MICROBIAL MOLECULAR BIOLOGY & GENETICS

A) Genes: Structure, Replication & Mutation

STEPS IN PROKARYOTIC DNA SYNTHESIS


 When the two strands of the DNA double helix are separated, each can
serve as a template for the replication of a new complementary strand.
This produces two daughter molecules, each of which contains two DNA
strands with an anti parallel orientation . This process is
called semi conservative replication because, although the parental
duplex is separated into two halves (and, therefore, is not “conserved” as
an entity), each of the individual parental strands remains intact in one of
the two new duplexes . The enzymes involved in the DNA
replication process are template-directed polymerases that can synthesize
the complementary sequence of each strand with extraordinary
fidelity. The reactions described in this section were first known from
studies of the bacterium Escherichia coli (E. coli), and the description
given below refers to the process in prokaryotes. DNA synthesis in
higher organisms is less well understood, but involves the same types of
mechanisms. In either case, initiation of DNA replication commits the cell
to continue the process until the entire genome has been replicated.
A. Separation of the two complementary DNA strands
      In order for the two strands of the parental double helical DNA to be
replicated, they must first separate (or “melt”) over a small region,
because the polymerases use only ssDNA as a template. In
prokaryotic organisms, DNA replication begins at a single, unique
nucleotide sequence—a site called the origin of replication . [Note: This is referred to as a consensus sequence,
because the order of nucleotides is essentially the same at each
site.] This site includes a short sequence composed almost exclusively
of AT base pairs that facilitate melting. In eukaryotes, replication
begins at multiple sites along the DNA helix .
Having multiple origins of replication provides a mechanism for
rapidly replicating the great length of the eukaryotic DNA molecules.



B. Formation of the replication fork:
  As the two strands unwind and separate, they form a “V” where
active synthesis occurs. This region is called the replication fork. It
moves along the DNA molecule as synthesis occurs. Replication of
dsDNA is bidirectional—that is, the replication forks move in opposite
directions from the origin, generating a replication bubble:
1. Proteins required for DNA strand separation:  Initiation of DNA
replication requires the recognition of the origin of replication by a
group of proteins that form the prepriming complex. These proteins
are responsible for maintaining the separation of the
parental strands, and for unwinding the double helix ahead of the
advancing replication fork. These proteins include the following:
a. DnaA protein: DnaA protein binds to specific nucleotide
sequences at the origin of replication, causing short, tandemly
arranged (one after the other) AT-rich regions in the origin to
melt. Melting is ATP-dependent, and results in strand separation

with the formation of localized regions of ssDNA

b. DNA helicases: These enzymes bind to ssDNA near the replication
fork, and then move into the neighboring doublestranded
region, forcing the strands apart—in effect,
unwinding the double helix. Helicases require energy provided
by ATP . [Note: DnaB is the principal helicase of replication in E.coli. Its binding to DNA requires DnaC.
c)  Single-stranded DNA-binding (SSB) proteins:  These proteins
bind to the ssDNA generated by helicases .
They bind cooperatively—that is, the binding of one molecule
of SSB protein makes it easier for additional molecules of SSB
protein to bind tightly to the DNA strand. The SSB proteins are
not enzymes, but rather serve to shift the equilibrium between
dsDNA and ssDNA in the direction of the single-stranded
forms. These proteins not only keep the two strands of DNA
separated in the area of the replication origin, thus providing
the single-stranded template required by polymerases, but
also protect the DNA from nucleases that degrade ssDNA.

2)  Solving the problem of supercoils:  As the two strands of the double
helix are separated, a problem is encountered, namely, the
appearance of positive supercoils (also called supertwists) in the
region of DNA ahead of the replication fork. The accumulating positive supercoils interfere with further unwinding
of the double helix. [Note: Supercoiling can be demonstrated by
tightly grasping one end of a helical telephone cord while twisting
the other end. If the cord is twisted in the direction of tightening
the coils, the cord will wrap around itself in space to form positive
supercoils. If the cord is twisted in the direction of loosening the
coils, the cord will wrap around itself in the opposite direction to
form negative supercoils.] To solve this problem, there is a group
of enzymes called DNA topoisomerases, which are responsible
for removing supercoils in the helix.
a. Type I DNA topoisomerases:  These enzymes reversibly cut
one strand of the double helix. They have both nuclease
(strand-cutting) and ligase (strand-resealing) activities. They
do not require ATP, but rather appear to store the energy from
the phosphodiester bond they cleave, reusing the energy to
reseal the strand . Each time a transient “nick” is
created in one DNA strand, the intact DNA strand is passed
through the break before it is resealed, thus relieving (“relaxing”)
accumulated supercoils. Type I topoisomerases relax
negative supercoils (that is, those that contain fewer turns of
the helix than relaxed DNA) in E. coli, and both negative and
positive supercoils (that is, those that contain fewer or more
turns of the helix than relaxed DNA) in eukaryotic cells.
b. Type II DNA topoisomerases:  These enzymes bind tightly to
the DNA double helix and make transient breaks in both
strands. The enzyme then causes a second stretch of the DNA
double helix to pass through the break and, finally, reseals the
break . As a result, both negative and positive
supercoils can be relieved by this ATP-requiring process. Type
II DNA topoisomerases are also required in both prokaryotes
and eukaryotes for the separation of interlocked molecules of
DNA following chromosomal replication. DNA gyrase, a Type II
topoisomerase found in bacteria and plants, has the unusual
property of being able to introduce negative supercoils into
relaxed circular DNA using energy from the hydrolysis of ATP.
This facilitates the future replication of DNA because the negative
supercoils neutralize the positive supercoils introduced
during opening of the double helix. It also aids in the transient
strand separation required during transcription. 


Cited By Kamal Singh Khadka
Msc Microbiology,TU
Assistant Professor In PU, RE-COST, LA, NOVEL ACADEMY, PNC.

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