DNA Replication in Eukaryotes

n eukaryotes there are only two different types of DNA polymerases in

contrast with DNA polymerase I, II and III of prokaryotes.
Furthermore the DNA of eukaryotes is a long linear molecule with
several replication units. A diploid mammalian cell contains on an
average about 6 pg of DNA in the G phase. This much DNA is
equivalent to a length of 2 metres of a linear DNA molecule.

If a single replication unit were to move along this length of DNA, it

could complete replication within the 8 hour S phase only if its rate of
movement is about 4 mm/min. This is obviously a very fast rate.

The replicating fork actually moves at a slower speed (0.5 to 2.0

micron/min.) in eukaryotes adding about 2,600 bases per minute. In
E. coli it moves faster adding about 6,000 bases per minute. It is,
therefore, necessary that in eukaryotes replication be initiated at
several points of origin.

Auto-radiographic studies on labelling patterns of individual

metaphase chromosomes have shown that multiple adjacent units
initiate replication simultaneously. The most convincing
demonstration however, came from similar observations in giant
polytene chromosomes.

Here tritiated thymidine is incorporated simultaneously into a large

number of different bands. By the same technique the egg in
Drosophila is shown to have 6,000 replication forks and all the DNA
synthesis is completed within 3 minutes.

The unit of replication is the replicon. The size of the replicon is

estimated from the distance between adjacent initiation points
(centre-to-centre distance). By autoradiography it has been found that
units within the same cell are not uniform in size but fall within the
range of 15-60 micron.
Replicons in rapidly growing cells with short S phases are smaller than
those in cells growing more slowly with longer S phases. Blumenthal
(1973) has estimated that in Drosophila melanogaster replicons in
embryonic cells are as short as 3-4 micron, whereas in a cell line of the
same species they were about 13 micron long.

Experimental studies on cultured mammalian (Chinese hamster) cells

have shown that the rate of DNA synthesis is not constant throughout
the S phase, Kleveroz (1975) found that synthesis is slow at the
beginning of S phase, thereafter it increases. About 50% of replication
occurs during the last hour of the 5.5 hour long S phase.

The occurrence of multiple adjacent units has led to the concept that
replication units exist in clusters. All units in a cluster do not replicate
simultaneously, some being late replicating. In mammalian cells there
are about 100 replicating units in a cluster.

The essential features of DNA replication are similar in eukaryotes and

prokaryotes. After replication begins at a central point of origin in
each unit, it proceeds in both directions away from the initiation site.
Chain growth occurs by means of fork-like growing points. Electron
micrographs therefore show a number of ‘eyes’ or ‘bubbles’, each
formed between two replicating forks along the linear molecule.

It appears that there are no specific term in DNA for stopping

replication. The forks travel towards each other and the newly
synthesised chains meet and fuse with chains synthesised on adjacent
units (Fig. 14.11). In this way long DNA duplexes characteristic of
eukaryotic chromosomes are produced.
As in prokaryotes, the first step in DNA synthesis in eukaryotes is the
formation of a primer strand of RNA about 10 nucleotides in length—
catalysed by the enzyme RNA polymerase. After that DNA polymerase
takes over and adds deoxyribonucleotides to the 3′ end of the primer
RNA.

The Okazaki fragments thus formed are shorter in eukaryotes (about

100-150 nucleotides long) than in prokaryotes (1,000 to 2,000
nucleotides). The gaps between the fragments are filled up against the
parent DNA template and their ends are joined by DNA ligase enzyme.
The RNA primer is digested, starting from its 5′ end by the
exonuclease activity of DNA polymerase.

Significance of the RNA Primer in DNA Synthesis:

Why should DNA replication be initiated by the enzyme RNA
polymerase and formation of RNA strand take place? Detailed analysis
of DNA polymerase enzymes have revealed the fact that each
polymerase enzyme can add nucleotides only to an already existing
polynucleotide chain.

These enzymes are not able to initiate new DNA chains. The point of
origin in a DNA duplex is perhaps recognised by RNA polymerase, the
enzyme which catalyses the synthesis of RNA on a DNA template. In
other words, RNA polymerase is required for both RNA and DNA
synthesis.
Synthesis of RNA primer on the DNA template continues until a stop
signal is reached. The enzyme is then released and the RNA chain
serves as a primer for addition of DNA nucleotides by DNA
polymerase enzyme. However, the molecular mechanism which
initiates DNA replication is not fully known.

DNA Replication in Bacteria

The work of J. Cairns demonstrated DNA replication in bacteria in an
elegant way. He used high resolution autoradiography for observing
the distribution of radioactive label in the “chromosomes” of dividing
E. coli cells.

