1. Atta ur Rahman School of Biological Science National University of Sciences & Technology MUTAGENESIS 31-Dec-2012 CMB-234:Molecular Biology

2. Mutagenesis Mutagenesis is a process by which the genetic information of an organism is changed in a stable manner, resulting in a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using laboratory procedures.

3. Significance In nature mutagenesis can lead to cancer and various heritable diseases, but it is also the driving force of evolution. Mutagenesis as a science was developed based on work done by Hermann Muller, Charlotte Auerbach and J. M. Robson in the first half of the 20th century.

4. Mechanisms Mutagenesis may occur endogenously through spontaneous hydrolysis through normal cellular processes that can generate reactive oxygen species and DNA adducts through error in replication and repair as a result of the presence of environmental mutagens that induces changes to the DNA Mechanisms

5. 1-Spontaneous hydrolysis Under physiological conditions the glycosidic bond may be hydrolyzed spontaneously and 10,000 purine sites in DNA are estimated to be depurinated each day in a cell. Numerous DNA repair pathway exist for the DNA, however, if the apurinic site failed to be repaired, misincorporation of nucleotide may occur during replication. Adenine is preferentially incorporated by DNA polymerases in an apurinic site. Cytidine may also become deaminated to uridine at one five-hundredth of the rate of depurination and can result in G to A transition.

6. 2-Modification of bases Many compounds may form reactive oxygen species catalyzed by cytochrome P450. These metabolites form adducts with the DNA, which can cause errors in replication Bulky aromatic adducts may form stable intercalation between bases and block replication. The adducts may also induce conformational changes in the DNA. Some adducts may also result in the depurination of the DNA, Bases may be modified endogenously by normal cellular molecules. For example DNA may be methylated by S- adenosylmethionine, and glycosylated by reducing sugars.

7. 3-Intercalation between bases The planar structure of chemicals such as ethidium bromide and proflavine allows them to insert between bases in DNA. This insert causes the DNA's backbone to stretch and makes slippage in DNA during replication more likely to occur since the bonding between the strands is made less stable by the stretching. Forward slippage will result in deletion mutation, while reverse slippage will result in an insertion mutation.

8. 4-DNA slippage example normal 5'-GGGGGCAT-3' 3'-CCCCCGTA-5' slip (just used the underscore to take up space) 5-‘ GGGGCAT-3' 5'-CCCCCCGTA-3' When top slip strand is replicated, the G that is slipped out won't be replicated leading to a deletion of a single base.. Mutated: ?

9. Cont…

10. Ethidium bromide contains a tricyclic phenanthridine ring system that is able to intercalate between the stacked base pairs of double-stranded DNA. After insertion into the helix, the drug lies perpendicular to the helical axis and makes van der Waals contacts with the base pairs above and below. Whereas the planar ring system of the drug is buried, its peripheral phenyl and ethyl groups project into the major groove of the DNA helix. At saturation in solutions of high ionic strength, approximately one ethidium molecule is intercalated per 2.5 base pairs independent of the base composition of the DNA. J. Sambrook and D.W. Russell, 2001 Molecular Cloning (3rd edition) A9.3

11. 5-Backbone damage Ionizing radiations may produce highly reactive free radicals that can break the bonds in the DNA. Double- stranded breakages are especially damaging and hard to repair, producing translocation and deletion of part of a chromosomes.

12. 6-Insertional mutagenesis Transposon and virus may insert DNA sequence into coding region or functional elements of a gene and result in inactivation of the gene.

13. 7-Error in replication While most mutagens produce effects that ultimately result in error in replication, some mutagens may affect directly the replication process. Base analog such as 5-bromouracil may substitute for thymine in replication. Some metals such as cadmium, chromium, and nickel may alter the fidelity of DNA replication.

14. Mutagenesis as a laboratory technique Mutagenesis in the laboratory is an important technique whereby DNA mutations are deliberately engineered to produce mutant genes, proteins, or strains of organism.

15. 1-Site Directed mutagenesis Technique in which a mutation is created at a defined site in a DNA molecule Result -> library of wild- type and mutated DNA (site-specific)

16. 2-Random Mutagenesis A non-directed change of one or more nucleotide pairs in a DNA molecule. Result -> library of wild- type and mutated DNA (random) A real library -> many variants -> screening !

17. DNA shuffling x x x x x x x x DNAse I In shuffling, the products are degraded to random small fragments with DNAse I. 3-DNA SHUFFLING

18. x x x x Then full-length sequences are re-assembled by enzymatic DNA synthesis Denature, reanneal, enzymatic DNA synthesis Some products consist of full-length sequences containing several mutations. Recombinants with improved functions are selected.

19. EXAMPLES

20. Engineering Ca-independency of subtilisin: Serine protease Require calcium Calcium induces conformation change necessary for activity Modify amino acid sequence to achieve conformation and stability without calcium. Mutant: 10x more stable than native enzyme in absence of Ca

21. Protein Engineering Generating proteins with modified structures that confer novel properties such as higher catalytic specificity or thermal stability. It is a young discipline, with much research currently taking place into the understanding of protein folding and protein recognition for protein design principles.

22. Protein is NOT at an optimum for that function Sequence changes without disruption of the structure (otherwise it would not fold) New sequence is not TOO different from the native sequence (otherwise loss in function of protein) Protein Engineering Assumption : Natural sequence can be modified to improve a certain function of protein. This implies:

23. Thank you!