Transcript DNA polymerase I

Introduction to
Microbial Genetics
Microbiology 221
A Historical Overview
The scientists who provided the clues to
the nature of DNA
Friederich Meischer – DNA isolated
Luria and Delbruck – Bacteriophages
Stanely Giffiths( 1928) The idea of the
transforming “ substance” – Avery,
MacLoed, and McCarty( 1944) – the
nature of transformation
Hershey and Chase – Bacteriophage –
DNA as the hereditary material
Chargaff – A= T and C=G
Maurice Wilkins and Rosalind Franklin – xray crystallography of DNA
Watson and Crick – Double helix
Griffiths
Luria and Delbruck at Cold
Spring Harbor in 1953
Luria and Delbruck
studied bacterial
mutations and
resistance to
infection with
bacteriophages
The characterized the
virus and its life cycle
Alfred Hershey and Martha Chase
and the Blender Experiment
Hershey and Chase
wanted to verify that
DNA was the
hereditary material
They used a
bacteriophage for
their study
They labeled the DNA
with Radioactive P(
P32) and the protein
with radioactive
sulfur( S35)
Results of the
Experiment
Proved that the radioactivity from the labeled
DNA was present in the progeny phage produced
from infection of the bacteria.
The Race for the Double
Helix
Rosalind Franklin
and Maurice
Wilkins at Kings
College
Studied the A and
B forms of DNA
Rosalind’s famous
x-ray
crystallography
picture of the B
form held the
secret, but she
didn’t realize its
significance
The Race for the Double
Helix
Watson and Crick
formed an unlikely
partnership
A 22 year old PhD and
a 34 year old “want to
be” PhD
embarked on a model
making venture at
Cambridge
Used the research of
other scientists to
determine the nature
of the double helix
Nucleic Acid Composition
DNA and RNA
a.
b.
c.
d.
DNA – Basic Molecules
Purines – adenine and guanine
Pyrmidines – cytosine and thymine
Sugar – Deoxyribose
Phosphate phosphate group
http://www.dnai.org/index.htm - DNA background
Double Helix
Two polynucleotide strands joined by
phosphodiester bonds( backbone)
Complementary base pairing in the center of the
molecule
A= T and C
G – base pairing. Two hydrogen
bonds between A and T and three hydrogen bonds
between C and G.
A purine is bonded to a complementary pyrimidine
Bases are attached to the 1’ C in the sugar
At opposite ends of the strand – one strand has
the 3’hydroxyl, the other the 5’ hydroxyl of the
sugar molecule
DNA Structure
http://www.johnkyrk.com/DNAanatomy.html - DNA
structure
Double helix
( continued)
The double helix is right handed – the
chains turn counter-clockwise.
As the strand turn around each other
they form a major and minor groove.
The is a distance of .34nm between each
base
The distance between two major grooves
is 2.4nm or 10 bases
The diameter of the strand is 2nm
Complementary Base
Pairing
Adenine pairs
with Thymine
Cytosine pairs
with Guanine
The end view of DNA
This view shows
the double
helix and the
outer backbone
with the bases
in the center.
An AT base
pair is
highlighted in
white
Double helix and antiparallel
DNA is a directional molecule
The complementary strands
run in opposite directions
One strand runs 3’-5’
The other strand runs 5’ to 3’
( the end of the 5’ has the
phosphates attached, while the
3’ end has a hydroxyl exposed)
RNA structure
Polynucleotide – nucleic acid Single stranded molecule that
can coil back on itself and
produce complementary basepairing ( t- RNA)
Four bases in RNA are Adenine
and Guanine ( purines) and
Cytosine and Uracil(
pyrimidines)
Sugar – ribose
Phosphates
RNA
a.
b.
c.
d.
Three types of RNA
Messenger
Transfer
Ribosomal
nc- non coding RNA’s
Prokaryote DNA
Tightly coiled
Coiling maintained by molecules
similar to the coiling in eukaryotes
Circular ds molecule
Borrelia burgdoferi ( Lyme Disease
)has a linear chromosome
Other bacteria have multiple
chromosomes
Agrobacterium tumefaciens (
Produces Crown Gall disease in
plants) has both circular and linear
Prokaryote
chromosomes
Circular DNA
E. coli – most often
studied in molecular
biology
of
prokaryotes
The genes of E. coli are located on a circular
chromosome of 4.6 million basepairs. This 1.6
mm long molecule is compressed into a highly
organized structure which fits inside the 1-2
micrometer cell in a format which can still be
read by the gene expression machinery.
