Genetic Material-DNA

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Genetic Material-DNA

• 6 November 2003
• ReadingThe Cell Chapter 5,
• pages 192-201

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DNA Repair

• In the living cell, DNA undergoes frequent
  chemical change, especially when it is being
  replicated. Most of these changes are quickly
  repaired.
• A failure to repair DNA produces a mutation
• The human genome has already revealed 130 genes
  whose products participate in DNA repair.

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Agents that Damage DNA

• Certain wavelengths of radiation
• ionizing radiation such as gamma rays and x-rays
• ultraviolet rays, especially the UV-C rays (260
  nm) that are absorbed strongly by DNA but also
  the longer-wavelength UV-B that penetrates the
  ozone shield.
• Highly-reactive oxygen radicals produced during
  normal cellular respiration as well as by other
  biochemical pathways.

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Agents that Damage DNA

• Chemicals in the Environment
• many hydrocarbons, including some found in
  cigarette smoke
• some plant and microbial products
• Chemicals used in chemotherapy, especially
  chemotherapy of cancers

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Types of DNA Damage

• All four of the bases in DNA (A, T, C, G) can be
  covalently modified at various positions.

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Types of DNA Damage

• Spontaneous damage to DNA.
• One of the most frequent is the loss of an amino
  group ("deamination") - resulting, for example,
  in a C being converted to a U.

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Types of DNA Damage

• Spontaneous damage to DNA.
• Depurination cleavage of the bond between the
  purine bases and the sugar, leaving apurinic site
  (AP) in DNA

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Types of DNA Damage

• DNA damage induced by radiation and chemicals.
• Formation of pyrimidine dimers.

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Types of DNA Damage

• Alkylation addition of methyl or ethyl groups to
  various positions on the DNA bases. Instead of C,
  T is put to complement G.

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Types of DNA Damage

• Reaction with carcinogens many carcinogens
  results in the addition of bulky groups to the
  DNA molecule

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What can be done to repair the damage?
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DNA Repair

• Direct reversal of of the chemical reaction that
  causes DNA damage
• Removal of the damaged base.

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Types of DNA Damage

• DNA damage induced by radiation and chemicals.
• Formation of pyrimidine dimers.

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Direct Reversal of Base Damage

• Pyrimidine dimers
• UV-induced damage causes skin cancers.
• Cyclobutane ring results from the saturation of
  the double bonds between carbons 5 and t.
• Formation of such dimers distort DNA structure
• Photoreactivaton provides energy to break the
  cyclobutane ring. Humans lack this mechanism.

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Types of DNA Damage

• Alkylation addition of methyl or ethyl groups to
  various positions on the DNA bases. Instead of C,
  T is put to complement G.

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Direct Reversal of Base Damage

• Alkylated guanine residues results from exposure
  to alkylating agents.
• They can transfer methyl or ethyl groups to DNA.
• O6 -methylguanine transferase transfers a methyl
  group from DNA to a cysteine residue in its
  active site. Humans have this mechanism.

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Excision Repair

• General means to repair DNA.
• Damaged DNA is recognized and removed as free
  bases or as nucleotides.
• The resulting gap is filled.
• Uracil is occationally incorporated in place of
  Tymine and should be removed.
• Uracil can be formed by deamination of cytosine.

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Base Excision Repair

• Removal of the damaged base. Base excision
  repair. This is done by a DNA glycosylase.
• Removal of its deoxyribose phosphate in the
  backbone, producing a gap.
• Replacement with the correct nucleotide. This
  relies on DNA polymerase ?,
• Ligation of the break in the strand with DNA
  ligase. This requires ATP to provide the needed
  energy.

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Nucleotide excision repair

• Widespread form of DNA repair.
• Damaged bases are removed as part of an
  oligonucleotide containing the lesion.
• UV induced pyrimidine dimers and bulky group
  addition can be repaired by this mechanism.

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Nucleotide excision repair

• The damage is recognized by one or more protein
  factors that assemble at the location.
• Cuts are made on both the 3' side and the 5' side
  of the damaged area so the tract containing the
  damage can be removed.
• DNA synthesis - using the intact (opposite)
  strand as a template - fills in the correct
  nucleotides.
• A DNA ligase covalent binds the fresh piece into
  the backbone

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In E.coli

• Three genes, uvrA, uvrB, uvrC.
• What happens if these genes are mutated?
• The bacteria become highly sensitive to UV (gets
  damaged by it).
• UvrA-recognizes the damaged DNA and recruits UvrB
  and UvrC to the damaged area.
• UvrB and UvrC then cleave the 3 and 5 sides of
  the damaged site.
• UvrABC comples is called exinuclease (excise an
  oligonucleotide).
• Helicase is needed to remove the damaged area
  gap is filled with polymerase and ligase.

