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DNA Damage and Repair. Why do we care? Genetic diseases Cancer. Cellular Responses to DNA Damage. Reversal of DNA Damage Enzymatic photoreactivation Ligation of DNA strands Repair of photoproduct Tolerance of DNA Damage

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slide1

DNA Damage and Repair

  • Why do we care?
    • Genetic diseases
    • Cancer
cellular responses to dna damage
Cellular Responses to DNA Damage
  • Reversal of DNA Damage
    • Enzymatic photoreactivation
    • Ligation of DNA strands
    • Repair of photoproduct
  • Tolerance of DNA Damage
    • Replicative bypass of template damage with gap formation and recombination (gap repair)
  • Excision of DNA Damage
    • Base excision repair
    • Nucleotide excision repair
    • Mismatch repair
slide4

Mutagens and Carcinogens

  • Essentially all mutagens are carcinogens
  • Most carcinogens are mutagens
slide5

Somatic vs. germ line mutations

  • Somatic mutations can lead to cancer
  • Germ line mutations can lead to birth defects
  • Most mutations cause neither
    • Some fall in non-coding DNA
    • Others are silent
slide7

Types of substitutions

  • Missense
    • Results in an amino acid substitution
  • Nonsense
    • Results in a stop codon (TAG, TAA, TGA)
  • Same sense
    • No effect (silent mutation)
slide9

Types of mutations

  • Multisite
  • Point mutations
slide10

Multisite mutations

  • Cause gross chromosome abnormalities
  • Involve large regions of DNA
  • Arise during meiosis
slide11

Types of multisite mutations

  • Inversions: ACBDEF
  • Duplications ABCDEEF
  • Deletions: ABCDF
  • Insertions: ABCDSEF
  • Substitutions: ATCDEF
slide12

Point mutations

  • Involve only one or a few nucleotides
  • Arise during DNA replication
  • Require two errors
    • An error during DNA replication
    • Failure to correct that error
slide13

Types of point mutations

  • Substitutions: GATC CATC
  • Insertion: GATC GGATC
  • Deletion: GATC GTC
  • Duplication: GATC GAGATC
  • Inversion: GATC GTAC
slide14

What is the first defense against mutations?

  • 3’ to 5’ exonuclease activity of the polymerases
slide15

Natural causes of mutations

  • Base tautomerization
  • UV damage
  • Spontaneous deamination
slide19

Spontaneous deamination

  • Three of the four bases have exocyclic amino groups
  • Adenosine produces hypoxanthine
  • Guanine produces xanthine
  • Cytosine produces uracil
slide25

Answer

  • The reason cells use thymine in their DNA
    • Is to allow recognition of uracil formed from cytosine
  • But what about RNA?
    • RNA is short lived and in many copies.
slide26

Chemical mutagens

  • Chemicals that accelerate the deamination reaction
  • Base analogues
  • Alkylating agents
  • Intercalation agents
slide27

Base analogues

  • 5-bromouracil
  • Goes in as T
  • Can base pair with A but also G to a smaller degree
slide29

Intercalation

  • Flat aromatic compounds
  • Acridine dyes
  • Ethidium bromide
  • Cause frame-shifting
slide30

Repair mechanisms

  • We are exposed to mutagens all the time
    • you would expect repair mechanisms to exist
  • A number of different repair mechanisms do exist
slide31

Repair Mechanisms

  • In mismatch repair
    • Incorrect base is identified
    • On short section of a newly synthesized DNA
    • Removed, and replaced
      • by DNA synthesis directed by the correct template.
  • In excision repair
    • bulky lesions in DNA
      • exposure to UV light
      • removed by specialized nuclease systems
      • DNA polymerase fills gap
      • DNA ligase joins the free ends.
slide33

Intro to DNA Mismatch Repair

  • Mismatch Repair Genes
    • recognition and repair of certain types of DNA damage or replication errors
  • Function to help preserve the fidelity of the genome
    • through successive cycles of cell division
slide34

Mismatch repair

  • Occurs just after replication
  • Improves accuracy 102 - 103 fold
  • Must distinguish the parent from the daughter strand
slide35