Bacterial DNA is supercoiled by DNA gyrase.
Chemical inhibition of gyrase without allowing
the cells to reprogram gene expression relaxes
supercoiling and expands the nucleoid,
suggesting that supercoiling is one of the tools
used to compress the genome
Coiling
Coiling maintained by Gyrase
Relaxation of the coils by
Topoisomerase
Nucleosome formation
DNA is more highly
organized in
eukaryote cells
The DNA is
associated with
proteins called
histones.( eukaryotes)
These are small basic
proteins rich in the
amino acids lysine
and/or arginine
There are five
histones in eukaryote
cells, H1, H2A,
H2B,H3 and H4.
.
Beads on a String
The DNA coils around the ellipsoid
approximately 1 ¾ turns or 166 base pairs
before proceeding to the next.
The DNA + the histone proteins arranged
in this formation are referred to as a
nucleosome.
The stretch of DMA between the beads
varies in length from 14 to 100 base pairs.
H1 appears to associate with the linker
regions to enable the nucleosome to
supercoil
When folding of the structure reaches a
maximum, the chromosomes can be
visualized
Chromosome structure
http://www.johnkyrk.com/chromo
somestructure.html
Eukaryote replication
The nature of
DNA
replication was
elucidated by
Meselson and
Stahl
1.
Meselson and Stahl
experiment
Grew bacteria in heavy Nitrogen – N-15
2. Transferred bacteria to N-14
3. Before bacteria reproduce in new media, all bacteria
contain heavy DNA
4. Samples were taken after one round of replication and
two round of replication
Semiconservative
replication
Each original strand
serves a template or
pattern for the
replication of the new
strand.
The new strand
contains one original
and a newly
synthesized strand
Eukaryote replication
Multiple linear chromosomes
Each chromosome has more than one origin of
replication
Approximately 1400 x as long as bacterial DNA
Multiple replicons on a chromosome
Oris along the length – every 10 to 100 um
Replication forks and bubbles are formed.
Replication proceeds bidirectionally until the
bubbles meet
This shortens the length of time necessary to
replicate eukaryote chromosomes
The process of elongation occurs at a speed of
50-100 base pairs/minute as compared to 750 to
1000 base pairs/ minute
http://www.johnkyrk.com/DNAreplication
The origin of replication
and replication forks
Eukaryote replication
During the S phase, there are 100
replication complexes and each one
contains as many as 300 replication forks.
These replication complexes are
stationary. The DNA threads through
these complexes as single strands and
emerges as double strands.
DNA Polymerases
Fourteen DNA polymerases
have been observed in human
beings as compared to three in
E. coli.
Prokaryote Replication
Enzyme
Type
Source
Properties
Bacterial topoisomerase I
(v protein)
I
E. coli
Relaxation of negative but
not positive supercoils
Vaccinia virus
topoisomerase I
I
Vaccinia virus
Relaxation of positive and
negative supercoils
Eukaryotic topoisomerase
I
I
Calf thymus
Relaxation of positive and
negative supercoils
Reverse gyrase
I
Thermophilic bacteria
Introduces positive
supercoils into DNA
Topoisomerase V
I
Hyperthermophilic
bacteria
Relaxation of positive
supercoils
DNA gyrase
II
E. coli
Introduces negative
supercoils into DNA
Topoisomerase IV
II
E. coli
DNA relaxation and
potent decatenation
T4 topoisomerase II
Eukaryotic topoisomerase
II
II
II
Bacteriophage T4
Relaxation of positive and
negative supercoils and
decatenation
S. cerevisiae
Relaxation of positive and
negative supercoils and
decatenation
Bidirectional replication
There is an origin
of replication
Two replication
forks are formed
Replication occurs
around the circle
until they have
opened and copied
the entire
chromosome
Replicon- contains
an origin and is
replicated as a
unit
Ori – Origin of replication
Characteristics used to define Origins:
The position on the DNA at which replication
start points (see right) are found.
