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Encyclopedia of Physical Science and Technology en010k-502 July 16, 2001 16:56

864 Nucleic Acid Synthesis

Different replicons of prokaryotes and eukaryotes uti- DnaB is the key helicase for replication of the genome
lize distinct mechanisms which vary in complexity, de- E. coli. However, other helicases such as Rep and PriA are
pending on the complexity of the organisms. A common also involved in replication and interact with other com-
feature of replication initiation control in E. coli genomes ponents of the replication complex called the replisome.
and plasmids is the presence of repeats of A•T rich se- Replication requires a large number of proteins, includ-
quences which facilitate unwinding of DNA and one or ing the holoenzyme of Pol III which includes, in addition
multiple repeats of a “dnaA box” to which the initiator to the catalytic polymerase cores, ten or more pairs of
DnaA protein in E. coli or its functional homolog (called other subunits. The polymerase complex appears to have
Rep in other cases) binds to allow helical unwinding and a dimeric asymmetric structure in order to replicate simul-
primer synthesis. The level of DnaA protein regulates the taneously two strands with opposite polarity. The continu-
initiation frequency and, in turn, is controlled at the level ous leading strand synthesis should be processive without
of transcription of the dnaA gene. Thus, there are com- interruption,becauseperiodicRNAprimersynthesisisnot
plex negative autofeedback loops to control dnaA gene necessary once the leading DNA strand synthesis is initi-
expression. DnaA regulates its own gene, and its steady- ated. On the other hand, the discontinuous lagging strand
state level in the cell is determined by the cellular growth synthesis should not be processive, because repeated syn-
state. The frequency of replicon firing is dependent on the thesis of RNA primers is required to initiate synthesis of
growth rate of the bacteria. As mentioned before, rapidly each Okazaki fragment. The Pol III holoenzyme appears
growing cells can have multiple copies of the genome, to assemble in a stepwise fashion, with its key β-subunit
while cells with a very low growth rate have only one copy. dimer acting as a sliding clamp based on its X-ray crystal-
Furthermore, as expected in cells with multiple genome lographic structure of a ring surrounding the DNA. This
copies, the genes near the origin will have a higher average clamp is loaded on DNA by the γ -complex, accompanied
copy number than the genes located near the terminus of by ATP hydrolysis. The dimeric structure of the repli-
replication and, therefore, will be more transcriptionally cation complex is maintained by the dimeric subunit of
active. the holoenzyme. The β-clamp slides on the duplex DNA
In the case of multicopy plasmids, the control of copy template and thus promotes processivity. Proliferating cell
number is mediated by the synthesis of anti-sense RNA nuclear antigen (PCNA) is the sliding clamp homolog in
of the replication initiator protein Rep, which is copied eukaryotic cells and is also used in SV40 replication.
from the nontranscribed DNA strand and is thus comple- Much of the information about the composition of the
mentary to the normal RNA. Anti-sense RNA prevents E. coli Pol III holoenzyme, and DNA chain elongation,
synthesis of the Rep protein, which is required for initia- was generated from studies of the replication of small,
tion of DNA synthesis and whose concentration is the pri- single-stranded circular DNAs of bacterial viruses φX174
mary mechanism of controlling initiation frequency. Rep and M13 and also of laboratory-constructed plasmid
proteins encoded by plasmids bind to additional copies of DNA containing the ori (ori C)of E. coli. Asymmetric
binding sites called “iterons,” often present upstream of dimeric replication complexes have also been identified
the ori sequences in the plasmids. for larger E. coli viruses such as T4 with a linear genome
and for the mammalian SV40 virus with a double-strand
circular genome. In circular genomes, DNA synthesis is
D. DNA Chain Elongation and Termination
terminated at around 180 from the origin. In the case
◦
in Prokaryotes
of linear genomes, termination occurs halfway between
Once initiated, DNA replication proceeds by coordinated two neighboring replicons. The mechanism of termina-
copying of both leading and lagging strands. Although tion is not completely understood. Although, in the E. coli
both bacteria and eukaryotes have multiple DNA poly- genome, specific termination (ter) sequences are present,
merases, only one, named polymerase III (Pol III), is which bind to terminator proteins, such proteins act as
primarily responsible for replicative DNA synthesis in anti-helicases to prevent strand separation. However, the
E. coli. In eukaryotes, DNA polymerases δ and ε have termination may not be precise and occurs when the repli-
both been implicated in this process along with a sugges- cating forks collide.
tion that each of these two enzymes may be specific for
leading or lagging strand synthesis.
E. General Features of Eukaryotic
Replication involves separation of two DNA strands
DNA Replication
which are catalyzed by DNA helicases which hydrolyze
ATP during this reaction. ATP hydrolysis provides the Unlike the genomes in bacteria and plasmids (as well as in
energy needed for the unwinding process. All cells have mitochondria and chloroplasts) which consist of a circular
multiple DNA helicases for a variety of DNA transactions. duplex DNA, with a single ori sequence, the genomes of