DNA Replication

Semiconservative mechanism
Enzymes that catalyze the addition of deoxynucleotides are
called DNA polymerases
There are five different DNA polymerases in Escherichia coli,
called DNA polymerases I, II, III, IV, and V
DNA polymerases synthesize DNA in the 5 to 3 direction
To start a new chain, a primer, a nucleic acid molecule to
which DNA polymerase can attach the first nucleotide, is
required. In most cases this primer is a short stretch of RNA.

 Initiation
Elongation
termination

 Initiation of DNA Synthesis

Formation of replication fork ; The enzyme DNA helicase unwinds the
double helix, using energy from ATP, and exposes a short single
stranded region
Helicase moves along the DNA and separates the strands just in
advance of the replication fork. The single-stranded region is covered
by single strand binding protein. This stabilizes the single-stranded DNA
and prevents the double helix from re-forming
DNA gyrase travels along the DNA ahead of the replication fork and
inserts negative supercoils to cancel out the positive supercoiling
The origin of replication (oriC) consists of a specific DNA sequence of
about 250 bases that is recognized by initiation proteins, DnaA, which
binds to this region and opens up the double helix before helicase.
Next to assemble is the helicase (known as DnaB), which is helped onto
the DNA by the helicase loader protein (DnaC). Two helicases are
loaded, one onto each strand, facing in opposite directions.
Next, two primase and then two DNA polymerase enzymes are loaded
onto the DNA behind the helicases.
Primase adds RNA primer to begin DNA synthesis
Initiation of DNA replication then begins on the two single strands. As
replication proceeds, the replication fork appears to move along the
DNA

 Synthesis of the New DNA Strands

DNA synthesis replication always proceeds from 5 to 3
Leading strand and lagging strand
After synthesizing the RNA primer, primase is replaced by
Pol III

 Each polymerase is held on the DNA by a sliding clamp, which encircles and
slides along the single template strands of DNA.
Consequently, the replication fork contains two polymerase core enzymes
and two sliding clamps, one set for each strand.
However, there is only a single clamp-loader complex. This is needed to
assemble the sliding clamps onto the DNA. After assembly on the lagging
strand, the elongation component of Pol III, DnaE, then adds
deoxyribonucleotides until it reaches previously synthesized DNA At this
point, Pol III stops.
The next enzyme to take part, Pol I, it removes the RNA primer
When the primer has been removed and replaced with DNA, Pol I is
released. The last phosphodiester bond is made by an enzyme called DNA
ligase which seals the nicks in DNA

 Termination:
In E.coli, there are 10 replication termini (Ter) located in a
region opposite to the replication origin
The Ter sites interact with the replication terminator
protein called Tus, to stops DNA unwinding activity of DnaB
At the end, replication forms as catenated (two circular
chromosomes joined at ter region) ring.
Catenated rings are separated by topoisomerases IV
Topoisomerase IV transiently breaks both DNA strands of
one chromosome and allowing the other chromosome to
pass through the break

Plasmids

Plasmid Replication
Two methods
Theta formation
Rolling circle replication

Rolling circle replication

Ori

RepA

3 OH

RepA: Binds at the Ori, forms a nick on one

strand and remain attached to the 5 end of
the strand
Free 3 end with free OH group act as the
primer for Bacterial DNA polymerase III to
start replication of plasmid.
DNA PolII

Helicase

Helicase unwind the double strand,

simultaneously single strand binding protein
binds to the strand in which Rep A is attached
and stabilize the strand and progressively
peeled off from the plasmid.
Once the replication of the intact strand is
complete the RepA joins the two ends of the
nicked strand.

Ss binding
protein

DNA ligase seals the nick of the double

stranded DNA
Now the peeled single stranded DNA forms
loop like structure allowing RNA polymerase to
bind on it and prime the replication.
DNA polymerase starts the replication and
forms dsDNA

RNA poly
Ligase

Mobile DNA: Transposable Elements

Mobile DNA refers to discrete segments of DNA that move as units
from one location to another within other DNA molecules
The two major types of transposable elements in Bacteria are
insertion sequences (IS) and transposons.
Both elements have two important features in common:
They carry genes encoding transposase, the enzyme necessary for
transposition, and they have short inverted terminal repeats at their ends
that are also needed for transposition.

 Insertion sequences are the simplest type of transposable element.

They are short DNA segments, about 1000 nucleotides long, and
typically contain inverted repeats of 1050 bp.
Each different IS has a specific number of base pairs in its terminal
repeats. The only gene they possess is for the transposase.
Several hundred distinct IS elements have been characterized.

 Transposons are larger than IS elements, but have the same two
essential components: inverted repeats at both ends and a gene that
encodes transposase.
The transposase recognizes the inverted repeats and moves the
segment of DNA flanked by them from one site to another.
Consequently, any DNA that lies between the two inverted repeats is
moved and is, in effect, part of the transposon.