A DNA sequence that when added to a nonreplicating DNA causes it to replicate.
A DNA sequence whose mutation abolishes
replication.
A DNA sequence that in vitro is the binding
target for enzyme
Topoisomerases
Topoisomerase
When the double helix of DNA, which is
composed of two strands, separates,
helicase makes these two strands rotate
around each other.
The DnaB protein is the helicase most
involved in replication, but the n’ protin
may also participate in unwinding.
The single stranded binding proteins
SSBP help to keep the strand open
But there is a problem due to the
topological reason that the unreplicated
part ahead of the replication fork will
rotate around its helical axis when the
two strands separate at the replication
fork
Topoisomerase action
It causes strong strain in the helix
(1). Thus, it is impossible to unlink
the double helical structure of
DNA without disrupting the
continuity of the strands.
In order to perform unraveling of a
"compensating winding up" DNA,
enzymes are required (1).
Topoisomerase changes the linking
number as well as catalyzes the
interconversionn of other kinds of
topological isomers of DNA (2).
Initiation
Initiation
a. oriC - origin of chromosomal replication
Recognized by DnaA protein - only
recognizes if GATC sites are fully
methylated
Binding of DnaA allows DnaB to open
complex
b. DnaB is the replication helicase
c. Strand separation by helicase
d. SSB (single-stranded binding) protein
keeps strands apart
e. DNA gyrase - a topoisomerase - puts
swivel in DNA which allows strands to
rotate and relieve strain of unwinding
Explanation
Recall that DNA double helix is tightly wound
structure and that bases lie between the two
backbones. If these bases are the template for
new strand, how do the appropriate enzymes
reach these bases? By the unwinding of the helix.
An enzyme called helicase catalyzes the unwinding
of short DNA segments just ahead of the
replication fork. The reaction is driven by the
hydrolysis of ATP.
Explanation continued
As soon as duplex is unwound, SSB
(single-stranded binding protein) binds to
each of the separated strands to prevent
them from base-pairing again. Therefore,
the bases are exposed to the replication
system.
The unwinding of the duplex would cause
the entire DNA molecule to swivel except
for the action of a topoisomerase (DNA
gyrase) which introduce breaks in the
DNA just ahead of the unwinding duplex.
These breaks are then rejoined after a
few revolutions of the duplex.
The need for a primer
When DNA template is exposed, DNA
synthesis must begin. But DNA
polymerases not only need a template but
also a primer for replication to proceed.
Where does the primer come from?
After observations that RNA synthesis is
required for DNA synthesis, it was
discovered that the synthesis of DNA
fragments requires a short length of
RNA as a primer.
Primosome (complex of 20 polypeptides)
makes RNA primers in E. coli
Formation of the Primer
Primosome contains primase
Primosome moves along DNA duplex in
3'>5' direction (with respect to lagging
strand; follows replication fork) even
though primer is made in 5'>3' direction
(Note: The symbol ">" indicates the
direction; that is, the primer is made
from 5' to 3'.)
n' protein removes SSB in front of
primosome
DnaB protein organizes some components
of primosome and prepares DNA for
primase
Primase forms the primer
DNA POLYMERASE III
Holoenzyme
Complex that
synthesizes most of the
DNA copy contains the
DNA polymerase enzyme
and other proteins
The gamma delta
complex and the B
subunits of the
holoenzyme bind it to
the template and the
primer
The alpha subunit
carries out the actual
polymerization reaction
All of the proteins form
a huge complex called
the replisome
DNA polymerase III
This is a
stationary
complex that
probably
attached to the
plasma
membrane.
The DNA moves
through the
replisome and is
copied
Elongation of the chain
dCTP
dCMP +
PPi
Energy is
supplied for
biosynthesis by
the cleaving of
the phosphate
bond
Elongation( continued)
Elongation proceeds in 5' > 3'
direction and requires
1) all 4 deoxyribonucleoside 5'triphosphates (dATP, dGTP, dCTP,
dTTP),
2) Mg+ ions,
3) a primer made of nucleic acid,
and
4) a DNA template.
