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Principles of Genetics Lecture 15

Replication & Packaging


When phage inserts its linear DNA into the host cell, the DNA circularises, but
how is the linear DNA is produced from this circular molecular and then packaged
into phage heads.
During the lytic cycle, following adsorption to the host and injection of the phage
DNA, the DNA circularises and that is possible because of the presence of the
COS (cohesive) ends. The linear phage DNA has overhangs on each end that is
complementary to one another complementary sticky ends these ends fold
upon itself and forms a circular molecule.
Ligase then catalyses the formation of the phosphodiester thus the molecules
are truly covalently bound.

Circular molecule to linear DNA


Prior to packaging the DNA into phage heads, DNA undergoes replicative
rounds making numerous copies of the circular DNA, only after numerous copies
of the genome is made can they be individually packaged into phage heads.
There are 2 different types of DNA replication: Bidirectional Replication (
Replication) and Rolling circle ( replication).

Bidirection
Replication

Rolling Circle
Replication

DNA is melted at particular origins of replication


A replication bubble with 2 replication forks forms
New complementary DNA is built after DNA primers
anneal to origin of replication
This process is semi conservative and results in 2
circular DNA molecules are the end

After a few cycles of replication, switches to the


replication cycle

Rolling circle replication uses a different origin of


replication to that of bidirection replication
A single stranded knick is made by an enzyme that
has which cuts at the origin of replication
The 5 end is peeled off, giving a single stranded DNA
which can act as a template, the replication fork
occurs here and again, there is a leading and lagging
strand
Nothing happens to the 3 end, there is no replication
fork there. There are proteins that bind there, blocking
replication at that end

Because of the rolling nature, the DNA replication occurs

endlessly, making many copies of the genome joined end


to end, forming what is termed a concatamer.
Concatamer: genome joined end to end. Thus you
have gone from circular to linear DNA
The concatamer must be consolved to single genome
packages. produces an endonuclease (which cuts within
DNA) and this enzyme recognises COS sites and make
staggered cuts thus results in genomes with sticky COS sites
that can be packaged into new phage heads.
The cos ends are recognised by the head proteins, the DNA
is encapsulate the tail added.
Often, the endonuclease sits at the neck of the new phage
head and DNA is packaged at concatamer and when a COS
site is recognised, the enzyme will cut knowing the phage
has a genome equivalence. The free end then joins onto
another head. The offset cleavage of phage at COS sites
allows for a mechanism for the circularisation of the
chromosome immediately after infection. The
complementary COS ends are joined by DNA ligase.
The genes in genome are clustered according to function.
This is not the case for eukaryotes.
Temperate phages
It is possible for some phages, not , in which the DNA remains separate from the
host DNA but is also replicated with cell division.

T4 Lytic cycle
T4 injects its DNA into the host, the DNA then circularises. The DNA is replicated
via bidirection replication, the phage hijacks and uses host machinery to
replicate its DNA and ultimately reproduce.
Unlike in who uses rolling over, T4 forms concatamers via recombination of the
copies of the genome. The recombination of the various circular copies of the
phage DNA results in a concatamer. T4 doesnt make use of COS sites to package
DNA, instead DNA is packaged into phage by the headful method. DNA attaches
to the head and is packaged into T4 until it is full, enzymes then cut the DNA
regardless of the DNA sequence unlike COS enzymes.
However, the space that the T4 head has allows for a little more than just one
complete T4 genome, as a result the terminals of the DNA are redundant.

T4 genome is 166kb

But T4 phage head can package 171kb

There are always 2 copies of the terminal genes. In addition each particle starts a
little later in the genome sequence, the differences in the redundant terminals
lead to circular permutation, which is all due to the packaging of more than a
genomes equivalent of DNA.
T4 rapid lysis mutant
T4 usually makes small fuzzy plaques but a mutant strain causes large plaques
which are more lethal. There have been studies in which certain T4 plaques
exhibit a mixture of morphologies thus suggesting that they probably contain 2
alleles terminal redundancy.
The event of terminal redundancy also allows the possibility a phage may be
heterozygous in the region of the genome that is diploid. This then leads to the
possibility of a variety of phenotypes being observed.

Phage statistics

T4

Genome is 166kb
Head contains 171kb of DNA
Genome is terminally redundant
Genome is circularly permuted

Genome is 48kb
Head contains 48kb
No genome redundancy
No circular permutation due to
specific cleavage at COS sites
there is only 1 copy of each gene
because one Cos site in each
genome equivalent

Phage diversity

linear genome (48kb), cohesive ends, circularises, bidirectional


replication then rolling circle producing concatamers of the 48kb genome

T4 liner genome (166kb), terminally redundant ends, circularise,


bidirectional replication, heedful packaging, circularly permuted

P2 linear genome (34kb) cohesive ends, circularises, rolling circle but


terminates after 34kb

Single Phages
X-174

Single stranded, circular DNA, 5kb, the phages genome consists of a


single stranded plus strand.

The single stranded DNA after being injected into host cell uses the host
machinery which in turn leads to the synthesis of the minus
complementary strand. The resulting is a double stranded replicative form
(RF).

Early, the DNA undergoes bidirectional replication of the replicative form,


but later, it undergoes rolling circle replication but this is different to
rolling circle replication

X-174 rolling replication the 5 strand is peeled off but not replicated,
this strand is instead bound by singly stranded DNA binding proteins. The
coating of these proteins stops DNA polymerase from binding and
replicating the DNA. The 3 end is extended and it rolls out lengthening the
5 sing strand.

The single stranded end attaches to the head and is chopped off after one
genomes equivalent of the ssDNA is packaged. The linear DNA molecule
can be circularised.

Single stranded RNA phages


MS2 (male specific) thus only infects e.coli with the F factor. It has single
stranded linear RNA and makes use of RNA dependent RNA polymerase, in this
case, the e.coli doesnt make DNA, it makes RNA and uses RNA as a template.
Male bacteria are those that possess the F factor, which is a piece of circular DNA
which confers certain characteristics to the cells that have them. One of the
characteristics is that the cell can form a pilli on the surface and attach to cells
that dont have an F factor forming a conjugation tube which then can assist in
transfer of genetic material.
In MS2, the complementary strand is used as a template to produce copies of the
original strand of RNA. Many copies of the RNA is made and packaged into
heads. This bacterioiphage never makes DNA, it starts with RNA and makes more
RNA copies.