Download Microbial Genetics

Introduction to
Microbial Genetics
Microbiology 221
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
crystallograph
y picture of
the B form
held the
secret, but she
didn’t realize
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

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
 Three types of RNA
Messenger
b. Transfer
c. Ribosomal
d. nc- non coding RNA’s
a.
Prokaryote DNA
 Tightly coiled
 Coiling maintained by
molecules similar to the
coiling in eukaryotes
 Circular ds molecule
Some Special Cases
 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 and
Supercoiling
 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.
.
Chromosome
structure
 http://www.johnkyrk.com/c
hromosomestructure.html
Eukaryote replication
 The nature
of DNA
replication
was
elucidated
by Meselson
and Stahl
Meselson and Stahl
experiment
1. 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
 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/DNArep
lication.html
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
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-
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
non-replicating 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
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 aphosphate 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
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
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



5’
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
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
t- RNA
 Found in the cytoplasm
 Amino acyl t- RNA synthetase
is an enzyme that enables the
amino acid to attach to tRNA
 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
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
Approxima
te number
of
nucleotide
s
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 t-RNA 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
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
Base analogs are
Intercalating structurally similar to
normal nitrogenous bases
agents
and can be incorporated
Ionizing
into the growing
radiation
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
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