Rate of elongation = 750 - 1000
nucleotides per second
Rate of formation of initiation
complex = 1-2 minutes
Elongation
Elongation
DNA polymerase I, II and III in E .coli
DNA polymerase III holoenzyme - complex of 7
polypeptides
Replisome - primosome and 2 DNA polymerase III synthesizes DNA on both strands simultaneously
without dissociating from DNA
DNA polymerase III catalyzes the addition of
deoxyribonucleotide units to end of the DNA strand
with release of inorganic pyrophosphate (PPi)
(DNA)n residues + dNTP <> (DNA)n + 1 residues + PPi
Attachment of new units is by their a-phosphate groups
to a free 3'-hydroxyl end of preexisting DNA chain.
The lagging strand and
discontinuous replication
The replication on the 5’ to 3’ strand
differs
The template strand still must be read
from 3’ to 5’
The reading begins at the replication fork
Occurs at the same time as the synthesis
of the lagging strand
Same steps in synthesis of DNA
But DNA is synthesized in pieces about
1000 to 2000 bases in length. These are
known as Okazaki fragments
Okazaki fragments
After the lagging strand has been
duplicated by the formation of Okazaki
fragments, DNA Polymerase I or RNase
H removes the RNA primer. Polymerase I
synthesizes the complementary DNA to
fill the gap resulting from the RNA
delection.
The polymerase removes one nucleotide
at a time and then replaces it
AMP( RNA nucleotide) replaced by dAMP(
DNA nucleotide)
DNA ligase
Ligase can catalyze
the formation of a
phosphodiester bond
given an unattached
but adjacent 3'OH
and 5'phosphate.
This can fill in the
unattached gap left
when the RNA primer
is removed and filled
in.
The DNA polymerase
can organize the bond
on the 5' end of the
primer, but ligase is
needed to make the
bond on the 3' end.
The End of Replication
DNA replication stops when the
polymerase complex reaches a
termination site on the DNA in E. coli
The Tus protein binds to the ter site and
halts replication.
In many prokaryotes the replication
process stops when the replication forks
meet
Plasmid replication
ColE1 is a naturally occurring plasmid of E. coli.
Its replication is controlled independently of the
replication of the host chromosome.
Two plasmids with the same origin of replication
can not coexist in the same cell.
The ColE1 origin, defined by molecular genetic
methods, is in a region from which two RNAs are
transcribed.
An active RNase H gene is required for ColE1
replication. RNase H cleaves the RNA II
transcript. The remaining RNA serves as primer
for initiation of replication.
RNA I binds to 5' sequences of RNA II via
pseudoknots and regular complementary pairing.
This binding is stabilized by the ROP or ROM
protein.
The binding prevents changes in the conformation
of RNA II that would otherwise result in RNAse
H cleavage.
Rolling Circle Replication – Occurs in
Conjugation in E. coli.
How can one account for the high fidelity of
replication?
The answer is based on the fact that DNA
Polymerase absolutely requires 3'-OH end of
base-paired primer strand on which to add new
nucleotides.
DNA polymerase III has 3' > 5' exonuclease
activity. It was discovered that DNA polymerase
III actually proofreads the newly synthesized
strand before continuing with replication. When
incorrect nucleotide is incorporated, DNA
polymerase III, by means of the 3' > 5'
exonuclease activity, "backs up" and hydrolyzes
off the incorrect nucleotide. The correct
nucleotide is then added to the chain and
elongation is resumed.
All 3 DNA polymerases have 3'>5' exonuclease
activity
Proofreading ability - 1 error in 10 million
Exonucleases and repair
DNA polymerase I also has 5'>3'
exonuclease activity which removes RNA
primer and 5'>3' polymerase activity
which fills in the gap
This causes a single-stranded break in
the DNA - called a nick
DNA ligase repairs nick by creating a
phosphodiester bond
Genes and Gene
Expression
Genes are written in a code consisting of groups of three
letters called triplets.
There are four letters in the DNA alphabet. There are 64
possible arrangements of the four letters in groups of three
The triplets specify amino acids for the synthesis of proteins
from the information contained in the gene
Genes can also specify t- RNA or r- RNAs
The gene begins with a start triplet and ends with a stop. The
bases between the start and the stop are called an open
reading frame, ORF.
