DNA Replication Steps

DNA is known as the 'blueprint of life'. It was discovered by James Watson, a biologist from Indiana University and Fransis Crick, a physicist. The duo worked at the Cavendish Lab in Cambridge, England on the structure of DNA. In spite of many hurdles and difficulties, they managed to announce to the world, that they had "found the secret of life". This secret of life is what we all know as DNA. This molecule undergoes DNA replication process. Thus, DNA replication results in two DNA molecules. This article will cover eukaryotic DNA replication steps and prokaryotic replication steps. But, first let us understand the structure of DNA.

Structure of DNA
DNA (Deoxyribonucleic acid) is a double stranded structure, that has many chains of genetic matter in the form of chromosomes. There are two complementary strands that form a double helix. The two strands of DNA run anti-parallel to each other. One strand runs in the 5' → 3' direction and the other in the parallel direction of 3' → 5' direction. Each strand has a 5' phosphate end and a 3' hydroxyl end. The backbone of DNA molecule is the deoxyribose sugar. There are 5 carbons that are numbered 1', 2', 3', 4' and 5', to help them to be distinguished from the atoms of purine and pyrimidine rings. The backbone of DNA is formed by the deoxyribose sugar and a phosphate group along with the base. The DNA twists at specific lengths due to the bonding angles of the DNA backbone molecules. This forms a helical structure instead of a straight ladder. The steps of the twisted ladder are made by the base pairs.

The Base Pairs of DNA
The monomer unit of a DNA is called a nucleotide, that consists of a 5-carbon sugar, that is, deoxyribose, a nitrogen attached to the sugar and a phosphate group. There are four types of nucleotide molecules in the DNA structure that differ only in the nitrogenous base. These four nucleotides are as follows:

• Adenine
• Guanine
• Cytosien
• Thymine

The first letter of the nucleotide name is used as an abbreviation for the entire name. Adenine and Guanine are purines and form the larger base pairs in DNA. Cytosine and Thymine form the smaller base pair and are known as pyrimidines. These nucleotides are the base pairs that form hydrogen bonds with the complementary base on the other strand of DNA. Adenine forms a double bond with thymine (A = T) and cytosine forms a triple bond with guanine (C ≡ G).

This was the basic DNA structure. Now let us move on to our main topic of DNA replication steps. The eukaryotic DNA replication steps differ from the prokaryotic DNA replication steps. Both the complementary strands of DNA contain genetic information that is required for the development of a new cell or organism. These two strands serve as templates for the reproduction of the complementary strand. During DNA replication process, the template strand is conserved entirely, with a new strand assembled with the help of nucleotides. This DNA replication process is, therefore, called semi-conservative replication. Thus, a double stranded DNA molecule is manufactured, that is identical to the previous strands. This mechanism is fool-proof as there is proofreading and error checking process, to make sure there are least errors.

Difference Between the Eukaryotic and Prokaryotic DNA Replication Steps
As mentioned earlier, the eukaryotic DNA replication and the prokaryotic DNA replication steps have a different mechanism. The eukaryotic DNA replication steps are more complex than the prokaryotic DNA replication. Eukaryotic DNA is found in all complex organisms which includes plants and animals, whereas the prokaryotic DNA is present in 'simple' organisms like bacteria and cyanobacteria. The eukaryotic DNA is always present in combination with histone proteins and the prokaryotic DNA is a simple duplex that does not contain histones (that is, basic proteins). In the prokaryotic DNA replication steps, the DNA is replicated during the interval between the cell divisions. The eukaryotic DNA replication steps are highly regulated and the process takes place during the 'S' phase of the cell cycle, that precedes mitosis or meiosis I.

DNA Replication Process
DNA replication is a very complex process, that requires many proteins to act together. These proteins known as replication proteins, are clustered together in the cell and that unit of the cell can be called the 'Replication Factory'. Here the DNA replication, results in two DNA molecules. The DNA replication proteins each have a specific function in the production of new DNA strand. The six proteins arranged in a ring shape known as Helicase, unwind the double stranded DNA helix into single strands. The tetramers, that is, the single stranded binding proteins, cover the single-stranded DNA. This prevents the DNA strands from re-annealing and forming the double stranded molecule. The RNA polmerase known as primase, synthesizes short RNA primers that initiate the DNA replication process. DNA polymerase threads the nucleotides together, forming a DNA strand. The DNA polymerase is held on to the DNA strand during the DNA replication step, by an accessory protein called the sliding clamp. 'RNAse H' helps in removing the RNA primers that initiate the DNA strand synthesis. The long continuous DNA strand is created by the linking of short stretches of DNA by DNA ligase. Know more on difference between DNA and RNA.

The Replication Fork
The replication fork is a structure that is formed during the DNA replication process. The fork is made with the action of helicase, that breaks the hydrogen bonds, that hold the two DNA strands together. This results in a structure that has two branching 'prongs' of a single strand DNA each.