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In eukaryotes

• RAD genes (radiation sensitivity) mutants have
  increased sensitivity to UV exposure.
• Inherited diseases that result from deficiencies
  in ability to repair DNA damage.
• Xeroderma pigmentosum (XP)-sensitive to UV,
  develop skin cancers. They cant carry out
  nucleotide excision repair.
• XPA to XPG (seven repair genes) highly homologous
  to yeast RAD genes.

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Mismatch Repair

• Mismatch repair deals with correcting mismatches
  of the normal bases that is, failures to
  maintain normal Watson-Crick base pairing (A.T,
  C.G)
• Many of the mismatched bases are removed during
  replication by the proofreading activity of DNA
  polymerase. Missed ones are subject to mismatch
  repair!!!
• Mutations in either of these genes predisposes
  the person to an inherited form of colon cancer.
  (Do not forget to read the box _at_ page 198.

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How could the mismatched base be understood?
GGTACGATG CCATTCTAC
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Mismatch repair in E. coli

• Scans newly replicated DNA, if found enzymes of
  this system can identify and repair the
  mismatched base from newly replicated DNA.
• In E.coli, methylation indicates parental strand
  Adenine residues in the sequence GATC forms
  6-methyladenine. Methylation occurs after
  replication.

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Mismatch repair in E.coli

• MutS protein initiates repair because it
  recognizes the mismatch and forms a complex with
  two other proteins MutL and MutH.
• MutH is an endonuclease that can cleave the
  unmethylated DNA strand.
• MutL and MutS then excise the DNA between the
  strand break and gap is filled with Pol and
  ligase.

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Mismatch Repair in E.coli
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Mismatch Repair in mammalian cells
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Mismatch repair in mammalian cells

• The old and new strands of DNA is distinguished
  by a different mechanism than methylation.
• Presence of single strand breaks indicate newly
  replicating DNA or associations between MutS and
  MutL homologs also indicate which strand is new.

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Colon Cancer

• Cancers of the colon and rectum (colorectal
  cancers).
• 140,000 cancer cases per year (10 of total
  cancer cases).
• Mostly non inherited.
• Inherited cases
• Familial adenomatous polyposis (rare, 1)
• Heretidary nonpolyposis colorectal cancer (15).

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Molecular Basis

• Mutated genes involved in cell proliferation,
  leading to uncontrolled growth.
• Mutations occur sporadically in somatic cells.
• In hereditary cases, inherited germ-line
  mutations predispose the individual to cancer.

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The gene

• Human homology of E.coli MutS gene involved in
  mismatch repair of DNA is responsible for 50 of
  HNPCC.
• Three other genes also involved in repair may be
  responsible.
• Defects in these genes result in high frequency
  of mutations in other cells.

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Symptoms

• Development of the outgrowth of small benign
  polyps, which eventually become malignant.
• Polyps can be removed surgically. Early
  diagnosis is important.

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Postreplication Repair

• Recombinational repair relies on replacement of
  damaged DNA by recombination with an undamaged
  molecule.
• Happens during replication.

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Recombinational Repair

• Normal replication is blocked with a TT dimer.
• Downstream of the damage replication goes on.
• Undamaged parental strand (which has been
  replicated) is then used as a template, new
  strand is synthesized based on this.
• TT dimer later is dealth with an excision repair
  mechanism.

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Double strand breaks

• X-rays induce double strand breaks on the
  chromosomes.
• Ligate the ends of the chromosomes (risky,
  possible errors (loss of bases at the ends).
• Homologous recombination provides new templates
  at the site of the double strand break.

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Error-prone repair

• Reversal and excision repair systems act to
  correct DNA damage before replication.
• Replicative DNA synthesis requires an undamaged
  DNA strand as a template.
• What about the damage at the replciation fork,
  when TT dimers for example block the replication.
• Cells have specialized Polymerases to replicate
  across a damaged site but these polymerases lead
  to a lot mistakes.

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Error-prone polymerases

• In E. coli Polymerase V is induced in response to
  UV irradiation and can synthesize a new DNA
  strand across from a thymine dimer.
• E. coli Pol II and Pol IV are induced by DNA
  damage.
• Characteristically error-prone DNA polymerases
  exhibit low fidelity (100 to 10,000 times higher
  than replicative polymerases E.coli PolII and
  eurkaryotic epsilon).
• Error prone polymerases lack 3? 5 proofreading
  activity.