DNA Replication
DNA Replication (Prokaryotes and Eukaryotes)
The E. coli chromosome begins its DNA replication at a single origin
of replication called, oriC and proceeds bidirectionally to a
termination site located approximately halfway around the circular
chromosome. In order for replication to proceed, the DNA strands of the
double helix must be both unwound and separated. DNA replication is
initiated when a protein encoded by the gene dnaA binds repetitive
9-mer sequences at the origin.
Subsequently, helicases specified by dnaB and inhibitory proteins
encoded by dnaC bind repetitive 13-mer sequences. As helicase
progresses 5` to 3`, dissociation of protein DnaC allows the helicase
to unwind the DNA. The unwinding produces positive superhelical turns
in the rest of the DNA, making it energetically favorable to continue
unwinding the strands. To unwind the DNA, positive superhelical turns
have to be removed by cutting the DNA and allowing it to relax or by
introducing negative superhelical turns to compensate for the positive
ones. The introduction of negative superhelical turns requires energy
and an enzyme called DNA gyrase (a topo-isomerase). DNA gyrase is an
enzyme that can both remove positive supercoils or introduce negative
supercoils into the DNA and thereby make strand separation
energetically more favorable.
Presumably the DNA gyrase binds ahead of the unwound DNA during
replication. Single-stranded binding proteins (SSBPs) act to
temporarily stabilize the unwound state. DNA replication begins with
the synthesis of a 30 nucleotide long RNA primer by an RNA polymerase
called primase (specified by dnaG). The helicase and primase
subsequently form a complex enzyme system known as the primosome, which
synthesizes primers after DNA synthesis begins. Two catalytic subunits
of DNA polymeraseIII (PolC) associate with the templates and the 3`
ends of the primers and begin to polymerize deoxyribonucleotides into
DNA. DNA gyrase continues to remove positive supercoils and/or
introduces negative supercoils ahead of the primosome that is opening
the two strands of DNA. At various intervals, the template signals the
primase portion of the primosome to polymerize primer RNAs about 30
nucleotides long on only one template at the replication fork. DNA
polymerase III polymerizes DNA 5` to 3` from each of the primers at the
replication fork. One strands of DNA is polymerized toward the
replication fork and continues to be elongated as the DNA unwinds
further.
The second strand of DNA is polymerized away from the replication
fork. As the DNA unwinds further, a new primer is synthesized away from
the replication fork and the DNA polymerase synthesizes DNA from the
last primer toward the previous RNA primer. As the DNA polymerase reads
the template strand, it selects complementary nucleotides for the
nascent strand based on hydrogen bonding capability. The DNA
synthesized toward the replication fork is synthesized in a continuous
manner and is called the leading strand. The opposite DNA strand is
synthesized in a discontinuous manner away from the replication fork
and is referred to as the lagging strand. The leading and lagging
strands are synthesized halfway around the bacterial chromosome until
they encounter the lagging and leading strands synthesized at the other
replication fork. The RNA-DNA fragments that initially constitute the
lagging strand are known as Okazaki fragments, named after the
scientist who discovered them. The RNA primers are removed by a DNA
repair enzyme called DNA polymerase I speci?ed by polA. It uses
neighboring DNA as a primer and polymerizes DNA from it, displacing the
RNA primer. A DNA ligase removes nicks in the DNA by connecting the
fragments together. Topoisomerase IV is required to separate the two
daughter chromosomes.
DNA replication in eukaryotic chromosomes generally is initiated
from many origin of replication sites. Replication forks proceed in
both directions from these sites. The sites that comprise yeast origins
of replication are called autonomously replicating sequences (ARSs) and
consist of two regions that bind a distinct set of proteins that
destabilize the double helix. In one region, conserved, repeating
11-mers bind a multiprotein complex called the origin recognition
complex (ORC). When proteins also bind the other region, the DNA bends
by interaction of the proteins in the two regions.
This distortion of the DNA promotes the separation of paired DNA
strands at the origin and initiation of RNA primer synthesis. Enzymes
similar to those involved in bacterial DNA replication are found in
eukaryotes. Numerous topoisomerases, helicases, and RNA polymerases
have been found in eukaryotes. DNA topoisomerase II is involved in
relieving positive supercoils in the DNA, whereas a helicase activity
separates the two strands. At least five different DNA polymerases have
been found in eukaryotic cells. The primase (DNA pola) synthesizes
lagging strand DNA. DNA pola catalyzes leading strand synthesis. DNA
pole and DNA polb are responsible for replacing the nucleotide gaps
created when RNA primers are removed by endonucleases. A DNA ligase
repairs single stranded nicks (unconnected adjacent nucleotides) left
in the DNA. DNA polg performs DNA replication in the mitochondria. To
complete replication of a linear chromosome, RNA primers at each end of
the chromosome have to be removed and replaced by DNA. Although RNA
primers can be removed by exonucleases, none of the usual DNA
polymerases are able to replace the RNA without a DNA primer. An
unusual type of DNA polymerase known as telomerase consists of protein
and an RNA template that the protein portion copies repetitively into
DNA in order to extend one strand of the telomere. Thus, telomerase is
responsible for maintaining the length of the chromosomes.
