Replication of viral DNA

Introduction
• Virus replication can be divided into eight stages.
• Regardless of their hosts, all viruses must undergo each of these stages in
some form to successfully complete their replication cycle.
• Stages of viral replication
1. Attachment
2. Entry
3. Uncoating
4. Genome Replication
5. Assembly
6. Release
Attachment
• The attachment phase of replication comprises specific binding of a virus-
attachment protein (or ‘antireceptor’) to a cellular receptor molecule.
• Virus receptors on cell surfaces may be proteins (usually glycoproteins) or
carbohydrate residues present on glycoproteins or glycolipids.
• Some complex viruses (e.g., in the Poxviridae or Herpesviridae) use more
than one receptor and therefore have alternative routes of uptake into
cells. Most bacteriophage receptors are on the bacterial cell wall, although
certain phages use cellular appendages (pili, flagella) as primary adsorption
sites.
• Attachment is an automatic docking process and the kinetics of receptor
binding are controlled by the chemical and thermodynamic characteristics
of the molecules involved, that is, their relative concentrations and
availability.
Entry

• Entry of the virus particle into the host cell normally occurs a short
time after attachment of the virus to the receptor.
• Unlike attachment, cell entry is generally an energy-dependent
process, that is, the cell must be metabolically active for this to occur.
• This occurs through contraction of the tail sheath, which acts like a
hypodermic needle to inject the viral genome through the cell wall
and membrane. The phage head and remaining components remain
outside the bacteria.
Uncoating
• Uncoating describes the events which occur after host cell entry,
during which the virus capsid is partially or completely degraded or
removed and the virus genome exposed, usually still in the form of a
nucleic acid–protein complex. Uncoating occurs simultaneously with
or immediately after entry.
• Cell enzymes (from lysosomes) strip off the virus protein coat. This
releases or renders accessible the virus nucleic acid or genome.
• The removal of a virus envelope during membrane fusion is the initial
stage of the uncoating process for enveloped viruses. Uncoating may
occur inside endosomes, being triggered by the change in pH as the
endosome is acidified, or directly in the cytoplasm..
• Entry into the endocytic pathway is a hazardous process for viruses
because if they remain in the vesicle too long, they will be irreversibly
damaged by low pH or lysosomal enzymes.
Genome Replication
• The replication mechanism depends on the viral genome. DNA viruses
usually use host cell proteins and enzymes to make additional DNA that is
transcribed to messenger RNA (mRNA), which is then used to direct protein
synthesis.
• RNA viruses usually use the RNA core as a template for synthesis of viral
genomic RNA and mRNA. The viral mRNA directs the host cell to synthesize
viral enzymes and capsid proteins, and to assemble new virions. Of course,
there are exceptions to this pattern.
• If a host cell does not provide the enzymes necessary for viral replication,
viral genes supply the information to direct synthesis of the missing
proteins. Retroviruses, such as HIV, have an RNA genome that must be
reverse transcribed into DNA, which then is incorporated into the host cell
genome.
• To convert RNA into DNA, retroviruses must contain genes that
encode the virus-specific enzyme reverse transcriptase, which
transcribes an RNA template to DNA. Reverse transcription never
occurs in uninfected host cells; the needed enzyme, reverse
transcriptase, is only derived from the expression of viral genes within
the infected host cells.
Assembly
• A virion is simply an active or intact virus particle. In this stage, newly
synthesized genome (nucleic acid), and proteins are assembled to
form new virus particles.
• This may take place in the cell's nucleus, cytoplasm, or at plasma
membrane for most developed viruses.
Release
• The viruses, now being mature are released by either sudden rupture
of the cell, or gradual extrusion (force out) of enveloped viruses
through the cell membrane.
• The new viruses may invade or attack other cells, or
remain dormant in the cell. In the case of bacterial viruses, the
release of progeny virions takes place by lysis of the infected
bacterium. However, in the case of animal viruses, release usually
occurs without cell lysis.
Prokaryotic genome
• There are specific nucleotide sequences called origins of
replication where replication begins.
• The origin of replication is approximately 245 base pairs long. This
sequence of base pairs is recognized by certain proteins that bind to
this site.
• An enzyme called helicase unwinds the DNA by breaking the
hydrogen bonds between the nitrogenous base pairs. ATP hydrolysis
is required for this process because it requires energy. As the DNA
opens up, Y-shaped structures called replication forks are formed.
• Two replication forks are formed at the origin of replication and these
