1. Mutation Point Mutations Repair of “point” mutations
Covered Spontaneous and Induced last time
Ames Test for Mutagens (because some mutagens = carcinogens)
Repair of “point” mutations
Transposon as mutagens

2. Example Results of the Ames Test
Ames test measures rate that mutagen reverts mutant phenotype to wild type
Wild type Salmonella typhimurium can make histidine, so it can grow on medium lacking histidine (histidine protoroph).
S. typhimurium mutants contain different types of mutations in a His biosynthesis gene, they cannot make histidine and cannot grow on medium lacking histidine (histidine auxotrophs).
Part of wild type His+ amino acid sequence: Cys His Ala Ser Met
Part of wild type His+ gene sequence: TGT CAC GCC TCT ATG
TA100 [His to Arg missense (nonconserv. substitution)] TGT CGC GCC TCT ATG
TA1538 [frameshift mutation] TGT TCA CGC CTC TAT G
His+ revertant of TA 1538 (pseudorevertant) TGT TCC GCC TCT ATG
TA w [Ser to Pro missense (nonconserv. substitution] TGT CAC GCC CCT ATG
TA x [Cys to Tyr missense (noncoserv. substituion] TAT CAC GCC TCT ATG
TA y [Met to Ile missense (conservative substitution] TGT CAC GCC TCT ATA
TA z [Cys to Trp missense (nonconserv. substitution] TGG CAC GCC TCT ATG
Possible mechanisms of mutation from His+ to His- = ?
Revertants = His- strains mutated back to His+ phenotype
True Revertants: change back to the wild type sequence (G back to A in TA100)
Pseudorevertants: change that compensates for first mutation without fixing it (suppressor mutation)
A. deletion of A in TA1538 fixes frame shift, results in a conservative substitution
B. a second missense mutation in TA100 ( TA x or TA y or TA z) that allows protein to fold properly
C. a mutation in a different gene - not relevant to Ames test.

3. Repair Processes Direct Reversal of DNA lesion
photolyase repair of UV lesions
alkyltransferase repair of alkylations
Homology-dependent repair processes
Pre-replication repair
General nucleotide excision repair of bulky lesions
pyrimidine dimers, insertion or deletions loops
defects cause Xeroderma pigmentosa
Nucleotide excision repair starting with specific glycosidases
specific base modifications, removal of Uracil
Post-replication repair
Mismatch repair
Defects cause Hereditary Nonpolyposis Colon Cancer
DNA replication at sites of DNA damage
Recombinational repair (error free)
SOS system (error prone)

4. Light-dependent repair by photolyase
When inducing mutations using UV – keep cells in the dark until after DNA replication in order to avoid this

5. Base-excision repair
Base-excision repair and general nucleotide excision repair are similar after the initial steps
Both involve exonucleolytic removal of many nucleotides on either side of the lesion
General nucleotide excision repair can remove bulky lesions pre-replication
Base-excision repair differs in that more subtle lesions can be recognized
- C to U by deamination
- damaged bases
- each base is removed by a specific glycosylase
- then AP endonuclease…
- then exonucleolytic removal and fill in synthesis – see next slide

6. Base-excision repair

7. Mismatch excision repair
Mutations in mismatch repair genes contribute to development o f colon cancer
Mismatch is repaired to the correct sequence by recognizing methylated vs nonmethylated DNA
Step 3 and 4 are similar to other excision repair pathways

8. Assay of UV sensitivity of cells cultured from Xeroderma Pigmentosa patients
Each cell line is a different complementation group
- complementation groups defined by testing heterokaryons for UV sensitivity
- if a heterokaryon made by fusing Cell A with Cell B (A B heterokaryon) is UV sensitive, then A and B have mutations in the same comp. group (same gene).
- if heterokaryon is no longer UV sensitive, then mutations are in different complementation groups (different genes)
Many of these mutation have been mapped to a unique chromosomal location, confirming different complementation groups = different genes
Genes identified with XP groups related to excision repair genes important for removing pyrimidine dimers due to UV light.

9. Getting past a replication block by Recombinational Repair
RecA protein recognizes ss DNA region skipped during replication
Can’t use damaged strand as template
Promotes recombination event removing strand from other replication fork
Template exists to replace this stand of DNA
Lesion causing the block is still present and must be removed (excision repair)

10. Error-prone SOS-mediated repair
If block to replication is not fixed by recombinational repair – then repair or die pathway kicks in – SOS repair
when SOS vs recombinational? High levels of damage, defects in recombination repair…
Random sequence is added to the new strand opposite the DNA lesion

11. Transposons as mutagens
ALSO:
Insertion of TN near a Gene can cause Change regulation Of gene expression
different from inactivation of regulatory sequence
here, adding new regulatory sequences (from the TN)
For example, can cause gene to be expressed in cell types that normally do not express the gene – gain of function mutation

12. Transposons DNA transposons TNs Retrotransposons
Transpose through DNA – no RNA intermediate
Contain inverted repeat sequences at end
Autonomous TNs encode transposase
Mechanims of transposion: conservative or replicative
10-20 bps are duplicated at the site of insertion
Can identify sites of TN insertions even when TN is no longer there
Nonautonomous TNs contain repeats but don’t encode active transposase
Retrotransposons
Transpose through an RNA intermediate (require reverse transcriptase)
Contain direct repeats at ends that contain transcriptional signals
Can be autonomous or nonautonomous
Major class of TNs in human genome

14. Example showing duplication of “target site” = site of insertion

15. Transposons (repetitive DNA elements)
Mobile Genetic Elements in Humans
DNA transposons
transpose either by conservative or replicative mechanism
many defective copies exist
most (all?) are now inactive
retrotransposons (move by reverse transcription)
LINEs (long interspersed elements)
6 to 8 kb, 20 to 40 thousand copies/genome
Autonomous elements encode all proteins required for transposition
SINEs (short interspersed elements)
Deletion derivates from LINEs
Alu elements (AluI = restriction enzyme site)
similar to 7SL RNA = part of SRP
Pseudogenes
SINEs, Alu, and pseudogenes are thought to use reverse
transposition proteins of LINE elements
Autonomous vs Nonautonomous Transposition
autonomous transposons encode their own transposition enzymes
One type of LINE element in humans is still active
- example of a hemophilia caused by an apparent + of a LINE element into a X-linked blot clotting factor gene during gametogenesis in a woman
An active SINE element = Alu element -