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Introduction to Microbial Genetics

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Title: Introduction to Microbial Genetics


1
Introduction to Microbial Genetics
  • Microbiology 221

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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 CG
  • Maurice Wilkins and Rosalind Franklin x-ray
    crystallography of DNA
  • Watson and Crick Double helix

4
Griffiths
5
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

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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)

8
Results of the Experiment
  • Proved that the radioactivity from the labeled
    DNA was present in the progeny phage produced
    from infection of the bacteria.

9
The Race for the Double Helix
  • Rosalind Franklin and Maurice Wilkins at Kings
    College
  • Studied the A and B forms of DNA
  • Rosalinds famous x-ray crystallography picture
    of the B form held the secret, but she didnt
    realize its significance

10
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

11
Nucleic Acid CompositionDNA 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

12
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 3hydroxyl, the other the 5 hydroxyl of the
    sugar molecule

13
DNA Structure
http//www.johnkyrk.com/DNAanatomy.html - DNA
structure
14
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

15
Complementary Base Pairing
  • Adenine pairs with Thymine
  • Cytosine pairs with Guanine

16
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

17
Double helix and anti-parallel
  • 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)

18
RNA structure
  • Polynucleotide nucleic acid - Single stranded
    molecule that can coil back on itself and produce
    complementary base-pairing ( t- RNA)
  • Four bases in RNA are Adenine and Guanine (
    purines) and Cytosine and Uracil( pyrimidines)
  • Sugar ribose
  • Phosphates

19
RNA
  • Three types of RNA
  • Messenger
  • Transfer
  • Ribosomal
  • nc- non coding RNAs

20
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

21
Prokaryote chromosomes
  • Circular DNA

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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

24
Coiling
  • Coiling maintained by Gyrase
  • Relaxation of the coils by Topoisomerase

25
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.
  • .

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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

28
Chromosome structure
  • http//www.johnkyrk.com/chromosomestructure.html

29
Eukaryote replication
  • The nature of DNA replication was elucidated by
    Meselson and Stahl

30
Meselson and Stahl experiment
  • Grew bacteria in heavy Nitrogen N-15
  • Transferred bacteria to N-14
  • Before bacteria reproduce in new media, all
    bacteria contain heavy DNA
  • Samples were taken after one round of replication
    and two round of replication

31
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

32
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.html

33
The origin of replication and replication forks
34
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.

35
DNA Polymerases
  • Fourteen DNA polymerases have been observed in
    human beings as compared to three in E. coli.

36
Prokaryote Replication
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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

39
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

40
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

41
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).

42
Initiation
  • Initiationa. oriC - origin of chromosomal
    replicationRecognized by DnaA protein - only
    recognizes if GATC sites are fully
    methylatedBinding of DnaA allows DnaB to open
    complexb. DnaB is the replication helicasec.
    Strand separation by helicased. SSB
    (single-stranded binding) protein keeps strands
    aparte. DNA gyrase - a topoisomerase - puts
    swivel in DNA which allows strands to rotate and
    relieve strain of unwinding

43
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.

44
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.

45
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

46
Formation of the Primer
  • Primosome contains primase
  • Primosome moves along DNA duplex in 3'gt5'
    direction (with respect to lagging strand
    follows replication fork) even though primer is
    made in 5'gt3' direction(Note The symbol "gt"
    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 primasePrimase
    forms the primer

47
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

48
DNA polymerase III
  • This is a stationary complex that probably
    attached to the plasma membrane.
  • The DNA moves through the replisome and is copied

49
Elongation of the chain
  • dCTP dCMP
  • PPi
  • Energy is supplied for biosynthesis by the
    cleaving of the phosphate bond

50
Elongation( continued)
  • Elongation proceeds in 5' gt 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
    secondRate of formation of initiation complex
    1-2 minutes

51
Elongation
  • ElongationDNA polymerase I, II and III in E
    .coliDNA 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 ltgt (DNA)n 1
    residues PPiAttachment of new units is by
    their a-phosphate groups to a free 3'-hydroxyl
    end of preexisting DNA chain.

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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

55
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)

56
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.

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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

60
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.

61
Rolling Circle Replication Occurs in
Conjugation in E. coli.
62
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' gt 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' gt 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'gt5' exonuclease
    activity
  • Proofreading ability - 1 error in 10 million

63
Exonucleases and repair
  • DNA polymerase I also has 5'gt3' exonuclease
    activity which removes RNA primer and 5'gt3'
    polymerase activity which fills in the gap
  • This causes a single-stranded break in the DNA -
    called a nickDNA ligase repairs nick by creating
    a phosphodiester bond

64
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

65
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)

66
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

67
The structure of a prokaryote gene
68
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

69
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

70
RNA POLYMERASE
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  • 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

73
Codon chart
74
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.

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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

77
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

78
t- RNA
  • Common Features
  • a CCA trinucleotide at the 3' end, unpaired
  • four base-paired stems, and
  • One loop containing a T-pseudoU-C sequence and
    another containing dihydroU.

79
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

80
Ribosomal genes
  • Very similar to the structure of protein genes

81
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

82
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

83
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.    

84
Prokaryote ribosomal RNA

85
Prokaryote ribosomes polysomes- the process of
translation
86
Prokaryote transcriptionand 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

87
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

88
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, 5UAG3 forms a
    temporary bond to begin translation

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From Gene to polypeptide
93
E. Coli Gene Map
94
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

95
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

96
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?

97
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

98
Spontaneous mutations
  • Types
  • 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
  • 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.
  • Insertions or deletions cause frame shifts the
    code shifts over the number of bases inserted or
    deleted

99
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

100
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

101
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

102
Mutagenesis
  • 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.
103
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

104
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

105
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

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The selection of auxotorph revertants
  • The lysine auxotrophs ( Lys-) are treated with a
    mutagen such as nitroquanidine or uv light to
    produce revertants

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Ames Test
  • Developed by Bruce Ames
  • Used to test for carcinogens
  • A mutational reversion assay based upon mutants
    of Salmonella typhimurium

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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

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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

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Dark repair of mutations
  • Dark repairThree types1) 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'gt3' direction and ligase
    seals.

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The Effects of uv light
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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

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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
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