get extended bi-directionally as replication proceeds. Single-strand
binding proteins coat the single strands of DNA near the replication
fork to prevent the single-stranded DNA from winding back into a
double helix.
• DNA pol III, which adds nucleotides one by one to the growing DNA
chain. The addition of nucleotides requires energy; this energy is
obtained from the nucleotides that have three phosphates attached
to them.
• ATP structurally is an adenine nucleotide which has three phosphate
groups attached; breaking off the third phosphate releases energy. In
addition to ATP, there are also TTP, CTP, and GTP. Each of these is
made up of the corresponding nucleotide with three phosphates
attached.
• When the bond between the phosphates is broken, the energy
released is used to form the phosphodi ester bond between the
incoming nucleotide and the existing chain.
• DNA polymerase is able to add nucleotides only in the 5′ to 3′
direction (a new DNA strand can be only extended in this direction). It
requires a free 3′-OH group (located on the sugar) to which it can add
the next nucleotide by forming a phosphodi ester bond between the
3′-OH end and the 5′ phosphate of the next nucleotide. This
essentially means that it cannot add nucleotides if a free 3′-OH group
is not available. The problem is solved with the help of a primer that
provides the free 3′-OH end
• RNA Primer that provides the free 3′-OH end. Another enzyme, RNA
primase, synthesizes an RNA primer that is about five to ten
nucleotides long and complementary to the DNA. RNA primase does
not require a free 3′-OH group. Because this sequence primes the
DNA synthesis, it is appropriately called the primer. DNA polymerase
can now extend this RNA primer, adding nucleotides one by one that
are complementary to the template strand
• The replication fork moves at the rate of 1000 nucleotides per
second. DNA polymerase can only extend in the 5′ to 3′ direction,
which poses a slight problem at the replication [Link] DNA double
helix is anti-parallel; that is, one strand is in the 5′ to 3′ direction and
the other is oriented in the 3′ to 5′ direction. One strand, which is
complementary to the 3′ to 5′ parental DNA strand, is synthesized
continuously towards the replication fork because the polymerase
can add nucleotides in this direction.
• This continuously synthesized strand is known as the leading strand.
The other strand, complementary to the 5′ to 3′ parental DNA, is
extended away from the replication fork, in small fragments known
as Okazaki fragments, each requiring a primer to start the synthesis.
• Okazaki fragments are named after the Japanese scientist who first
discovered them. The strand with the Okazaki fragments is known as
the lagging strand.
• The leading strand can be extended by one primer alone, whereas the
lagging strand needs a new primer for each of the short Okazaki
fragments. The overall direction of the lagging strand will be 3′ to 5′,
and that of the leading strand 5′ to 3′.
• A protein called the sliding clamp holds the DNA polymerase in place
as it continues to add nucleotides. The sliding clamp is a ring-shaped
protein that binds to the DNA and holds the polymerase in place.
• Topoisomerase prevents the over-winding of the DNA double helix
ahead of the replication fork as the DNA is opening up; it does so by
causing temporary nicks in the DNA helix and then resealing it.
• As synthesis proceeds, the RNA primers are replaced by DNA pol I,
which breaks down the RNA and fills the gaps with DNA nucleotides.
The nicks that remain between the newly synthesized DNA (that
replaced the RNA primer) and the previously synthesized DNA are
sealed by the enzyme DNA ligase that catalyzes the formation of
phosphodiester linkage between the 3′-OH end of one nucleotide and
the 5′ phosphate end of the other fragments.
DNA replication in eukaryotes
• DNA replication in eukaryotes occurs in three stages: initiation,
elongation, and termination, which are aided by several enzymes.
Initiation

• Eukaryotic DNA is bound to proteins known as histones to form structures

called nucleosomes. During initiation, the DNA is made accessible to the
proteins and enzymes involved in the replication process. There are specific
chromosomal locations called origins of replication where replication
begins. In some eukaryotes, like yeast, these locations are defined by
having a specific sequence of base pairs to which the replication initiation
proteins bind.
• In other eukaryotes, like humans, there does not appear to be a consensus
sequence for their origins of replication. Instead, the replication initiation
proteins might identify and bind to specific modifications to the
nucleosomes in the origin region.
• Certain proteins recognize and bind to the origin of replication and then
allow the other proteins necessary for DNA replication to bind the same
region. The first proteins to bind the DNA are said to “recruit” the other
proteins. Two copies of an enzyme called helicase are among the proteins
recruited to the origin. Each helicase unwinds and separates the DNA helix
into single-stranded DNA.

