There is a major difference between DNA polymerase and RNA polymerase: the RNA polymerase can synthesize a new strand whereas the DNA polymerase can only extend an existing strand. Therefore, to synthesize a DNA molecule, a short RNA molecule (~ 5 – 12 nucleotides) must be synthesize first by a special enzyme. The initiating RNA molecule is known as a primer, and the enzyme is called primase.
In addition to DNA polymerase and primase, DNA replication requires helicase and single strand binding protein (SSB protein). The role of helicase is to unwind the duplex DNA. SSB proteins can bind to both separated strands, preventing them from annealing (reconstitution of double-stranded DNA from single strands).
The replication mechanisms in both bacteria and eukaryotes are similar. However, eukaryotic DNA polymerases do not contain a subunit similar to the E. coli b subunit. They use a separate protein called proliferating cell nuclear antigen (PCNA) to clamp the DNA.
Figure 7-B-2. Structure of PCNA which is formed by three identical subunits. PDB ID = 1AXC.
DNA polymerases can extend nucleic acid strands only in the 5′ to 3′ direction. However, in the direction of a growing fork, only one strand is from 5′ to 3′. This strand (the leading strand) can be synthesized continuously. The other strand (the lagging strand), whose 5′ to 3′ direction is opposite to the movement of a growing fork, should be synthesized discontinuously.
Figure 7-B-3. Steps in the synthesis of the lagging strand.
(a) Comparison between the leading strand and the lagging strand.
(b) The primase first synthesizes a new primer which is about 10 nucleotides in length. The distance between two primers is about 1000-2000 nucleotides in bacteria, and about 100-200 nucleotides in eukaryotic cells.
(c) DNA polymerase elongates the new primer in the 5′ to 3′ direction until it reaches the 5′ end of a neighboring primer. The newly synthesized DNA is called an Okazaki fragment.
(d) In E. coli, DNA polymerase I has the 5′ to 3′ exonuclease activity, which is used to remove a primer.
(e) DNA ligase joins adjacent Okazaki fragments.
The whole lagging strand is synthesized by repeating steps (b) to (e).