As discussed above, initiation of DNA replication involves loading and activation of DnaB helicase. DnaB directs other necessary enzymes (particularly DnaG primase and DNA pol III
HE) to the template and help in DNA elongation. These elongation proteins and the mechanism involved in elongation will be briefly described in this section.
DnaB is the major helicase, although other helicases have been discovered. It unwinds the template in 5'-3' direction during oriC
replication in vitro,
at a rate of 700bp/sec. The direction of unwinding requires DnaB to be placed on lagging strand, ahead of the polymerase on the leading strand. It also activates primer synthesis by DnaG primase.
The primase (DnaG) synthesizes a unique oligonucleotide primer on SSB coated single stranded phage G4 DNA. The sequence recognized by primase is over lOOnt (nt = nucleotides) long containing a secondary structure. Since primer synthesis requires activation by DnaB, the latter is often described as a 'mobile promoter'
of primer synthesis.
DNA polymerase III holoenzyme. DNA pol III
HE is the large multiprotein complex responsible for elongation of DNA replication in E. coli.
It has at least 10 subunits, each with specific function (Table 26.3). The holoenzyme consists of a 'core polymerase'
and its 'accessory factors',
which are analogous to the components of phage T4 and eukaryotic enzymes. In the holoenzyme, β subunit is twice as abundant as the other subunits, and in the cell its abundance is 300 times that of the polymerase assembly. It
has a 'sliding clamp'
mode of DNA binding. It has similarity with T4
'gene 45' protein and human PCNA (see later).
Other proteins for DNA elongation.
Other proteins involved in DNA elongation are (i) DNA gyrase
(a topoisomerase), which removes the topological strain associated with DNA unwinding; (ii) SSB (single stranded DNA binding) protein,
that binds tightly to single stranded DNA thus stabilizing the unwound DNA; (iii) RNase H, which removes RNA primers; (iv) DNA pol
I, which is used for filling the gap created due to removal of RNA primers and (v) DNA ligase,
which converts Okazaki fragments
into a continuous strand.