The SOS Regulatory Network

In gram-negative bacterium Escherichia coli (E. coli), DNA damage and replication
perturbations results in the SOS response, a genetic program that transcriptionally up-regulates
over 50 unlinked genes. The term “SOS” was coined by Miroslav Radman in 1974 when he
postulated the existence of the pathway on the basis of a set of physiological responses induced
by DNA damage whose regulation was controlled by the lexA+ and recA+ gene products.
Radman defined “SOS” as a distress signal used to sense DNA damage or replication fork
blockages. Since the original hypothesis, the distress signal has been shown to be the
accumulation of single stranded DNA (ssDNA). The SOS response is wired to allow for high
fidelity repair to take place before giving way to a more mutagenic mode that allows for cell
survival. When the SOS response is induced the first set of genes to be expressed are gene
products involved in high fidelity DNA repair. Further into SOS induction, sulA gene expression
is induced and this protein causes a DNA damage checkpoint by inhibiting cell division. The
SulA-dependent checkpoint allows cells time to repair their DNA before damaged chromosomes
are segregated into daughter cells. Late in the SOS response, umuC and umuD genes are
expressed and these gene products assemble into a translesion polymerase that has mutagenic
potential, as high fidelity repair gives way to lower fidelity damage toleraence. This lower
fidelity DNA damage tolerance pathway, is so named because the damage is not removed, but
instead tolerated.
The genetics of SOS regulation
The SOS response is a genetic circuit that is regulated by the LexA and RecA proteins. LexA is a
transcriptional repressor that occupies its cognate operator binding site (SOS box) as a
homodimer thereby blocking RNA polymerase (RNAP) binding and transcription. LexA has a
cryptic autocleavage activity that is activated when LexA interacts with a RecA/ssDNA
nucleoprotein filament. Expression of recA+ and lexA+ gene products are regulated in an SOS
dependent fashion, and RecA is rather abundant in the non-induced state. Considerable in vitro
and in vivo evidence has shown that when bacterial DNA is damaged, ssDNA is generated. RecA
binds ssDNA forming a nucleoprotein filament. Interaction between the RecA/ssDNA
nucleoprotein filament and LexA activates LexA auto-digestion, thereby inactivating LexA as a
repressor and leading to the transcription of LexA repressed genes. RecA, a key player in DNA
repair, is required for homologous recombination, SOS induction, and translesion synthesis
(TLS).
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Bacteriophage T4 DNA replication
Bacteriophage T4 encodes its own DNA polymerase for replication. Other proteins that function
in viral DNA replication such as primases and helicases are also encoded by the T4 genome.
Besides encoding its own replication machinery, the T4 genome has another unusual feature: In
a population of T4 virions, although each copy of the genome contains the same set of genes,
they are arranged in a different order. This is a phenomenon called circular permutation, which
is a feature of many virus genomes. The term circular permutation is derived from the fact that
DNA molecules that are circularly permuted appear to have been linearized by opening identical
circular genomes at different locations. Circularly permuted genomes are also terminally
redundant, meaning that some DNA sequences are duplicated on both ends of the DNA molecule
as a result of the mechanism that generated them. The T4 genome is first replicated as a unit and
then several genomic units are recombined end to end to form a long DNA molecule called a
concatemer. When the T4 DNA is packaged into capsids, the concatemer is not cut at a specific
sequence; instead linear segments of DNA just long enough to fill a phage head are generated.
This is called headful packaging, and is common among bacteriophages.
The bacteriophage T4 encodes 10 proteins, known collectively as the replisome, that are
responsible for the replication of the phage genome. The replisomal proteins can be subdivided
into three activities; the replicase, responsible for duplicating DNA, the primosomal proteins,
responsible for unwinding and Okazaki fragment initiation, and the Okazaki repair proteins. The
replicase includes the gp43 DNA polymerase, the gp45 processivity clamp, the gp44/62 clamp
loader complex, and the gp32 single-stranded DNA binding protein. The primosomal proteins
include the gp41 hexameric helicase, the gp61 primase, and the gp59 helicase loading protein.
The RNaseH, a 5' to 3' exonuclease and T4 DNA ligase comprise the activities necessary for
Okazaki repair. The T4 provides a model system for DNA replication.
Figure: A cartoon model of leading and lagging strand DNA synthesis by the Bacteriophage T4
Replisome
The helicase (gp41) and primase (gp61) interact to form the primosome with the assistance of the
helicase loader (gp59). The primosome complex encircles the lagging strand DNA, unwinding
duplex DNA while synthesizing RNA primers for use by the lagging strand polymerase (gp43).
DNA synthesis on both strands is catalyzed by a holoenzyme complex formed by a polymerase
(gp43) and a trimeric processivity clamp (gp45). The clamp is loaded onto the DNA by the
clamp loader complex (gp44/62). The leading and lagging strand holoenzymes interact to form a
dimer. Single-stranded DNA formed by the helicase is coated with single-stranded DNA-binding
protein (gp32). Figure 1. A model of the T4 bacteriophage DNA replisome. Replication of T4
genomic DNA is accomplished by a replication complex composed of eight proteins. The
helicase (gp41) and primase (gp61) interact to form the primosome with the assistance of the
helicase loader (gp59). The primosome complex encircles the lagging strand DNA, unwinding
duplex DNA while synthesizing RNA primers for use by the lagging strand polymerase (gp43).
DNA synthesis on both strands is catalyzed by a holoenzyme complex formed by a polymerase
(gp43) and a trimeric processivity clamp (gp45). The clamp is loaded onto the DNA by the
clamp loader complex (gp44/62). The leading and lagging strand holoenzymes interact to form a
dimer. Single-stranded DNA formed by the helicase is coated with single-stranded DNA-binding
protein (gp32).

