In this article we will discuss about the DNA replication in eukaryotes.
In 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.