Mutation, DNA Repair andRecombination

BIT 220Chapter 14

Mutation

• Heritable change in genetic material– change in gene– change in chromo #– change in chromo

structure

• Germinal Mutation– may be passed to

progeny

• Somatic Mutation– NOT passed to

progeny offspring

What causes mutation?

• SPONTANEOUS– No known cause– maybe be indeed a

causative agent, but has not been identified

• INDUCED– Caused by MUTAGEN

• (physical or chemical agents which alter DNA)

• nicotine• UV light• drugs• ionizing radiation

Phenotypic Effects

• Neutral Mutation- NO Effect on phenotype» ISOALLELES

• Null Allele - No functional protein

• Recessive Lethal - deadly null mutation in both alleles

Conditional Lethal MutationsDefine:

Lethal in one environment (restrictive condition)

Viable in second environment (permissive condition)

1. Auxotrophs Mutants - can’t synthesize metabolite

2. Temperature-Sensitive mutants - grow in one T, not another T

3. Suppressor-sensitive mutants - will grow in presence of suppressor;suppressor protein compensates for mutation

Replica Plating• Figure 14.3

• Non-Selective Plates -no antibiotic

• Selective plates- antibiotic in medium

• Mutation does NOT occur in response to selective agent

Frameshift Mutations

Base pair addition or deletion•alter reading frame

•always result in non-functional protein

Forward and Reverse Mutations

• Figure 14.4• Forward: wild-type to mutant phenotype• Reverse: 2nd mutation restores earlier

phenotype; reverse occurs by back mutation or suppressor mutation

• Summary of mutation process: Figure 14.7• Example (sickle-cell) Figure 14.8

TransposonsFigure 14.24

Pieces of DNA that can move from one location of genome to another

No specific target site

Effects:•help bacteria to adapt to new environments -develop antibiotic resistance

•can interrupt functional genes•can carry another gene

sit behind promoter and upregulate expression of gene it carries

•variegation of color in corn

DNA Repair Mechanisms

Enzymes constantly scan DNA looking for errors.

Repair can occur during or after replication

Mutations which cripple the repair systemXeroderma pimentosumcan not correct UV-induced damage to DNA

Mechanisms of RepairA. DNA Polymerase corrects mistakes during replication

3’-5’ exonuclease activity

B. Mismatch Repairwrong NT (non complementary base)

added during synthesiscorrected after replicationdifferentiates between methylated strand and unmethylated

C. Excision Repair Figure 14.27 and Figure 14.28errors made from mutagens (UV rays, X rays)NUCLEASE cuts out errorPOLYMERASE replaces NTLIGASE seals upcorrection made post replication

Mutagenesis:Why Mutate?Native proteins are not well suited for industrial application

Native proteins are not optimized for medicinal purposes

•Increase the efficiency of enzyme-catalyzed reactions• Eliminate the need for cofactor in enzymatic reaction•Change substrate binding site to increase specificity •Change the thermal tolerance•Change the pH stability•Increase proteins resistance to proteases (purification)

Protein Engineering

Enzymes Used in Industrial Applications

amylase beer productionlipase cheese productionpapain meat tenderizerprotease detergents

ProblemsConditions of process inactivate the enzyme

high temperaturepH rangessolvents

Specific Changes at the Protein Level •Add Disulfide Bonds•Changing Asparagine to Other amino Acids

Adding Disulfide Bonds•Usually found in extracellular proteins, not intracellular

•Cross link between chains or in chains formed by oxidation of cysteine residues

connective tissuefibrin blood clots

• Why would we add disulfide bonds?-Artificial addition may increase stability of protein

• As with all engineering AVIOD active site (enzyme)

XYLANASEused to treat wood pulp in paper productionneeds to function at high temp

Aspargine Changes

At high T, asparagine and glutamine deaminate•ammonia is released•amino acids convert to aspartic acid and glutamic acid•Protein may refold•LOSE ACTIVITY

Use Computer modeling to predict where mutation is preferred

Test stability/activity/stability of enzyme in vivo/in vitro

Reducing Free SulfhydrylResidues

Cysteine residues may cause dimerizationthrough intermolecular disulfide bonding

Convert Cys to another amino acid reduce dimerizationmaintain activity of enzyme

Enzyme Activity and Specificity

•Increase enzymatic activity by increasing affinity for enzyme•change sequences in substrate binding site

•Change substrate of enzyme

Other Examples

tPA tissue plasminogen activator•dissolves blood clots•would like to decrease clearance•decrease non-specific bleeding•increase specificity for fibrin in blood clot

Eliminate need for cofactorsSerine protease are used in laundry detergentsrequire calciummodify enzymes ability to bind calcium