One-step growth curve of virus replication

The growth response during virus replication is form of a one-step growth curve, so named
because a time course of virion numbers in the culture medium shows essentially no increase
during the replication cycle until cells burst and release their newly synthesized virions. In the
first few minutes after infection, the virus enters the eclipse phase, during which the viral
genome and proteins will be replicated and translated, respectively. The maturation phase begins
as newly synthesized viral nucleic acid molecules become packaged inside their capsids. During
the maturation phase, the number of infectious virions inside the host cell rises dramatically. At
the end of maturation, mature virions are released, either as a result of cell lysis or by budding or
excretion, depending on the virus. The number of virions released per cell, called the burst size,
varies with the particular virus and the particular host cell, and can range from a few to a few
thousand.

Detecting and Counting Viruses: The Plaque Assay

A viral suspension can be quantified to estimate the number of infectious virions present per
volume of fluid, a quantity called the titer. This is typically done using a plaque assay. When a
virus infects host cells growing on a flat surface, a zone of cell lysis called a plaque forms and
appears as a clear area in the lawn of host cells.
The procedure requires the use of a Double-Layer Agar (DLA) technique also known as double
agar overlay method, in which the hard agar serves as a base layer (to form gel), and a mixture
of few phage particles (diluted stock) and a very large number of host cells in a soft agar forms
the upper overlay. When the plates are incubated, susceptible E. coli cells multiply rapidly and
produce a lawn of confluent growth on the medium. When one phage particle adsorbs to a
susceptible cell, penetrates the cell, replicates and release new phage particles which infect other
bacteria in the vicinity of the initial host cell. The growth or spread of the new viruses is then
restricted or limited to the neighboring cells by the gel. This cycle is repeated until large numbers
of bacteria have been destroyed. The destroyed cells produce single circular, non-turbid areas
called plaques in the bacterial lawn, where there is no growth of bacteria because the phage
progeny originating from single virus particles have multiplied sufficiently to kill bacteria over
an easily visible area. Eventually the plaque becomes too large to be visible to our naked eye.
Each plaque represents the lysis of a phage-infected bacterial culture and can be designated as a
plaque-forming unit (PFU) and is used to quantitate the number of infective phage particles in
the culture. Dyes that stain the living cells are frequently used to enhance the contrast between
the plaques and the living cells. Therefore the dead cells in the plaque will appear as unstained
against the colored background. Only viruses that have the ability to cause visible damage of
cells can be assayed using this way.