Molecular Mechanisms of Mutations and DNA Repair

1. Molecular Mechanisms of Mutations and DNA Repair Genetics Spring 2014

2. Outline

3. Types of Mutation and their Molecular Basis • Mutations can be classified in a variety of ways and can be heritable (germ-line) or nonheritable (somatic) leading to a mosaic genotype and phenotype (plant exception): • Origin • Cell type • Expression • Effect on function • Molecular change • Effect on translation Duplication: dynamic mutations

4. Expression • Conditional mutations are expressed in restrictive, but not permissive conditions (ex. temperature-sensitive mutation). • Siamese cats display a form of albinism, temperature-sensitive albinism leading to “point” coloration. • The gene encoding tyrosinase is mutated, not generating melanin in warm temperature.

6. Cell type • Somatic mutations occurs in somatic cells and can lead to mosaicism. • Two populations of cells with different genotypes in one individual who has developed from a single fertilized egg. • Proteus syndrome is an example of such mutation where somatic activating mutation (c.49G→A, p.Glu17Lys) in the oncogene AKT1, encoding the AKT1 kinase, an enzyme that mediates processes such as cell proliferation and apoptosis.

7. Panel A shows severe orthopedic manifestations, including scoliosis, overgrowth with a resultant discrepancy in leg length, and valgus deformity and distortion of the skeleton, in Patient 53. Panels B and C show the characteristic cerebriform connective-tissue nevus and overgrowth and distortion of the hands and feet. Cutaneous vascular anomalies are present on the dorsum of the foot.

8. Nevus Verrucous epidermal nevus: A) Brown verrucous plaques following the Blaschko lines (typo 1b); B) Brown papules and plaques distributed linearly along the Blaschko lines  Giant congenital melanocytic nevus. Plaque pattern, crossing the dorsal and ventral midlines 

9. Heterochromia iridis

10. Molecular change • Dynamic mutations are duplication (amplification) mutations that arise at short or long repeated sequences and are called dynamic mutations because the size of the array is unstable from generation to generation. • Expansion of short (triplet) sequence repeats is associated with several human genetic diseases: myotonic muscular dystrophy, fragile-X syndrome, Kennedy's disease and Huntington's disease.

11. Fragile-X syndrome is caused by very large expansions of CCG repeats outside the coding sequence. • Leading cause of X-linked mental retardation • Once a threshold number of repeats is reached, disease phenotype becomes visible. fragile X‑associated tremor ataxia syndrome (FXTAS).

13. Model: Replication slippage leads to trinucleotide expansion

14. Transposable Elements – Ds/Ac system • Study of the genetics of kernel mottling in maize (Zea mais) revealed a genetic element (Dissociation Ds) involved in mottling but also in chromosome breakage. • Transposition: Ds element is not fixed but moves to new position causing chromosome breakage at new sites. • Ds only moves if a second element (Activator Ac) is present in the same genome. • Transposons are moveable elements that can be mutagenic.

15. The Ac - Ds system in maize: genetics of jumping genes generating plants with a mosaic of cell patches In the presence of an Activator (Ac) element, a Dissociator (Ds) element can cause chromosome breaks. (a) In the absence of Ac element, Ds is not transposable and chromosome breaks do not occur. The W allele is expressed as expected. (b) In the presence of an Ac element, the Ds element can be transposed elsewhere in the chromosome. This transposition can cause a chromosome break at the point of insertion, so that the Ds element and any downstream loci are lost. In a W / W+ heterozygote, loss of the W allele would allow expression of the alternative W+ allele, producing an unexpected dark phenotype (c) Ds transposition can also cause the element to jump into the middle of the W allele, disrupting its normal function and again allowing expression of the alternative W+ allele. Still under the influence of Ac, Ds may subsequently jump back out of W, restoring function of W. The plant will then be a mosaic of cell patches, expressing either W or W+.

16. Transposase moves copy of element; Insertion creates short direct repeats flanking the transposition.

17. Transposable Elements – LTR and non-LTR Retrotransposons • LTR retrotransposons (Long terminal repeat) have terminal direct repeats present in in the same or reversed orientation at both ends of the element. • LTR retrotransposons transpose through an RNA intermediate where reverse transcriptase uses the RNA intermediate as a template for cDNA synthesis. • Non-LTR retrotransposons such as LINEs and SINEs do not have terminal repeats. The life cycle of LTR retrotransposons. IN, integrase; PR, protease; RT, reverse transcriptase; VLP, virus-like particle. Black triangles represent the LTRs.

18. The human genome has many transposable elements About 50% of genomic DNA is transposable elements They don’t move much anymore

19. Spontaneous Mutations • Mutations are statistical random events, which are increased by environmental agents (UV, chemicals, viruses) or mutations of repair genes. • Mutation rate can be calculated (event/109 bp/min -> 1 x 10-9 per minute = 10,000 damaged sites per cell per 24 hours). • Whether a mutation happens is unrelated to any adaptive advantage it may confer (a mutation does not arise because an organism needs it). • Mutations can be repaired. • Selective techniques select mutants already present in a population. Replica Plating: For the detection of mutants, cells are transferred onto successive plates

20. Mutational hotspots are specific DNA sequences where mutations such as cytosine methylations are more likely to happen (single nucleotides and short tandem repeats). • Methylated cytosines are in turn highly mutable through deamination.

21. Mutagens Mutagensare agents that causes an increase in the rate of mutation above a spontaneous background (X-ray, UV, chemicals, viruses and etc ….) Mechanisms include: • Depurination • Oxidation • Base analogs • Alkylating agents • Intercalating agents • Ultraviolet radiation • Ionizing radiation

22. Ultraviolet light causes thymine dimers. Structural view of formation of thymine dimer. distortion of DNA helix caused by two thymines moving closer together when joined in a dimer.

23. Ionizing radiation from radioactive compounds (X-rays, b-particles, a-particles) cause single and double-strand DNA breaks and alterations in nucleotides.

24. Mutagens Ionizing radiation from radioactive compounds (X-rays, b-particles, a-particles) cause single and double-strand DNA breaks and alterations in nucleotides. Radiotherapy Paradox

25. Annual exposure of human beings in the United States to various forms of ionizing radiation. The relationship between the percentage of X-linked recessive lethals in D. melanogaster and x-ray dose, obtained from several experiments.

28. Mechanisms of DNA Repair • Xeroderma pigmentosum is a genetically heterogeneous autosomal recessive disorder characterized by increased sensitivity to sunlight with the development of carcinomas at an early age. • The excision repair mechanism, involved in DNA repair is impaired, due to mutations in the gene encoding XP genes.

30. Review Problems