The information in the gene is transcribed by RNA
polymerase.
It reads the gene from 3’ to 5’
The template strand is now referred to as the CRICK strand
and the nontemplate strand is now known as the WATSON
strand
DNA sequences are stored in data bases as the WATSON
strand
Reference - COLD SPRING HARBOR - 2003
Promoters are at the beginning of the
Gene
RNA polymerase recognizes a binding site in front
of the gene. This is referred to as upstream of
the gene.
The direction of transcription is referred to as
downstream
Different genes have different promoters. IN E.
coli the promoters have two functions
The RNA recognition site for transcription which
is the consensus sequence for prokaryotes is
5’ TTGACA3’ ( Watson strand) which means on the
reading strand 3’ AACTGT5’ ( Crick strand)
The Pribnow Box and Shane Dalgarno
The RNA binding site has a consensus sequence of
5’ TATAAT 3’ ( -) and 3’ ATATTA 5’ (+)
This is where the DNA begins to become unwound
for transcription
The initially transcribed sequence of the gene
may not reflect doing but may be a leader
sequence.
The prokaryotes usually contain a consensus
sequence known as the Shane Delgarno which is
complememtary to the 16s rRNA on the ribosome
( small subunit )
The leader sequence also may regulate
transcription
The structure of a
prokaryote gene
Prokaryote Genes are
Continuous
They do not contain introns like
eukaryote genes
The gene consists of codons that will
determine the sequence of amino acids in
the protein
At the end of the gene there is a
terminator sequence rather than an
actual stop
The terminator may be at the end of a
trailer sequence located downstream
from the actual coding region of the gene
The Gene begins with
DNA is read 3’ to 5’ and m RNA is
synthesized 5’ to 3’
3’ TAC is the start triplet
This produces a complementary
mRNA message 5’ AUG 3’ –
Groups of three bases in the
messenger RNA formed are
referred to as CODONS
RNA POLYMERASE
Wobble
•There is wobble in
the DNA code – This
is a protection from
mutations
•More than one codon
can specify the same
amino acid
• Note arginine CGU, CGC,CGA, CGG
all code for arginine –
only the third base in
the codon changes
•There are two
additional codons for
arginine as well AGA
and AGG these
reflect the
degenerate nature of
the code
Codon chart
Genes for t RNAs and r
RNAs
The genes for t RNAs have a
promoter and transcribed leader
and trailer sequence that are
removed prior to their utilization in
translation. Genes coding for tRNA
may code for more than a single
tRNA molecule
The segments coding for r RNAs
are separated by spacer sequencs
that are removed after
transcription.
t-RNA
The acceptor stem
includes the 5' and 3'
ends of the tRNA.
The 5' end is
generated by RNase P
The 3' end is the site
which is charged with
amino acids for
translation.
Aminoacyl tRNA
synthetases interact
with both the
acceptor 3' end and
the anticodon when
charging tRNAs.
The anticodon
matches the codon on
mRNA and is read
3’ to 5’
t- RNA
Found in the cytoplasm
Amino acyl t- RNA synthetase
is an enzyme that enables the
amino acid to attach to t-RNA
Also activates the t- RNA
Clover leaf has a stem for
attachment to the amino acid
and an anticodon on the bottom
of the clover leaf
t- RNA
Common Features
a CCA
trinucleotide at
the 3' end,
unpaired
four base-paired
stems, and
One loop
containing a TpseudoU-C
sequence and
another containing
dihydroU.
tRNA
tRNAs attach to a
specific amino
acid and carry it
to the ribosome
There are 20
amino acids
61 different
codons for these
amino acids and 61
tRNAs
The anticodon is
complementary to
the codon
Binds to the codon
with hydrogen
bonds
Ribosomal genes
Very similar to the structure of
protein genes
tRNA and rRNA genes
The genes for rRNA are also similar to the
organization of genes coding for proteins
All rRNA genes are transcribed as a large
precursor molecule that is edited by
ribonucleases after transcription to yield the
final r RNA products
Ribosomal RNA
Combines with specific
proteins to form ribosomes
Serves as a site for protein
synthesis
Associated enzymes and
factors control the process of
translation
Prokaryote ribosomes
Ribosomes are small, but
complex structures,
roughly 20 to 30 nm in
diameter, consisting of
two unequally sized
subunits, referred to as
large and small which fit
closely together as seen
below.
