Spontaneous mutation

Spontaneous mutations occur naturally without any apparent cause. It arises from a variety of sources- Errors in DNA replication, Spontaneous lesions or by Transposable genetic element. These mutations results in several human diseases.

1. Spontaneous mutation Presented by: Megha Varshini Gowda B R

2. Content Introduction Sources of spontaneous mutation Errors in DNA replication Tautomeric shift Insertion and deletion of base Trinucleotide repeat expansion (TNRE) Spontaneous lesions Depurination Deamination Oxidatively damaged bases Transposable genetic element Conclusion Reference

3. Introduction Spontaneous mutations occur naturally without any apparent cause. Salvador Luria and Max Delbrück in 1943 designed fluctuation test to illustrate random mutation. Replica plating developed by Joshua and Esther Lederberg in 1952 to demonstrate the presence of mutants before selection. Strand-slippage hypothesis was given by George Streisinger in 1966. The rate of spontaneous mutation for a particular gene is 10 -5 to 10 -9 per cell generation. It arises from a variety of sources- Errors in DNA replication Spontaneous lesions Transposable genetic element

4. Errors in DNA replication It occurs when an illegitimate nucleotide pair forms in DNA synthesis, leading to base substitution. Tautomeric shift: Each bases in DNA can appear in one of several forms- tautomers . Tautomers are the isomers that differ in the positions of their atoms and in the bonds between the atoms. Base In normal state pairs with In tautomeric state pairs with A T C T A G G C T C G A Table 1: Formation of base pairs in normal and tautomeric state.

5. Amino form of A denine and C ytosine are more stable Keto form of G uanine and T hymine are more stable Figure 1: Tautomeric forms of (a) Adenine and Cytosine (b) Guanine and Thymine. a. b.

6. Tautomeric shift in incoming base (substrate transition) or the base already in the strand(template transition) results in mispairing . Fig.2: Hydrogen bonded A:C and G:T base pairs.

7. Mismatch could be repaired via proofreading activity of DNA Polymerase or via mismatch repair system. If it fails, one of the progeny will be a mutant type. Fig.3: Mechanism by which tautomeric shifts in the bases in DNA cause mutations.

8. Insertion and deletion If a newly synthesized strand loops out it results in the addition of an extra nucleotide and looping of template strand results in deletion of nucleotide base in newly synthesized strand. Region with repeated sequences are prone to such errors . Fig.4: Strand slippage during replication results in addition or deletion of base pairs.

9. Insertion or deletion of one or more nucleotides during replication leads to frameshift mutation. As the reading frame shifts the outcome of frameshift mutation is complete alteration of the amino acid sequence of a protein. Fig.5: Frameshift mutation by nucleotide insertion.

10. Trinucleotide repeat expansion (TNRE) TNRE are the hotspots of mutation. It is a phenomenon in which a repeated sequence of three nucleotides can readily increase in number from one generation to another. DNA polymeraese slips off the DNA after the repeat sequence is synthesized. A hairpin is quickly formed and DNA polymerase hops back onto the DNA and continues with DNA replication. Depending on how the DNA is repaired, this may result in trinucleotide repeat expansion. TNRE results in human diseases such as Spinal and bulbar muscular atrophy-(CAG), Huntington disease-(CAG), Fragile X syndrome(CGG, GCC)

11. Fig.6: Possible mechanism for trinucleotide expansion.

12. Spontaneous lesions Naturally occurring damage to the DNA (spontaneous lesions) causes mutation. Depurination : Refers to removal of purine base from its position. The space formed after removal is called apurinic (AP) site. A mammalian cell spontaneously loses about 10,000 purines from its DNA in a 20-hour cellgeneration period at 37°C. In replication, the apurinic sites cannot specify a base complementary to the original purine . If repair system fails, during replication any of the four bases are added to the new strand, leading to mutation. The chance of causing mutation is 75%.

13. Fig.7: Depurination of DNA

14. Deamination : Removal of amino group from the base. Deamination of Cytosine produces Uracil . Uracil -DNA glycosylase recognizes the Uracil residue in DNA and excises them. Fig.8: Deamination of Cytosine. H 2 O + NH 3

15. Deamination of 5-methylcytosine generates thymine (5-methyluracil), which is not recognized by the enzyme uracil -DNA glycosylase and thus is not repaired. Therefore, C → T transitions generated by deamination are seen more frequently at 5-methylcytosine sites, because they escape this repair system. Fig.9: Deamination of 5-methylcytosine. H 2 O + NH 3

16. Oxidatively damaged bases: Oxidative DNA damage refers to change in DNA structure that are caused by ROS. Reactive Oxygen Species(ROS) such as hydrogen peroxide (H 2 O 2 ), superoxide (O 2 ·), hydroxyl radical (OH·) are products of metabolism in all aerobic organisms. DNA bases, particularly Guanine are vulnerable to oxidation which leads to oxidized guanine products. Guanine on oxidation forms 8-Oxo-7-hydrodeoxyguanosine (8-oxo dG ) which base pairs with adenine during DNA replication. Fig.10: Oxidatively damaged bases.

17. Transposable genetic element Transposable genetic element are DNA elements that can move from one site in the genome to another site. The insertion of a transposon into a gene will often render the gene nonfunctional. If the gene encodes an important product, a mutant Phenotype is likely to result. Fig.11: Mutation caused by insertion of transposable genetic element.

18. Crossing over between homologous transposons located at different positions in a chromosome. If two transposons in the same orientation pair and cross over, the segment between them will be deleted . Fig.12: Formation of a deletion by intrachromosomal recombination between two transposons in the same orientation.

19. Common causes of Spontaneous mutation Description Errors in DNA replication A mistake by DNA polymerase may cause point mutation Tautomeric shift Spontaneous changes in base structure can cause mutations if they occur immediately prior to DNA replication Depurination The linkage between purines and deoxyribose can spontaneously break Deamination Cytosine and 5-methylcytosine can spontaneously deaminate to create Uracil and Thymine Toxic metabolic products Reactive oxygen species are chemically reactive and can alter the structure of DNA Transposable elements Transposable elements can insert themselves into the sequence of a gene. Summary and Conclusion Spontaneous mutations can be generated by different sources. Replication errors and spontaneous lesions generate most of the base-substitution and frameshift mutations. These mutations results in several human diseases.

20. References Snustad , D.P., Simmons, M.J. and Jenkins, J.R. (1997). Principles of Genetics. 6 th edn . John Wiley & Sons, Inc. N.Y. Pp.320-324, 497-498. Tamarin , R.H. (1985). Principles of Genetics. 7 th edn . TATA McGRAW HILL edn . Pp.328-332. Griffiths A.J.F, Miller J.H, Suzuki D.T, et al.(2000). An Introduction to Genetic Analysis. 7th edition. W. H. Freeman, New York. Pp.422-438 Brooker R.J. (2004). Genetics –analysis and principles. 2 nd edn . Addison Wesley Longman Inc. California. Pp.318-325 https://www.nature.com/scitable/topicpage/dna-replication-and-causes-of-mutation-409