DNA Replication

 

Living organisms perpetuate their kind or increase their number through reproduction. The reproduction may be simple duplication like cell fission as in bacteria or complex modes of sexual reproduction as in higher plants and animals. In all cases, however, reproduction entails the faithful transmission of the genetic information of the parents to the progeny. Since the genetic information is stored in DNA, the duplication or the replication of DNA is the central to all of biology.

 

Each time a cell divides into two daughter cells, all the DNA molecule must be duplicated. The

process by which a DNA molecule produces its identical copies is described as DNA replication.

It is a type of self duplication or self reproduction of DNA, where two daughter molecules are

formed from a single DNA molecule.

Theoretically, three possible modes of DNA replication are possible. They are:

1. Dispersive replication:

The two strands of parent DNA break randomly and produce several Pieces. These pieces replicate and reunite to form new daughter DNA molecules. These new DNA molecules create a mixture of old and new nucleotides scattered along, the chains. The daughter molecules can be described as hybrids. This mechanism is not accepted nor proved  experimentally.

 

2. Conservative replication:

After replication, one daughter DNA contains the original two strands of the parent molecule. While, the other daughter molecule contains two newly synthesized strands. This method is also not accepted.

3. Semi-conservative replication:

This method of DNA replication was proposed by Watson and Crick. Because of specificity of         base pairing, the sequence of bases along one chain automatically determines the base sequence along the other.

Half of the DNA is conserved i.e., only one strand is synthesized and the other half of the original DNA is retained.

The evidence for semi conservative replication of DNA molecules were provided by Meselson and Stahl using ¹⁵N - a heavy isotope of ¹⁴N. 

The purine and pyrimidine bases in DNA contain nitrogen, thus, the DNA of cells grown on medium containing ¹⁵N will have greater density than the DNA of cells grown on medium containing ¹⁴N.

They grew E.coli on the culture medium containing ¹⁵N – for many generations, so that the nitrogen present in DNA bases of these cells was ¹⁵N.

Then they transformed the E.Coli cells to a medium containing normal ¹⁴N. after allowing the cells to grown in the presence of ¹⁴N for varying periods of time, the DNA was extracted and its density was determined by ultracentrifugation on a cesium chloride gradient.

All the DNA isolated from cells after one generation of growth in medium containing ¹⁴N had a density halfway between the densities of ‘heavy’ and ‘light’ DNA.The DNA had only one DNA peak corresponding to the heavy-light molecule. This intermediate density usually referred to as ‘hybrid’ density has two heavy- light hybrid molecule i.e., one strand is labelled with ¹⁴N, the other with ¹⁵N.

         At the  second generation two peaks of DNA appear – one in which the two strands contain light ¹⁴N  and other still corresponding to hybrid molecules containing heavy-light molecules.

These findings can be readily explained on the basis of semi-conservative replication of DNA. The DNA from E.coli cells grown on ¹⁵N medium had ¹⁵N in both the strands, therefore it was heavier than the normal DNA.

When these E.coli cells were grown in ¹⁴N medium, each of the DNA molecules would have one heavy and one light strand. Therefore, these DNA had two have intermediate density.

 

J Cairns demonstrated the semi – conservative mode of replication of bacterial chromosome using         autoradiography technique.

 

Enzymes required for Replication:

Topoisomerase:

These enzymes can change the topological form or shape of DNA. Responsible  for initiation of the unwinding of the DNA. The tension holding the helix in coiled and supercoiled structure can be broken by nicking a single strand of DNA. DNA topoisomerases introduce a nick on only one of the DNA strands. This allows the molecule to roatate around the phosphodiester bond on the opposite strand as if it were a swivel. The topoisomerases  introduce negative supercoils  and relieve strains in the double helix at either end of the bubble. 

 The Helicase:

 Unwinds and unzips the DNA helix by breaking the hydrogen bonds between the  base pairs, thus allowing the two strands to separate. The two strands very much  want to bind together  because of their hydrogen bonding affinity for each other, so the helicase activity requires  energy (in the form of ATP) to break the strands apart.

 DNA Polymerase:

 Chief enzyme of DNA replication. Discovered by Kornberg in 1956. All the DNA polymerase require the following:

i.                     A template DNA strand

ii.                    A short primer (either RNA or DNA)

iii.                    A free 3’ – OH in the primer.

They add one nucleotide at a time to the free 3’OH of the primer, and extend the primer chain in 5’ → 3’ direction.

DNA polymerase is actually an aggregate of several different protein subunits, so it is  often called a holoenzyme. The holoenzyme also has proofreading activities, so that it can make sure that it inserted the right base and nuclease (excision of nucleotides) activities so that it can cut away any mistakes it might have made.

 DNA polymerase I :

            This enzyme was first purified by Kornberg in 1956. Hence, it is also called Kornberg enzyme.  This enzyme has three activities, which appear to be located in different parts of the molecule.

A polymerase activity, which catalyses chain growth in the 5’ → 3’ direction

A 3’ → 5’ exonuclease activity, which removes mismatched base (DNA proof reading

A 5’ → 3’ exonuclear activity, which degrades double stranded DNA (excision repair).

A exonuclease digests nucleic acids from one end (it does not cut DNA internally).

