Atta ur Rahman School of Biological ScienceNational University of Sciences & Technology

MUTAGENESIS

31-Dec-2012

CMB-234:Molecular Biology

Mutagenesis

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.

Mechanisms

Mechanisms

1-Spontaneous hydrolysis

2-Modification of bases

Bases may be modified endogenously by normal cellular molecules. For example DNA may be methylated by S-

adenosylmethionine, and glycosylated by reducing sugars.

3-Intercalation between bases

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: ?

Cont…

Cont…

• 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 

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.

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.

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.

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.

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)

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 !

DNA shuffling

x

x

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x

x

x

x

DNAse I

In shuffling, the products are degraded to random small fragments with DNAse I.

3-DNA SHUFFLING

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.

EXAMPLES

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

Protein Engineering

It is a young discipline, with much research currently taking place into the understanding of protein folding and protein recognition for protein design principles.

Protein Engineering

Assumption : Natural sequence can be modified to improve a certain function of protein. This implies:

Thank you!