA strands.

Sources

Molecular Biology of the cell, 5th edition, Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter, chapters 4 and 5.

Lehninger; principles of biochemistry by Michael M. Cox and David E. nelson

https://en.wikipedia.org/wiki/DNA_replication

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