The bacterial cells were grown on a medium containing tritiated

thymidine. The labelled DNA was extracted from the cells and spread
on EM grids. A very thin layer of the photographic emulsion was laid
on the grids which were stored in the dark.

Whatever radioactive thymidine was present in the DNA, it reduced

the grains of silver halide located directly above it. When the grids
were developed and examined in the electron microscope, a track of
reduce silver grains (black dots) was observed.

Cairns study revealed that the chromosome of E. coli was a closed

circle of duplex DNA; that replication began at a single point of origin
and proceeded in a unidirectional manner round the whole circle,
until it reached the point of origin.

However, later studies showed that after initiation at the point of

origin, replication was bidirectional and proceeds stepwise as follows.
First of all an endonuclease enzyme introduces a break or nick in one
strand of the duplex at the point of origin thus allowing the two
strands to untwist.

The separation of strands is aided by an unwinding protein which

binds to a small portion of the nicked strand. The successive binding
of additional molecules of this protein leads to opening up of the
duplex just before the replication fork. An E. coli cell contains about
10,000 copies of the unwinding protein. The break is perhaps joined
by a ligase enzyme.

Replication begins and proceeds in both directions; it is visible in the

electron microscope in the form of a replicating “bubble” or “eye” at
the point of origin. Both strands of parental DNA are replicated
simultaneously (Fig. 14.8), by movement of two replicating forks away
from each other.

A little later the circular replicating DNA appears like the Greek letter
theta (θ). The process of unwinding of the circular double helical DNA
creates torque which results in supercoiling. An untwisting protein,
also called swivelase appears to relieve tension by nicking one strand
and allowing some unwinding to take place.

Both strands of the double helix are replicated in short fragments. The
DNA polymerase enzymes are not able to polymerise native double
stranded DNA. The enzyme DNA dependent RNA polymerase which
takes part in transcription of DNA to RNA is able to recognise specific
initiation points or chromosomes.

Due to the activity of this enzyme DNA first forms a number of short
strands of complementary RNA called primer RNA, each about 100
nucleotides long in the 5′ to 3′ direction. The 3′ end of the primer RNA
serves as a starting place for DNA polymerase to add nucleotide units
of DNA. The DNA chain is replicated in the 5′ to 3′ direction and is
complementary to a single strand of the parent- DNA duplex.
After the DNA chain has attained a length of about 1000 to 2000
nucleotides, an exonuclease enzyme cuts off primer RNA from the 5′
end of DNA. In this way a number of DNA fragments, known as
Okazaki fragments are generated during replication instead of one
continuous linear strand (Fig. 14.9).
The gaps between the DNA fragments are filled by DNA polymerase I
which adds nucleotides complementary to the parental strand in the 5′
to 3′ direction. After the gaps are filled, the remaining nicks are
removed by the enzyme ligase, which joins adjacent nucleotides by 3′
5′ phosphodiester linkages.

In addition to its polymerizing function, DNA polymerase enzyme also

performs exonuclease activity by removing bases in 3′ to 5′ direction.
If during the synthesis of a DNA chain an incorrect base is added, the
DNA polymerase turns back, removes the wrong base in 3′ to 5′
direction, and again starts adding bases in 5′ to 3′ direction.
The idea that DNA replication takes place by first forming Okazaki
fragments received strong support from studies on E. coli mutants
deficient in DNA ligase enzyme. In such cells the fragments were seen
to accumulate in very large numbers.

Cell division in bacteria is accomplished by replication of the single

circular DNA chromosome into two identical daughter duplexes. The
replication fork in E. coli moves at the rate of about 20-30 µm per
minute. Replication of the entire genome is completed in about 40
minutes. This is followed by partitioning of cell cytoplasm into two
daughter cells.

The nuclear membrane is absent; there is no mitosis or spindle

formation as in eukaryotes. It seems likely that there is some
association between the replicating DNA duplex and the plasma
membrane. Electron micrographs of Bacillus subtilis have shown that
the plasma membrane invaginates to form a mesosome, and the
daughter chromosomes become attached to either side of this
invagination.

There is some evidence from in vivo DNA transformation experiments

that both the origin and terminal end of replicating DNA are
preferentially bound to the membrane. However, as yet, the
association between replicating DNA and membrane remains unclear.
Further growth of the mesosome partitions the parent cell into two
daughter cells.

Modification of Replicated DNA:

After synthesis the bacterial DNA becomes modified to a conformation
characteristic for that bacterium. There is a class of modification
enzymes known as DNA methylases which add a methyl group to a
newly synthesised adenine (methylate A), or sometimes to a cytosine.
There is a specific DNA methylase for each species.