Synthesis of New Strands
The two single DNA strands act as templates individually, that are used for producing two complementary DNA strands. The double helix consists of two anti-parallel DNA strands with complementary 5' to 3' strands. The polymerase enzymes synthesize nucleic acid strands only in the 5' → 3' direction. It hooks the 5' phosphate group of an incoming nucleotide onto the 3' hydroxyl group at the end of the growing nucleic acid chain. Thus, the chain grows on to the extension of the 3' hydroxyl group and the strand synthesis proceeds in the 5' to 3' direction. DNA polymerase cannot begin synthesizing the DNA strand initially. It needs a nucleic chain in the beginning to begin copying the strand. Thus, RNA polymerase called primase, copies the first short stretch of the DNA strand. Thus, creating a complementary RNA segment, that is 60 nucleotides long and known as a primer. This gives the DNA polymerase the required platform to begin copying the DNA strand. It begins at the 3' end of the RNA primer and, with the reference from the old DNA strand, it synthesis the new complementary DNA strand. Two simple DNA replication enzymes are required for each parental DNA strand. The two polymerase enzymes move in opposite direction of the two strands.

During the synthesis, only one polymerase remains on the DNA template and copies the DNA in a continuous strand. The other polymerase copies only a short stretch of DNA, before running into the primer of the initially sequenced fragment. Thus, it needs to release the DNA strand and slide further up-stream and start the extension of another RNA primer. The sliding clamp holds the DNA in its place as it moves through the DNA replication process.

The strand that is synthesized continuously is called the leading strand and the strand that is synthesized in short pieces is called the lagging strand. The short pieces of synthesized DNA,, that make up the lagging strand, are called the Okazaki fragments.

Synthesis of the Lagging Strand
The lagging strand is the DNA strand of the replication fork, that is opposite to the leading strand. It is synthesized in the opposite direction, that is, 5' to 3' instead of the 3' end as in the leading strand. The DNA polymerase cannot synthesize the strand 5' → 3' as explained above. Thus, the strand is synthesized in short fragments forming a lagging strand known as the Okazaki fragment. Primase builds RNA primers in short bursts over the lagging strand, which is synthesized in the 5' → 3' by DNA polymerase. The RNA primers are removed and new deoxyribonucleotides are added to the gaps, where the RNA was present. These strands are joined by DNA ligase, thus, completing the synthesizis of lagging strand.

Note: The removal of RNA fragments from the strands, have different mechanism in eukarytotic DNA replication steps and prokaryotic DNA replication steps.

Synthesis of the Leading Strand
The DNA strand that is read in the 3' → 5' and synthesized in the 5' → 3' direction continuously, is known as the leading strand. The DNA polymerase III synthesis the DNA using the 3'- OH group, donated by the single RNA primer. The DNA replication continues in the direction of the replication fork, in a continuous manner.

Steps in DNA Replication
Now, that we have understood the basis of the DNA replication and the functions of the simple DNA replication enzymes, let us go through the DNA replication steps in action.

• The double helix structure is unwound by the helciase enzyme exposing two single stranded DNAs. This creates a replication fork, onto which DNA replication process occurs simultaneously on each fork. The proteins that are involved in the DNA replication process are collected in one location of the cell. This shows that the proteins do not move along the length of DNA, but the DNA is fed through the protein factory or area just like a film is fed into a projector.
• The single-stranded binding proteins (SSBs) cover the DNA strands and thus, preventing them from annealing into a double strand. These SSBs are easily moved by the DNA polymerase enzyme.
• The original DNA strand is used as a template to synthesize the DNA strand in the 5' → 3' direction with the help of an extension formed by RNA primer. DNA polymerase can synthesize the strand in 5' → 3' only, thus, one strand is synthesized in short bursts, joined together later on.
• RNAse H and DNA polymerase I (exonuclease) recognizes the RNA polymers that are bound to the DNA template and removes the primers by RNA hydrolysis.
• The gaps formed due to hydrolysis of RNA are filled in by the DNA polymerase.
• The DNA pieces or nicks are filled with deoxyribonucleotides and joined together by the enzyme ligase, thus, completing the DNA replication process.

The human DNA is up to 80 million base pair long in a chromosome. Thus, the DNA is unwound at multiple places along its length and DNA replication steps are carried out simultaneously at many places.

Fact File: The human DNA is copied at about 50 base pairs per second. The multiple location of DNA replication process takes about 1 hour to complete. If this were not the case, then it would take about a month to finish replicating the entire DNA strand!

The DNA replication process is almost error free with the help of DNA polymerase and other simple DNA replication enzymes, that proofread the nucleotides being added to the strand. If the nucleotides are not found to be complementary, then they are removed and a new nucleotide is synthesized. Thus, creating an error free DNA strand.

Fact File: A billion nucleotides have less than one mistakes. This means that copying 100 dictionaries with 1000 pages word to word, page to page and symbol to symbol, with only one mistake!

This is all about the DNA replication steps, involved in copying a new DNA strand. A lot of DNA research and DNA sequencing has been carried out to know these minute processes in a cell. The DNA replication process is essential for the survival of all life forms. DNA replication steps occur during the inter-phase and are copied before cell division. Specialized cells like the muscles and nerve cells do not divide, thus, there is no DNA replication process carried out here. DNA is a marvelous coding chip, that has all the information required for the function of a cell and the organisms. Nature has thought of every detail that helps in the growth of a species. This error free factory of nature, is one of the unmatched manufacturing units, that help produce organisms of the highest quality.