A subunit is composed
of a complex between
RNA molecules and
proteins; each subunit
contains at least one
ribosomal RNA (rRNA)
subunit and a large
quantity of ribosomal
proteins.
The subunits together
contain up to 82 specific
proteins assembled in a
precise sequence.
Prokaryote ribosomal RNA
Type of
rRNA
Approximat
e number of
nucleotides
Subunit
Location
16s
1,542
30s
5s
120
50s
23s
2,904
50s
Prokaryote ribosomes –
polysomes- the process of
translation
Prokaryote transcription
and translation
Prokaryote transcription and
translation take place in the
cytoplasm
All necessary enzymes and
molecules are present for the
transcription and translation to
take place
Translation
A molecule of messenger RNA
binds to the 30S ribosome
( small ribosomal unit) at the
Shine Dalgarno sequence
This insures the correct
orientation for the molecule
The large ribosomal sub unit
locks on top
The Ribosome
There are four significant
positions on the ribosome
EPAT
When the 5’ AUG 3’ of the
mRNA is on the P site the tRNA with the anticodon,
5’UAG3’ forms a temporary
bond to begin translation
From Gene to
polypeptide
E. Coli Gene Map
Mutations in DNA
May be characterized by their
genotypic or phenotypic change
Mutations can alter the phenotype
of a microorganisms in different
ways
Mutations can involve a change in
the cellular or colonial morphology
Types of Mutations
Conditional mutations are those mutations
that are expressed only under specific
environmental conditions ( temperature)
Biochemical mutations are those that can
cause a change in the biochemistry of the
cell
( these may inactivate a biochemical
pathway)
These mutants are referred to as
auxotrophs because they cannot grow on
minimal media
Prototrophs are usually wild type strains
capable of growing on minimal media
Two types of mutations
Spontaneous mutations – These
occur without a causative agent
during replication
Induced mutations are the result
of a substance referred to as a
mutagen
Cairns reports that a mutant E. coli
strain unable to use lactose is able
to regain its ability to use the
sugar again – should this be
referred to as adaptive mutation?
Hypermutation
One possible explanation is
hypermutation
A starving bacterium has the
ability to generate multiple
mutations with special mutator
genes that enable them to
form bacteria with the ability
to metabolize lactose
This is an interesting theory
still under investigation
Spontaneous mutations
Types
1.
A purine substitutes for a purine or a
pyrimidine substitutes of a pyrimidine. This
type of mutation is referred ta as a transition.
Most of these can be repaired by proofreading
mechanisms
2.
A pyrimidine substituted for by a purine is
referred to as a transversion. These are rarer
due to steric problems in the DNA molecule
such as pairing purines with purines.
3.
Insertions or deletions cause frame shifts –
the code shifts over the number of bases
inserted or deleted
Mutation Types
Erors in replication
due to base
tautomerization
AT and CG pairs are
formed when keto
groups participate in
hydrogen bonds
In contrast enol
tautomers produce AC
and GT base pairing
Spontaneous mutations –
another cause
Depurination
A purine nucleotide can lose its
base
It will not base pair normally
It will probably lead to a
transition type mutation after
the next round of replication.
Cytosine can be deaminated to
uracil which can then create a
problem
Frame Shifts
Additions and
deletions change
the reading frame.
The hypothetical
origin of deletions
and insertions may
occur during
replication
If the new strand
slips an insertion
or addition may
occur
If the parental
slips a deletion
may occur
Mutagenesis
a.
b.
c.
d.
Any agent that
directly damages
DNA, alters its
chemistry, or
interferes with
repair mechanisms
will induce mutations
Base analogs
Specific mispairing
Intercalating agents
Ionizing radiation
Base analogs are structurally
similar to normal nitrogenous
bases and can be
incorporated into the
growing polynucleotide chain
during replication.