History of MMR

  • System first discovered in bacteria
  • Partially homologous system in yeast
  • Marked homology between yeast and higher order organisms
  • Human MMR genes first described 1993.
slide36

DNA Mismatches

  • Damage to nucleotides in ds-DNA
  • Misincorporation of nucleotide
  • Missed or added nucleotides
slide37

Acquired DNA Damage

M

-C-A- -T-A-

-G-T- -G-T-

Demethylation

slide38

Nucleotide Misincorporation

-C-A-G-C-T-

-G-T-C-C-A-

CT substitution

-C-A-G-C-T-

-G-T-T-C-A-

-C-A-G-C-T-

-G-T-C-C-A-

-C-A-G-C-T-

-G-T-C-C-A-

correctly copied

slide39

nucleotide added

-C-A-G-C-T-

-G-T-C C-A-

-C-A-G-C-T-

-G-T-C-C-A-

A

correctly copied

Added Nucleotides

-C-A-G-C-T-

-G-T-C-C-A-

-C-A-G-C-T-

-G-T-C-C-A-

slide40

Mismatch Repair Genes

  • Recognition and repair of mismatches
  • Other functions
    • Repair of branched DNA structures
    • Prevent recombination of divergent sequences
    • Direct non-MMR proteins in nucleotide excision and other forms of DNA repair
    • MSH4 & MSH5 involved (with MLH1) in meiotic crossover
slide41

Human Mismatch Repair Genes

  • MLH1 (3p21)
  • PMS1 (2q31-33)
  • PMS2 (7p22)
  • MSH2 (2p16)
  • MSH3 (5q3)
  • MSH6 (2p16) (=GT Binding Protein)
slide42

Mismatch Repair Function

  • MMR Proteins combine as heterodimers
  • Recognise and bind mismatches
  • ATP consumption
  • Recruit other proteins
  • Separate, destroy and resynthesise new DNA strand
  • Mechanism works for up to 20 base pairs
slide43

MSH Protein Complexes

  • MutS (MSH2-MSH6)
    • GT mispairs and short (1 base pair) loops/deletions
  • MutS (MSH2-MSH3)
    • Larger mispair loops and deletions
  • Some overlap in function
  • MSH2 loss is greater cancer risk
slide44

MLH Protein Complexes

  • MutL (MLH1-PMS2)
  • MutL (MLH1-PMS1)
    • No established function
    • Can bind other MMR proteins, MSH heterodimers and replication factors
  • As for MSH2, overlap means loss of MLH1 confers the greater cancer risk
slide45

Other MMR Proteins

  • DNA ligase
  • Replication protein A
  • Replication factor C
  • Proliferating Cell Nuclear Antigen
  • Exonucleases
  • DNA polymerase 
slide46

Defective Mismatch Repair

  • Defects in MMR Genes and Function
  • Microsatellite Instability
  • Cancer development
slide47

Defects in MMR Genes

  • Control sequences  Nonexpression
  • Premature stop codon  Truncated protein
  • Point mutations  Altered sequence
  • Insertions/Deletions  Frameshift effects
  • Somatic loss of second allele
slide48

Microsatellite Instability

  • Simple nucleotide repeat sequences
  • Length should be stable at any one locus
  • Poly-A and poly-CA repeat sequences particularly prone to mismatch errors
  • Alterations in length are a sign of deficient mismatch repair
  • Also called RER (Replication ERror)
slide49

Microsatellite Instability

-C-A-C-A-C-A-C-A-

-G-T-G-T-G-T-G-T-

shortened repeat

-C-A-C-A-C-A-

-G-T-G-T-G-T

-C-A-C-A-C-A-C-A

-G-T-G-T-G-T-G-T

-C-A-C-A-C-A-

-G-T-G-T G-T-

CA skipped

G-T

heteroduplex results

slide50

MI Positive Tumours

  • 90% of HNPCC colorectal cancers
  • 20% of sporadic colorectal cancers
  • 30% of sporadic uterine cancers
slide51

