Mutation and DNA Repair

Mutations are Random

• Mutations happen all the time (not in response to selective agent)
• Luria and Delbruk used what they called the "Fluctuation Test" to show that many mutations for phage T1 resistance were present in the culture before phage was added.
• • Evidence was variation in the number of resistant colonies in the different replicate cultures.

Various kinds of mutations:

• Point mutations
• • Transitions (purine->purine, pyrimidine -> pyrimidine)
  • Transversions (purine -> pyrimidine)
• Point mutations can be
• • silent (same amino acid)
  • neutral (similar amino acid, so protein functions normally)
  • missense (different amino acid)
  • nonsense (stop)
• Additions/Deletions cause frameshift mutations
• • (Chromosome rearrangements will be the subject of next lecture)

See Fig. 19.3

Reverse mutation (back mutation)

• Mutation changes mutant to wild type.
• Reversion to the wild type amino acid restores function.
• Reversion to another amino acid partly or fully restores function.

Suppressor mutation

• Occur at sites different from the original mutation and mask or compensate for the initial mutation without reversing it.
• Intragenic suppressors occur in the same gene
• • e.g., nearby addition restores a deletion
• Intergenic suppressors occur on a different gene
• • e.g. tRNA anticodon mutation can suppress a mutation to a stop codon

Sources of mutation

DNA replication errors

• Wobble-pairing (mis pairing of bases)
• • T-G, C-A, A-G, T-C
  • Normal pairing typically occurs in the next round of replication; frequency of mutants in F2 is 1/4.
• Additions and deletions
• • DNA loops out on template strand, DNA polymerase skips bases, and deletion occurs.
  • DNA loops out on new strand, DNA polymerase adds untemplated bases.

Spontaneous chemical changes

• Depurination
• • Common; A or G are removed and replaced with a random base.
• Deamination
• • Amino group is removed from a base (C ® U); if not replaced U pairs with A in next round of replication (CG ® TA).
  • Prokaryote DNA contains small amounts of 5MC; deamination of 5MC produces T (CG ® TA).
  • Regions with high levels of 5MC are mutation hot spots.

See Fig. 19.9, Deamination

.

Induced mutations: chemical mutagens

• Mutagenic intercalating agents (e.g., ethidium bromide) can cause insertions during DNA replication.
• Loss of intercalating agent can result in deletion.

Induced mutations: radiation

• Can cause breaks in DNA
• Can cause crosslinking of pyrimidines (especially T-T dimmers)

Cells have lots of repair mechanisms

• Proofreading
• Mismatch repair
• Base excision

Effectiveness of repair mechanisms

Normal polymerase error rate about 1/100,000

Mutations in repair enzymes increase error rate 1000x

We briefly reviewed Proofreading of DNA polymerase

Mismatch repair (MMR)

• Prokaryotes and eukaryotes use a similar mechanism with common structural features
• Defects in mismatch repair underlie human predisposition to colon and other cancers
• G:T and A:C mispairs and one base insertions are particularly well-recognized (they are also the most common polymerase errors)
• Short section of newly replicated DNA containing a mutation is removed and a new strand is re-synthesized using the template

How does it know which base is incorrect?

• Uses methylation pattern to identify the old strand; then cuts out the new strand
• In E. coli, the A in GATC is often methylated
• (Only works if repair occurs in the short time between synthesis and methylation of the new strand)

Base excision repair (BER)

• Major pathway for repair of modified bases, uracil misincorporation, oxidative damage
• recognize lesion and remove base from sugar backbone, thereby producing an "empty" site
• Then the sugar is cut out and patch refilled by DNA synthesis and ligation
• Types of lesions repaired by BER
• • Oxidative lesions; 8-oxo-G, highly mutagenic, mispairs with A, producing GC --> TA transversions
  • Deoxyuracil: from misincorporation of dU or deamination of dC-->dU
  • Spontaneous depurination (esp. G) yield empty sites that are repaired by second half of BER pathway

Pyrimidine dimers

• UV radiation can cause crosslinking between adjacent T's; Forms a characteristic bend in DNA
• Xeroderma pigmentosum (a human skin cancer) is caused by defect in repair of pyrimidine dimers

Nucleotide excision repair (NER)

• Recognizes bulky lesions that block DNA replication
• Incision on both sides of lesion
• Short patch of DNA excised, repaired by repolymerization and ligation
• In E. coli, mediated by UvrABCD
• Many more proteins involved in eukaryotes

Note that all of the different repair pathways do basically the same thing: they recognize a mis-paired base by the change in shape of DNA, cut out a short stretch of 1 to several nucleotides, and then refill the gap with DNA polymerase. Each repair pathway has its own set of enzymes, and they recognize slightly different kinds of DNA damage, but the basic mechanism is similar.

In your studying, concentrate on the general features of these repair pathways, not the specific details.