DNA Replication

DNA Replication

DNA replication is the process of producing two identical replicas of DNA from one original DNA molecule. It is the master molecule that controls activities of the cell.

It performs two major functions:

1. Autocatalytic i.e., DNA synthesizes another DNA molecule similar to it. This is also termed as DNA replication. One of the two strands of DNA functions as its own template and hence replication is semiconservative.

2. Heterocatalytic i.e, DNA also directs the synthesis of protein molecules dissimilar in structure compared to its own structure. This process is brought about through a special mechanism involving different types of RNA. This process is termed as protein synthesis.

DNA replication is semiconservative

The Watson Crick model of DNA suggests that the two strands of DNA should separate. Each separated stand also termed as parent strand now serves as a template for the formation of a new but complementary strand.

Thus, the new or daughter DNA molecules formed would be made of one old or parental strand and another newly formed complementary strand. This method of formation of new daughter DNA molecules is termed as semi conservative method of replication.

Enzymes in replication

Following are the various enzymes which play important role in DNA replication.

Topoisomerases:

These are helpful for initiation or unwinding of DNA. These relax and disentangle DNA coils and super coils.

Helicases:

These are helpful in unwinding the original double helix after the removal of supercoils by topoisomerases. These enzymes are helpful in breaking the hydrogen bonds between the two DNA strands.

Single stranded binding proteins (SSBs):

Once the DNA helix opens, single strands  bind to these proteins. Thus, preventing DNA from reforming the duplex.

DNA polymerases:

These are the most important group of enzymes. In prokaryotes DNA polymerase  ε (Escherichia coli), there are three different polymerases viz., I, II, III. Of these DNA polymerases I and II are concerned with DNA repair and DNA polymerase III takes part in actual DNA replication. In eukaryotes there are 5 types of DNA polymerases viz., DNA polymerase α ( also termed as cytoplasmic polymerase), DNA polymerase β (also termed as nuclear polymerase), DNA polymerase γ (mitochondrial polymerase), DNA polymerase δ and DNA polymerase ε.

DNA polymerase III in E. coli and DNA polymerase ε in eukaryotes are directly involved in the replication of nuclear DNA. DNA polymerases can synthesize DNA only in 5’→ 3′ direction meaning thereby that the new nucleotide always adds to 3′ hydroxyl end of the growing DNA chain.

Many prokaryotic and eukaryotic DNA polymerases also possess 3′ → 5′ exonuclease activity. This causes the removal of nucleotides from 3′ end of the polynucleotide chain to correct the occasional errors in base pairing (also termed as proofreading).

In E. coli DNA polymerase 1 acts as exonuclease and helps in removal of RNA primers of the lagging strand. DNA polymerases, however, cannot start the formation of new DNA strand from free nucleotides and therefore need a primer.

Primases:

Primer is a short piece of RNA required for free 3′ hydroxyl group. This piece is synthesized by primase in E. coli and DNA polymerase alpha in eukaryotes.

DNA ligases:

This enzyme joins the bits of DNA by forming phosphodiester bond between 3′ hydroxyl and 5′ phosphate group.

Mechanism of DNA Replication

DNA replication involves following four major stages

1. Initiation of DNA replication
2. Unwinding of helix
3. Formation of primer strand and
4. Elongation of new strand

Initiation of replication

Replication of DNA in E. coli always begins at a definite site termed as the replication origin. There is only one such origin of replication and bacteria and viruses. On the other hand, eukaryotes have several thousand origins of replication. These linearly arranged units are termed as replicons. In E. coli replication is due to multienzyme complex. It is termed as replication apparatus or repliosome.

Unwinding of helix

Enzyme helicase, which is also termed as rep protein is useful in the unwinding of the helix. This unwinding may result in formation of supercoils. The action of another enzyme, termed as DNA gyrase (topoisomerases) is helpful in the removal of the supercoils.

Unwinding of DNA molecule separates two strands to form Y-shaped replication fork. These single strands  stabilizes with the help of single strand binding protein (SSB) or the helix-destabilizing protein. The SSB protein holds open the two separated strands to form replication fork.

Formation of primer strand

A new strand is now synthesized opposite the parental strand that acts as a template strand. DNA polymerase III is the true replicase in E.coli but is incapable of initiating DNA synthesis ,i.e., it is unable to deposit the first nucleotide in a daughter strand. Hence another enzyme, termed as primase initiates the synthesis by producing a short primer strand of RNA. The process requires some other enzymes. These are collectively termed as primasomes. The primer strand then elongates. Once the initiation of DNA synthesis completes, removal of this primer RNA strand occurs .

Elongation of new strand

After the formation of primer strand, DNA replication occurs in 5’→ 3′ direction, i.e., deoxyribonucleotides adds only to the 3’OH end of the new stand. During this process, for example, deoxyadenosine triphosphate (dATP) adds to an elongating DNA strand opposite thymine base in template strand. This is brought about by enzyme DNA replicase (DNA polymerase). The phosphate of dATP forms a 3’→5′, phosphodiester bond with free 3’OH of the elongating strand releasing a molecule of pyrophosphate simultaneously. Thus the nucleotide at 3′ carbon of sugar is always the most recently added nucleotide to the chain.

As the DNA replication proceeds, of the two parental strands, synthesis of daughter or new stand occurs continuously along (5’p-3’OH) the upper stand  (5’p-3’OH). It is now termed as leading daughter strand. Synthesis of another daughter strand along the lower parental strand however, takes place in the form of short pieces. This is termed as lagging daughter strand. These short pieces of DNA are termed as okazaki fragments, segments or pieces and are about 1,000 to 2,000 nucleotides long. Discontinuous pieces of the lagging strand later joins together by an enzyme DNA ligase to form continuous daughter strand.

Thus, two DNA molecules form from one molecule. Each of these daughter DNA molecules consists of two strands of which one is old (parental that acts as a template) and other one is new.