Bacteriophage λ (lambda phage)
➔ The genome contains 48,500 base pairs of double-stranded, linear
DNA
➔ 12-base single-strand segments present at both 5' ends. These
two single-stranded segments are the "sticky ends" of what is
called the cos sites
➔ The cos site circularizes the DNA in the host cytoplasm.
Infection
Upon adsorption on the host cell, lambda
gDNA is injected through the tail which forms
a hollow tube through which the DNA passes
to the host bacterial cell.
The phage λ leads two life cycles, the lytic
cycle and the lysogenic cycle after injecting
its DNA into E.coli cell. Once inside the host
cell the linear phage DNA undergo
circularization by means of cos sites
In the lytic cycle, phage genes are expressed
(leading to production of phage proteins) and
DNA is replicated. The produced phage
proteins and replicated DNA are packaged,
resulting in production of several phage
particles (virions).
The lytic cycle ends with lysis of E.coli cells
and liberation of phage particles.
Replication
Lambda DNA replication
initiates at a single origin and
proceeds bidirectionally from
origin to generate theta (θ)
replication until several tens
of circular phage DNA
molecules accumulate in the
infected cell.
(theta mode of replication is a
type of replication that is seen
in circular DNA molecules
including bacterial
chromosomal DNA, that we’ve
discussed in class)
At that point replication
switches to rolling circle
mode to generate the
concatemeric DNA molecules
that are the substrate for DNA
packaging into virions
● Rolling circle replication (RCR) is a process of unidirectional nucleic acid
replication
● can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such
as plasmids, the genomes of bacteriophages etc.
● the name “rolling circle” is because the double-stranded portion of the replicating
DNA can be compared to a roll of toilet paper unwinding as it moves across the
floor.
● intermediate resembles Greek letter σ (sigma)
● The Rolling circle replication is completed in the following steps;
○ Initiation
○ Elongation
○ Termination
● Initiation of Rolling circle replication
● A protein called nicking enzyme starts the process of DNA
replication. Cuts (nicks) one strand of DNA at a specific origin
-called “Double-Strand Origin” (DSO) - only single strand nick
● The 3’ OH end is free to act as a primer for the DNA
polymerase enzyme
● Right after the nick is made, DNA polymerase attaches
itself to the complementary strand (the unnicked/ inner
strand)
● Then, a helicase called PcrA (Plasmid Copy Reduced)
that is made by the host moves the nicked strand.
(Pcr A unwinds the duplex DNA displacing the leading
strand of the plasmid and leaving a free 3′-OH end at the
DSO.)
As DNA synthesis goes on, it can make multiple single-stranded copies of the original
DNA in a series called a “Concatemer” that goes from head to tail.
Roll of toilet paper
unwinding as it moves
across the floor.
● Greek letter σ (in this fig it’s
upside down)
Molecules are produced by the rolling circle mechanism of replication, in which a continuous DNA
strand is ‘rolled off’ the template molecule. The result is a catenane consisting of a series of linear λ
genomes joined together at the cos sites.
● The DNA polymerase and associated factors begins to proceed to a strand
displacement synthesis, producing a concatemer linear single stranded DNA with
one genome copy per turn of replication.
● On the concatemer strand, Okazaki fragments are elongated after sequential RNA
primer synthesis by the primase, thus turning it into dsDNA.
● The concatemer strand RNA primer are removed and okazaki fragments ligated.
● The replication forks go on and produces a long linear concatemer which will be
processed into linear genomes and encapsidated.
Bacteriophage ϕX 174
● The genome is circular single stranded DNA
● 4.6 - 6.1 kb long genome
● Coding for 11 proteins
● Capsid is icosahedral i.e. spherical.
Replication of ϕX174
Replication of ϕX174 genome occurs in 3 stage
Stage 1
Since the genome is single stranded, (-) strand complementary to the (+) strand of
phage is synthesized by host enzymes to form the double stranded replicative form (RF)
Stage 2
Replication of the RF involves rolling circle replication and requires phage encoded
protein A to synthesize new plus strands.
These then serve as a templates for minus strand synthesis to generate the new RFs.
Stage 3
• Asymmetric replication of progeny ssDNA plus strand.
• RF synthesis continuous until sufficient structural proteins have been synthesized and
assembled into empty precursor particle.
DNA rolling circle replication similar to that
occurring in ϕX174
DNA rolling circle replication in general has five steps
1. The dsDNA in circular form will
be “nicked.”
2. Using “unnicked” DNA as the
template, the 3′ end is
lengthened; the 5′ end is
displaced.
3. A succession of Okazaki
fragments are used to convert a
lagging strand of DNA into a
double-stranded molecule.
4. Both “unnicked” and misplaced
ssDNA replication.
5. Displaced DNA circularises
The covalently closed single-stranded DNA is produced that can be subsequently converted to dsDNA
which involves the synthesis of an RNA primer at the sso by the RNA polymerase followed by DNA
synthesis by DNA polymerases I and III.
https://www.youtube.com/watch?v=ZDqsojQ8A5k
