Introduction to Microbial Genetics

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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

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Griffiths
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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)

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Results of the Experiment

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

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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

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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

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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

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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

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

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Complementary Base Pairing

• Adenine pairs with Thymine
• Cytosine pairs with Guanine

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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

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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)

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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

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RNA

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

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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

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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

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Coiling

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

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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

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Chromosome structure

• http//www.johnkyrk.com/chromosomestructure.html

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Eukaryote replication

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

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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

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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

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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

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The origin of replication and replication forks
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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.

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DNA Polymerases

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

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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

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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

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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

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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).

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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

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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.

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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.

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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

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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

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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

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DNA polymerase III

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

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Elongation of the chain

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

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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

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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

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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)

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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

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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.

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Rolling Circle Replication Occurs in
Conjugation in E. coli.
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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

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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

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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

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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)

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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

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The structure of a prokaryote gene
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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

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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

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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

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Codon chart
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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

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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

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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.

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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

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Ribosomal genes

• Very similar to the structure of protein genes

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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

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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

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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.    

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Prokaryote ribosomal RNA

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Prokaryote ribosomes polysomes- the process of
translation
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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

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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

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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
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E. Coli Gene Map
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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

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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

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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?

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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

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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

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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

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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

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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

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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.
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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

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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

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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