Cancer Development

  • Activation of Oncogenes
  • Inactivation of Tumour Suppressor Genes
  • Repeat sequences are common in both of these classes of gene
  • Susceptible to mutation
slide52

Cancer Genes

  • Cell Cycle Control
  • Cellular differentiation
  • Cell Death
  • Other Genes
slide53

Summary

  • Inherited mutations of MMR genes lead to high relative and absolute risk of cancer
  • Colorectal and endometrial cancers
  • Cancer at early age
  • High risk of further cancers
  • Identification of families and individuals
    • Amsterdam criteria
amsterdam criteria
Amsterdam criteria
  • One patient should be a first degree relative of the other two
  • At least two successive generations should be affected
  • At least one tumor should be diagnosed <50 years of age
  • Tumors should be verified by histopathology
slide56

How to distinguish the mother and daughter strands

  • Strands are methylated at GATC sequences on A
slide59

What happens when mismatch repair fails in humans?

  • Missing enzymes homologous to MutS and MutL
  • Patients usually die by age 30
  • Disease: hereditary nonpolyposis colorectal cancer (HPCC)
  • 1 in 200 people affected
excision repair in eukaryotes1
Excision Repair in Eukaryotes
  • Mechanism responsible for most of the DNA repair in eukaryotes
  • Damaged or incorrect bases are excised from the DNA and replaced with the correct nucleotide
  • 5-step pathway
base excision repair
Base excision repair
  • Base excision involves removal of DNA damage such as:
    • Deaminated bases
    • Alkylated or oxidized bases
    • Bases with open rings
    • Apurinic/apyrimidinic sites
  • Pathway removes the types of DNA damage that occur spontaneously in all living cells
steps in excision repair
Steps in excision repair
  • Damage recognition
  • Incision of the lesion containing the DNA strand on both sides of the lesion
  • Excision of the damaged nucleotide(s)
  • Synthesis of new DNA by a DNA polymerase using the complementary strand as template
  • Ligation
slide68

Direct repair (Nucleotide excision repair)

  • Pyrimidine dimers
    • Repaired only in the presence of light
    • Requires a DNA photolyase enzyme
  • M6Guanine
    • Requires a methyl transferase
slide69

Chemically modified bases, such as thymine-thymine dimers, are corrected by nucleotide excision repair

slide70

DNA damage and repair and the role in carcinogenesis

  • A DNA sequence can be changed by copying errors introduced by DNA polymerase during replication and by environmental agents such as chemical mutagens or radiation
  • If uncorrected, such changes may interfere with the ability of the cell to function
  • DNA damage can be repaired by several mechanisms
  • All carcinogens cause changes in the DNA sequence and thus DNA damage and repair are important aspects in the development of cancer
  • Prokaryotic and eukaryotic DNA-repair systems are analogous
slide73

Chemical carcinogens react with DNA directly or after activation, and the carcinogenic effect of a chemical correlates with its mutagenicity

slide74

Mutations in the DNA repair machinery

  • Almost all mutations in genes encoding repair proteins are lethal
  • There are a few rare exceptions to this and the best characterized is Xeroderma pigmentosa
slide75

Xeroderma pigmentosa

  • Rare skin disease in humans
  • Patients are very sensitive to sunlight
  • Usually they develop skin cancer and die before the age of 30
  • Frequent neurological abnormalities
slide76

Distribution of Xeroderma pigmentosa

  • 1 in 100,000 in Europe and the US
  • 1 in 40,000 in Japan
slide77

What causes Xeroderma pigmentosa?

  • Autosomal recessive trait
  • Fibroblasts from patients are unable to excise pyrimidine dimers
  • In one case, cells were missing the endonuclease activity
  • Mutations in seven other genes will cause the same effect
slide78

Mutations and Cancer

What is the evidence that many cancers arise from mutations -- usually multiple mutations?

  • Cancers are common in animals with damaged DNA repair machinery
  • Most cancers arise as clones of a single cell
  • Mutagens are carcinogenic
  • Most carcinogens are also mutagenic