DNA REPAIR

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DNA REPAIR

• BASE EXCISION REPAIR
• NUCLEOTIED EXCISION REPAIR
• MISMATCH REPAIR
• STRAND BREAK REPAIR

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DNA Repair

• Mutations are rare events,
• 1 per 109-1010 base pairs per cell division.
• misincorporation during DNA synthesis yields
  noncomplementary base pairs or mismatches
• Mutations can also arise via incorporation of
  chemically damaged nucleotides or by
  incorporation of an undamaged nucleotide opposite
  a damaged base within the template strand.
• Strand slippage or formation of unusual secondary
  structures within DNA, especially within
  repetitive sequences, can also result in
  mutations when processed aberrantly during
  replication, recombination, or repair.

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MisMatch Repair

• Ravi R. Iyer, Anna Pluciennik, Vickers Burdett,
  and Paul L. Modrich, DNA Mismatch Repair
  Functions and Mechanisms Chem. Rev. 2006, 106,
  302-323.
• Thomas Jascur and C. Richard Boland, Structure
  and function of the components of the human DNA
  mismatch repair system Int. J. Cancer 119,
  20302035 (2006)

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MisMatch Repair

• Genetic inactivation of the mismatch repair
  system elevates spontaneous mutability
  50-1000-fold.
• Mismatch repair defects lead to highly elevated
  rates of base substitution and frameshift
  mutations, permit illegitimate recombination
  between quasi-homologous sequences, and render
  mammalian cells resistant to the cytotoxic
  effects of several classes of DNA damaging
  agents.
• Inactivation of the human mismatch repair system
  is the cause of hereditary nonpolyposis colon
  cancer (HNPCC)and has been implicated in the
  development of a subset of sporadic tumors that
  occur in a variety of tissues.

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Mismatch repair

• MMR system is an excision/resynthesis system that
  can be divided into 4 phases
• (i) recognition of a mismatch by MutS proteins,
• (ii) recruitment of repair enzymes
• (iii) excision of the incorrect sequence,
• (iv) resynthesis by DNA polymerase using the
  parental strand as a template.

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Mismatch Repair in E.coli

• MutS is responsible for initiation of E. coli
  mismatch repair.
• 95 kDa polypeptide, which exists as an
  equilibrium mixture of dimers and tetramers
• recognizes mismatched base pairs.
• MutL, a 68 kDa polypeptide that is dimeric in
  solution, is recruited to the heteroduplex in a
  MutS- and ATP-dependent fashion.
• The MutL MutSheteroduplex complex is believed
  to be a key intermediate in the initiation of
  mismatch repair

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Methyl Directed MisMatch repair in E. coli
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Mismatch repair in humans
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Mammalian MMR is bidirectional
In addition it seeks out the nick to determine
which strand Is the template and which strand is
the synthesized one
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Mismatch repair
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Base excision Repair

• For correction of specific Chemical Damage in DNA
• Uracil
• Hypoxanthine
• 3-m Adenine
• Urea
• Formamidopyrimidine
• 5,6 Hydrated Thymine

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Base excision repair.

• Consist of DNA glycosylases and AP endonuclease
• The DNA glycosylases are specific
• Uracil glycosylase
• Hypoxanthine DNA glycosylase
• Etc

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Mechanism
1.DNA glycosylase recognizes Specific Damaged
base 2. Cleaves glycosl bond to remove Base 3. AP
endonuclease cleaves Backbone 4. DNA Pol removes
abasic site 5. Replacement of Base
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Nucleotide Excision Repair

• Used by the cell for bulky DNA damage
• Non specific DNA damage
• Chemical adducts
• UV photoproducts
• First identified in 1964 in E.coli.

Ludovic C. J. Gillet and Orlando D. Scharer
Molecular Mechanisms of Mammalian Global Genome
Nucleotide Excision Repair Chem. Rev. 2006, 106,
253-276
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Nucleotide Excision Repair

• Defects cause
• Xeroderma Pigmentosum
• 1874, when Moriz Kaposi used this term for the
  first time to describe the symptoms observed in a
  patient.13 XP patients exhibit an extreme
  sensitivity to sunlight and have more than
  1000-fold increased risk to develop skin cancer,
  especiallyin regions exposed to sunlight such as
  hands, face, neck
• Cockayne Syndrome
• Trichothiodystrophy

