the scaffold, usually coinciding with origins of replication. Many other DNA binding
proteins are also present, such as high mobility group (HMG) proteins, which assist in
promoting certain DNA conformations during processes such as replication or active
gene expression.5.5 Functions of nucleic acids
5.5.1 DNA replication
The double-stranded nature of DNA provides a means of replication during cell
division since the separation of two DNA strands allows complementary strands to
be synthesised upon them. Many enzymes and accessory proteins are required for
in vivoreplication, which in prokaryotes begins at a region of the DNA termed the
origin of replication.
DNA has to be unwound before any of the proteins and enzymes needed for
replication can act, and this involves separating the double-helical DNA into single
strands. This process is carried out by the enzyme DNA helicase. Furthermore, in order
to prevent the single strands from re-annealing small proteins termedsingle-stranded
DNA binding proteins(SSBs) attach to the single DNA strands (Fig. 5.12).
On each exposed single strand a short, complementary RNA chain termed aprimer
is first produced, using the DNA as a template. The primer is synthesised by an RNA
polymerase enzyme known as aprimasewhich uses ribonucleoside triphosphates and
itself requires no primer to function. ThenDNA polymeraseIII (DNApolIII) also uses
the original DNA as a template for synthesis of a DNA strand, using the RNA primer as
a starting point. The primer is vital since it leaves an exposed 3^0 hydroxyl group. This
is necessary since DNA polymerase III can only add new nucleotides to the 3^0 end and
not the 5^0 end of a nucleic acid. Synthesis of the DNA strand therefore occurs only in a
50 to 3^0 direction from the RNA primer. This DNA strand is usually termed theleading
strandand provides the means for continuous DNA synthesis.
Since the two strands of double-helical DNA are antiparallel, only one can be
synthesised in a continuous fashion. Synthesis of the other strand must take place
in a more complex way. The precise mechanism was worked out by Reiji Okazaki in
the 1960s. Here the strand, usually termed thelagging strand, is produced in rela-
tively short stretches of 1–2 kb termedOkazaki fragments. This is still in a 5^0 to 3^0
direction, using many RNA primers for each individual stretch. Thus, discontinuous
synthesis of DNA takes place and allows DNA polymerase III to work in the 5^0 to 3^0
direction. The RNA primers are then removed by DNA polI, which has a 5^0 to 3^0
exonuclease, and the gaps are filled by the same enzyme acting as a polymerase. The
separate fragments are joined together by DNA ligase to give a newly formed strand of
DNA on the lagging strand (Fig. 5.13).
The replication of eukaryotic DNA is less well characterised, involves multiple
origins of replication and is certainly more complex than that of prokaryotes; how-
ever, in both cases the process involves 5^0 to 3^0 synthesis of new DNA strands. The net
result of the replication is that the original DNA is replaced by two molecules, each152 Molecular biology, bioinformatics and basic techniques