DNA polymerase II:

            This enzyme repairs the damaged DNA. It has 5’ → 3’ polymerase and 3’→ 5’ exonuclease activities.

DNA polymerase III:

            This enzyme is responsible for DNA replication in vivo. It has 5’ → 3’ polymerase and 3’ → 5’ exonuclease  activities. DNA polymerase III is a complex enzyme containing seven different polypeptides, and 5'to 3'of these polypeptides must be present for proper replicative function. The 5' to 3' polymerase  activity and the 5'to 3'exonuclease activity are both     present on the  α polypeptide of DNA polymerase III.

The 3'to 5'proofreading activity of polymerase III is present on the ϵ polypeptide.

Primase:

Is a part of an aggregate of proteins called the primeosome. The primase (RNA polymerase) added to other  proteins (forming a Primesome) makes short pieces of RNA (RNA primers) that are recongnised by DNA polymerase III to initiate replication.

As DNA polymerase III exhibits outstanding proofreading capabilities, which prevent it to initiate a polynucleotide strand synthesis. Therefore it requires a primer, a short piece of RNA (RNA primer) that it can recognize and elongate. This RNA primer is eventually removed by RNase and the gap is filled in by DNA polymerase I.

Ligase:

            Catalyze the formation of a phosphodiester bond given an unattached but adjacent 3’OH and 5’ phosphate.  This can fill in the unattached gap left when the RNA primer is removed and filled in.

 DNA polymerase can organize the bond on the 5’end of the primer, but ligase is needed to make the  bond on the 3’ end.

The SSB  proteins (Single Strands Binding) stabilize the single strands thus preventing them to zip back together and to form hairpin loops. Single – stranded binding proteins are important to maintain the stability of the replication fork. Single-stranded DNA is very labile, or unstable, so these proteins bind to it while it remains single stranded and keep it from being degraded.

Mechanism of DNA Replication

DNA synthesis is semiconservative (i.e., one of the parental DNA strands is conserved). DNA replication begins at certain unique and fixed points called ‘Origin’ (ori).Two enzymes DNA gyrase and DNA helicase, bind to the origin points and induce the unwinding and separation of complementary strands of DNA double helix. This separation is known as DNA melting.

Unwinding of DNA produces Y-shaped replication forks. 

           DNA polymerases proceed only in the 5’ to 3’ direction, DNA polymerase require for their action the presence of a :

i. Template DNA

ii. A primer (RNA or DNA, but only RNA is used in vivo).DNA polymerases can only add nucleotides  to the 3’OH group of a pre- existent primer

 iii. Four deoxynucleoside triphosphates (dATP, dTTP, dGTP and dCTP), which are the building blocks of DNA

 As the two strands separate, the bases are exposed and RNA polymerase or primase initiates transcription of the strand (3' to 5') and generates a 10 -60 nucleotide long RNA primer in 5' to 3' direction.

The free 3'- OH of this RNA primer provides the initiation point for the synthesis of new DNA strand. DNA polymerase III adds deoxyribonucleotides to the 3'- OH group of the last ribonucleotide of the RNA primer.

DNA polymerase I catalyzes the removal of the RNA primers by the concerted action of its 5'to 3' exonuclease activity and its 5'to 3' polymerase activity.

DNA poly III progressively adds deoxyribonucleotides to the free 3'- OH of this growing polynucleotide chain according to the base pairing rules (A=T; G=C). Consequently, the replication of 3' to 5' strand of a DNA molecule proceeds continuously.   

Because both strands of a DNA molecule are antiparallel, i.e., run in opposite directions, this creates a problem. When the two strands unwind at the replication fork the leading strands faces the DNA polymerase in correct 5’ to 3’ direction, so that the synthesis of a long continuous complementary strand takes place.

 On the lagging strand this is not possible; therefore, replication proceeds in a discontinuous way,  synthesizing short segments of DNA (always in the 5’→3’ direction) called the okazaki fragments.

These segments are then joined together by the action of a DNA ligase. This short discontinuous DNA segments were discovered by T. Okasaki, who exposed bacteria to ³H -thymidine for a few seconds and found that fragments of DNA 1000 - 2000 nucleotides long. These fragments were called Okasaki  fragments after their discoverer. These are only 200 nucleotides long in eukaryotes.

 DNA polymerases can only elongate a primer molecule; they cannot start a new chain by themselves.  This poses a problem when initiating each Okasaki fragment, which is solved by synthesizing a short segment of RNA that acts as the primer.

 Each Okasaki fragment starts with an RNA segment. RNA is used (instead of DNA) because it  immediately provides a ‘tag’ indicating that this part of the molecule has not yet been subjected to the proofreading  mechanism and must be replaced.

This RNA segment is subsequently removed by repair enzymes, the gap is filled by DNA polymerase, and then joined to the neighbouring Okasaki fragment by a DNA ligase to form a long polynucleotide chain.

 The rate of movement of a replication fork in E.coli is about 10⁵  base pairs per minute. In eukaryotes the polymerases are much less active and the rate ranges from 500 to 5000 base pairs per minute.

 In E.coli, the termination is signalled by specific sequences called ter elements. They serve as binding site for protein Tus. The Tus protein binds to ter element and stops helicase enzymes from unwinding DNA. This stops the movement of the replication fork.