The expression of
mutations
Forward mutations – a mutation
from the wild type to a mutant
form is called a forward mutation
Reversion-If the organism regains
its wild type characteristics
through a second mutation
Back mutation – The actual
nucleotide sequence is converted
back to the original
Suppressor mutation – overcomes
the effects of the first mutation
More on mutations
Point mutations – caused by the
change in one DNA base
Silent mutations – mutations can
occur which cause no effect – this
is due to the degeneracy of the
code ( more than one base coding
for the same amino acid)
Missense mutation – changes a
codon for one amino acid into a
codon for another amino acid
Nonsense – In eukaryotes the
substitution of a stop into the
sequence of a normal gene
Detection and isolation of
mutants
Requires a sensitive system
Mutations are rare
One in about every 107 – 1011
Replica plating is a technique that is used
to detect auxotrophs
It distinguishes between wild type and
mutants because of their ability to grow
in the absence of a particular
biosynthetic end product
Replica plating allows plating on minimal
media and enriched media from the same
master plate
The selection of
auxotorph revertants
The lysine auxotrophs
( Lys-) are treated
with a mutagen such
as nitroquanidine or
uv light to produce
revertants
Ames Test
Developed by Bruce Ames
Used to test for carcinogens
A mutational reversion assay
based upon mutants of
Salmonella typhimurium
DNA repair mechanisms
Type I -Excision repair
Corrects damage which causes distortions in the
double helix
A repair endonuclease or uvr ABC endonuclease
removes the damaged bases along with some
bases on either side of thee lesion
The usual gap is about 12 nucleotides long. It is
filled by DNA polymerase and ligase joins the
fragments.
This can remove Thymine-Thymine dimers
A special type of repair utilizes glycosylases to
remove damaged or unnatural bases yielding the
results discussed above
Mutations and repair
Type II – Removal of lesion
Thymine dimers and alkylated bases are often
repaired directly
Photoreactivation is the repair of thymine
dimers by splitting them apart into separate
thymines with the aid of visible light in a
photochemical reaction catalyzed by the
enzyme photolyase
Light repair
-phr gene - codes for deoxyribodipyrimidine
photolyase that, with cofactor folic acid, binds
in dark to T dimer. When light shines on cell,
folic acid absorbs the light and uses the energy
to break bond of T dimer; photolyase then falls
off DNA
Dark repair of
mutations
Dark repair
Three types
1) UV Damage Repair (also called NER - nucleotide
excision repair)
Excinuclease (an endonuclease; also called
correndonuclease [correction endo.]) that can
detect T dimer, nicks DNA strand on 5' end of
dimer (composed of subunits coded by uvrA, uvrB
and uvrC genes).
UvrA protein and ATP bind to DNA at the
distortion.
UvrB binds to the UvrA-DNA complex and
increases specificity of UvrA-ATP complex for
irradiated DNA.
UvrC nicks DNA 8 bases upstream and 4 or 5
bases downstream of dimer.
UvrD (DNA helicase II; same as DnaB used during
replication initiation) separates strands to release
12-bp segment.
DNA polymerase I now fills in gap in 5'>3'
direction and ligase seals.
The Effects of uv light
Post replication repair
If T dimer not repaired, DNA Pol III can't make
complementary strand during replication.
Postdimer initiation - skips over lesion and leaves
large gap (800 bases). Gap may be repaired by
enzymes in recombination system - lesion remains
but get intact double helix.
Successful post replication depends upon the
ability to recognize the old and newly replicated
DNA strands
This is possible because the newly replicated DNA
strand lack methyl groups on their bases, whereas
the older DNA has methyl groups on the bases of
both strands.
The DNA repair system cuts out the mismatch
from the non- methylated strand
Recombination repair
The DNA repair for which there is no remaining
template is restored
RecA protein cuts a piece of template DNA from
a sister molecule and puts it into the gap or uses
it to replace a damaged strand
Rec A also participates in a type of inducible
repair known as SOS repair.
If the DNA damage is so great that synthesis
stops completely leaving many gaps, the Rec A will
bind to the gaps and initiate strand exchange.
It takes on a proteolytic funtion that destroys
the lexA repressor protein which regulates genes
involved in DNA repair and synthesis