Mutagenesis

What is mutagenesis

The process that create heritable changes in DNA sequences providing genetic variation that dirves evolution and alllows adaptation to stress. can be spontaneous or induced

Spontaneous mutations

occur without external mutagens and arise from normal metabolic processes or DNA replication errors

DNA replication errors

DNA polymerase occasionally inserts the wrong base suring replication and this can sometimes escape the proofreading. this produces base substitutions

types of base sustitutions

Transitions: purine ←→ purine pyrimidine ←→ pyrimidine

Transversions: purine ←→ pyrimidine

functional changes that can occur becuase of DNA replication errors

missense - amino acid swap nonsense - early stop codon silent - nothing

Tautomeric shifts

bases can temporarily change form and pair with the wrong base pair. If replication occurs during this time the muation becomes fixed

spontaneous chemical changes

Some bases naturally undergo chemical changes Depurination is the loss of a purine base which creates an abasic site and deamination is when a base changes identity e.g cytosine to urasil. these lesions cause mispairing or replication blocks leading to substitutions.

Errors in recombination

illegitimate recombination or slipped mispairing can lead to insertions deletions or frameshift mutations which are especially bad cause they change the whole reading frame

induced mutations

can occur when we expose cells to mutagens. used in lab settings for generating mutants with specific phenotypes

chemical mutagens

Base analogues

alkylating agents

deaminating agents

intercalating agents

Base analogues

These resemble normal bases and trick cell to incorporate them into DNA helix during replication. 5-bromouracil similar to thiamine, pairs with G instead of A causing transitions.

alkylating agents

add alkyl group to bases leading to faulty base pairs e.g. EMS nitrosoguanidine

Deaminating agents

remove amino groups from bases e.g. nitrous acid converts C→ U

Intercalating agents

Insert between base pairs causing insertion or deletions e.g. acridines, ethidium bromide these often produce frameshift mutations

physical mutagens

UV radiation

lonising radiation

UV radiation

When UV light hits DNA it cuases pyrimidine dimers. that distort DNA and block replication; error-prone repair introduces mutations

ionising rays (X-ray, Y-ray)

creates double strand breaks and highly reactive radicals leading to large deletions rearrangements and lethal damage

DNA repair and error-prone pathways

cells have repair systems that correct damage but sometimes introduce additional mutations

direct repair

base excision and nucleotide excision repair

mismatch repair

sos repair

direct repair

photolyase removes UV dimers using light energy

base excision and nucleotide excision repair

remove damaged bases or nucleotides and replace them using DNA polymerasemis

match repair

corrects replication errors shortly after DNA synthesis

SOS repair (error-prone repair)

when damage is overhwhelming bacteria activate the sos response. low fidelity polymerases replicate across lesions without proofreading increasing mutation rates. this helps survival but produces many mutations

Laboratory induction and isolation of mutants

mutagenesis is used widely in bacterial genetics to study gene function or create new phenotypes.

random mutagenesis

positive selection

negative selection

reversion analysis

random mutagenesis

chemicals used to create many mutations. large populations are screened for desired traits

positive selection

only mutants with specific phenotype survive; Example: selecting antibiotic-resistant mutants by plating on antibiotic-containing medium

 Negative selection

Used to isolate auxotrophs (mutants lacking a metabolic function).

Replica plating identifies colonies that fail to grow on minimal medium but grow on complete medium.

Reversion analysis

Mutations that restore wild-type function (true revertants or suppressor mutations) help map functional sites in genes.

Importance of Mutagenesis in Evolution and Microbial Genetics

Mutations are essential for:

• Adaptive evolution
  e.g., antibiotic resistance, metabolic innovation

• Pathogenesis
  antigenic variation and immune evasion

• Genetic mapping
  using mutants to identify gene order and function

• Biotechnology
  generating strains with improved production or altered metabolism

Mutagenesis creates the diversity that natural selection acts upon, making it a fundamental driver of microbial evolution.