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Nucleotide Excision Repair

• Defects cause
• Cockayne Syndrome
• A second disorder with UV sensitivity was
  reported by Edward Alfred Cockayne in 1936.
  Cockayne syndrome CS) is characterized by
  additional symptoms such as short stature, severe
  neurological abnormalities caused by
  dysmyelination, bird-like faces, tooth decay, and
  cataracts. CS patients have a mean life
  expectancy of 12.5 years but in contrast to XP do
  not show a clear predisposition to skin cancer.
  CS cells are deficient in transcription-coupled
  NER but are proficient in global genome NER.
• Trichothiodystrophy

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Nucleotide Excision Repair

• Defects cause
• Trichothiodystrophy
• A third genetic disease characterized by UV
  sensitivity, trichothiodystrophy (TTD,
  literally sulfur-deficient brittle hair), was
  reported by Price in 1980. In addition to
  symptoms shared with CS patients, TTD patients
  show characteristic sulfur-deficient, brittle
  hair and scaling of skin. This genetic disorder
  is now known to correlate with mutations in genes
  involved in NER (XPB, XPD, and TTDA genes). All
  of these genes are part of the 10-subunit
  transcription/repair factor TFIIH, and TTD is
  likely to reflect an impairment of
  transcriptional transactions rather than regular
  defect in DNA repair. This disorder is therefore
  sometimes referred as a transcriptional
  syndrome.

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Nucleotide Excision Repair

• Steps for NER
• Recognition of DAMAGE
• Excision of DAMAGE
• Replacement of excised DNA

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(No Transcript)
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Recognition
Model of the NER mechanism. (A) A lesion induces
DNA helix distortion (B) XPC-HR23B detects the
helix distortion and stabilizes the DNA bend (C)
XPC-HR23B recruits TFIIH at the site of the
lesion (D) upon ATP addition, TFIIH unwinds the
DNA helix, until one of its helicase subunit
(here XPD) encounters a chemically modified base
the second helicase subunit (here XPB) goes on
unwinding the DNA to create a 20-bp opened
bubble structure
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Excision
Model of the NER mechansm. (E) RPA, XPA, and XPG
are then recruited to assemble the preincision
complex (F) ERCC1-XPF joins the complex and the
dual incision (5 by ERCC1-XPF and 3 by XPG)
occurs (G) RPA remains bound to the ssDNA and
facilitates the transition to repair synthesis by
Pol d (or ?) supported by RFC and PCNA ligase I
finally seals the nick.
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Double strand break repair

• DNA double-strand breaks (DSBs) are the most
  hazardous lesions arising in the genome of
  eukaryotic organisms, and yet occur normally
  during DNA replication, meiosis, and immune
  system development. The efficient repair of DSBs
  is crucial in maintaining genomic integrity,
  cellular viability, and the prevention of
  tumorigenesis.
• Shaun P. Scott and Tej K. Pandita The Cellular
  Control of DNA Double-Strand Breaks Journal of
  Cellular Biochemistry 9914631475 (2006)

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Double strand break repair
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Homologous Recombination
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Non Homologous end joining
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Genes involved in DSB repair
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In Vitro Mutagenesis

• Site specific mutagenesis
• Zoller and Smith 1987 Methods of Enzymology
  154,329-350
• Won Smith the Nobel prize in 1993 for Chemistry

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In Vitro Mutagenesis
M13 single stranded phase DNA annealed to a
mutagenic Primer and then in vitro synthesize the
remaining DNA
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In Vitro Mutagenesis dut ung
The DNA template is obtained from plasmids or
phage grown in a dut ung mutant of E. coli. The
dut gene encodes dUTPase which normally degrades
dUTP. An elevated concentration of dUTP
accumulates in dut strains, resulting in
incorporation of U in place T at some positions
during DNA replication. The ung gene encodes
uracil N-glycosylase which normally removes U
from DNA. Thus, in the double mutant U is
occasionally incorporated into DNA and this error
is not repaired. Because U has the same base
pairing properties and the same coding properties
as T, incorporation of U into DNA in place of T
is not mutagenic.
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In Vitro Mutagenesis mutS
The mutS gene encodes an essential componant of
the methyl-directed mismatch repair system.
Thus, a mutS mutation in the recipient prevents
repair of the mutant base to the wild-type
sequence, so that after DNA replication about
50 of the plasmids will be mutant and 50 will
be wild-type . An very clever variation of the
mutS approach was developed by scientists at
Promega and is marketed as the "Altered Sites
Kit". A figure of this approach is shown below
and a more detailed description of this
technique can be found on the Promega WWW site.
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In Vitro Mutagenesis PCR