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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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Presentation Transcript
- 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
- Replicative bypass of template damage with gap formation and recombination (gap repair)
- Excision of DNA Damage
- Base excision repair
- Nucleotide excision repair
- Mismatch repair
- Essentially all mutagens are carcinogens
- Most carcinogens are mutagens
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
- Missense
- Results in an amino acid substitution
- Nonsense
- Results in a stop codon (TAG, TAA, TGA)
- Same sense
- No effect (silent mutation)
- Multisite
- Point mutations
- Cause gross chromosome abnormalities
- Involve large regions of DNA
- Arise during meiosis
- Inversions: ACBDEF
- Duplications ABCDEEF
- Deletions: ABCDF
- Insertions: ABCDSEF
- Substitutions: ATCDEF
- Involve only one or a few nucleotides
- Arise during DNA replication
- Require two errors
- An error during DNA replication
- Failure to correct that error
- Substitutions: GATC CATC
- Insertion: GATC GGATC
- Deletion: GATC GTC
- Duplication: GATC GAGATC
- Inversion: GATC GTAC
What is the first defense against mutations?
- 3’ to 5’ exonuclease activity of the polymerases
- Base tautomerization
- UV damage
- Spontaneous deamination
Generation of a mutation by the adenine tautomer- About every 1 in 104 bases
- Three of the four bases have exocyclic amino groups
- Adenosine produces hypoxanthine
- Guanine produces xanthine
- Cytosine produces uracil
- 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.
- Chemicals that accelerate the deamination reaction
- Base analogues
- Alkylating agents
- Intercalation agents
- 5-bromouracil
- Goes in as T
- Can base pair with A but also G to a smaller degree
- Flat aromatic compounds
- Acridine dyes
- Ethidium bromide
- Cause frame-shifting
- We are exposed to mutagens all the time
- you would expect repair mechanisms to exist
- A number of different repair mechanisms do exist
- 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.
- 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
- Occurs just after replication
- Improves accuracy 102 - 103 fold
- Must distinguish the parent from the daughter strand
- System first discovered in bacteria
- Partially homologous system in yeast
- Marked homology between yeast and higher order organisms
- Human MMR genes first described 1993.
- Damage to nucleotides in ds-DNA
- Misincorporation of nucleotide
- Missed or added nucleotides
-C-A-G-C-T-
-G-T-C-C-A-
CT 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
-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-
- 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
- MLH1 (3p21)
- PMS1 (2q31-33)
- PMS2 (7p22)
- MSH2 (2p16)
- MSH3 (5q3)
- MSH6 (2p16) (=GT Binding Protein)
- 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
- 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
- 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
- DNA ligase
- Replication protein A
- Replication factor C
- Proliferating Cell Nuclear Antigen
- Exonucleases
- DNA polymerase
- Defects in MMR Genes and Function
- Microsatellite Instability
- Cancer development
- Control sequences Nonexpression
- Premature stop codon Truncated protein
- Point mutations Altered sequence
- Insertions/Deletions Frameshift effects
- Somatic loss of second allele
- 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)
-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
- 90% of HNPCC colorectal cancers
- 20% of sporadic colorectal cancers
- 30% of sporadic uterine cancers
- Activation of Oncogenes
- Inactivation of Tumour Suppressor Genes
- Repeat sequences are common in both of these classes of gene
- Susceptible to mutation
- Cell Cycle Control
- Cellular differentiation
- Cell Death
- Other Genes
- 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
- 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
How to distinguish the mother and daughter strands
- Strands are methylated at GATC sequences on A
The parental strand is methylated the daughter strand is not yet methylated
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 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 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
- 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
Direct repair (Nucleotide excision repair)
- Pyrimidine dimers
- Repaired only in the presence of light
- Requires a DNA photolyase enzyme
- M6Guanine
- Requires a methyl transferase
Chemically modified bases, such as thymine-thymine dimers, are corrected by nucleotide excision repair
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
Chemical carcinogens react with DNA directly or after activation, and the carcinogenic effect of a chemical correlates with its mutagenicity
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
- 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
Distribution of Xeroderma pigmentosa
- 1 in 100,000 in Europe and the US
- 1 in 40,000 in Japan
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
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