• As the DNA opens up, Y-shaped structures called replication forks are
formed. Because two helicases bind, two replication forks are formed at
the origin of replication; these are extended in both directions as
replication proceeds creating a replication bubble. There are multiple
origins of replication on the eukaryotic chromosome which allow
replication to occur simultaneously in hundreds to thousands of locations
along each chromosome.
Elongation

• During elongation, an enzyme called DNA polymerase adds DNA

nucleotides to the 3′ end of the newly synthesized polynucleotide strand.
The template strand specifies which of the four DNA nucleotides (A, T, C, or
G) is added at each position along the new chain. Only the nucleotide
complementary to the template nucleotide at that position is added to the
new strand.
• DNA polymerase contains a groove that allows it to bind to a single-
stranded template DNA and travel one nucleotide at at time. For example,
when DNA polymerase meets an adenosine nucleotide on the template
strand, it adds a thymidine to the 3′ end of the newly synthesized strand,
and then moves to the next nucleotide on the template strand. This
process will continue until the DNA polymerase reaches the end of the
template strand.
• DNA polymerase cannot initiate new strand synthesis; it only adds new
nucleotides at the 3′ end of an existing strand. All newly synthesized
polynucleotide strands must be initiated by a specialized RNA polymerase
called primase. Primase initiates polynucleotide synthesis and by creating a
short RNA polynucleotide strand complementary to template DNA strand.
This short stretch of RNA nucleotides is called the primer. Once RNA primer
has been synthesized at the template DNA, primase exits, and DNA
polymerase extends the new strand with nucleotides complementary to
the template DNA.
• Eventually, the RNA nucleotides in the primer are removed and replaced
with DNA nucleotides. Once DNA replication is finished, the daughter
molecules are made entirely of continuous DNA nucleotides, with no RNA
portions.
The Leading and Lagging Strands
• DNA polymerase can only synthesize new strands in the 5′ to 3′ direction.
Therefore, the two newly-synthesized strands grow in opposite directions
because the template strands at each replication fork are antiparallel. The
“leading strand” is synthesized continuously toward the replication fork as
helicase unwinds the template double-stranded DNA.
• The “lagging strand” is synthesized in the direction away from the
replication fork and away from the DNA helicase unwinds. This lagging
strand is synthesized in pieces because the DNA polymerase can only
synthesize in the 5′ to 3′ direction, and so it constantly encounters the
previously-synthesized new strand. The pieces are called Okazaki
fragments, and each fragment begins with its own RNA primer.
Termination

• Eukaryotic chromosomes have multiple origins of replication, which initiate

replication almost simultaneously. Each origin of replication forms a bubble of
duplicated DNA on either side of the origin of replication. Eventually, the leading
strand of one replication bubble reaches the lagging strand of another bubble,
and the lagging strand will reach the 5′ end of the previous Okazaki fragment in
the same bubble.
• DNA polymerase halts when it reaches a section of DNA template that has
already been replicated. However, DNA polymerase cannot catalyze the
formation of a phosphodiester bond between the two segments of the new DNA
strand, and it drops off. These unattached sections of the sugar-phosphate
backbone in an otherwise full-replicated DNA strand are called nicks.
• Once all the template nucleotides have been replicated, the replication process is
not yet over. RNA primers need to be replaced with DNA, and nicks in the sugar-
phosphate backbone need to be connected.
• The group of cellular enzymes that remove RNA primers include the
proteins FEN1 (flap endonulcease 1) and RNase H. The enzymes FEN1
and RNase H remove RNA primers at the start of each leading strand
and at the start of each Okazaki fragment, leaving gaps of
unreplicated template DNA. Once the primers are removed, a free-
floating DNA polymerase lands at the 3′ end of the preceding DNA
fragment and extends the DNA over the gap. However, this creates
new nicks (unconnected sugar-phosphate backbone).
• In the final stage of DNA replication, the enyzme ligase joins the
sugar-phosphate backbones at each nick site. After ligase has
connected all nicks, the new strand is one long continuous DNA
strand, and the daughter DNA molecule is complete.