Genetic Diversity and Mutation

What are the three main components of a DNA nucleotide? (Slide 2)

• 5-carbon sugar (deoxyribose)

• Phosphate group attached to the 5′ carbon

• Nitrogenous base attached to the 1′ carbon

What forms the backbone of DNA? (Slide 2)

• Covalent bonds between phosphate and sugar

• Creates the backbone that gives structure

• Bases encode the genetic information

What is directionality in DNA strands? (Slide 3)

• DNA strands have chemically different ends

• 5′ end: free phosphate  3′ end: free hydroxyl group

• Nucleotides can be added only to the 3′ end

• DNA and RNA built in the 5′ → 3′ direction

What are the two classes of nitrogen bases? (Slide 4)

• Purines: Adenine (A) and Guanine (G)

• Pyrimidines: Cytosine (C), Thymine (T); Uracil (U) appears only in RNA

How are two DNA strands held together? (Slide 5)

• Hydrogen bonds between complementary bases

• A–T pairs and G–C pairs

• Base pairing allows complementarity – each strand predicts the other

Describe double-stranded DNA structure. (Slide 5)

• Two strands run antiparallel

• Twisted into a double helix

• Backbones → outside; bases → inside

What are the major and minor grooves? (Slide 6)

• Major groove: wider space between backbones

• Minor groove: narrower space

• Grooves alternate and serve as binding sites for proteins

How do bacteria reproduce? (Slide 7)

• By binary fission (asexual)

• Chromosome copied once per division

• Daughter cells are genetically identical clones

Where is the bacterial chromosome located? (Slide 8)

• In the nucleoid region

• Circular chromosome (no ends)

• Compact via supercoiling

What is supercoiling and why is it important? (Slide 8)

• DNA helix twisted around itself

• Reduces size and fits DNA inside the cell

• Organizes and stabilizes the chromosome

Where does replication begin in bacteria? (Slide 9)

• At the origin of replication (oriC)

• DNA must be unwound and separated

• Step is called initiation

Which protein initiates DNA replication? (Slide 9)

• DnaA binds multiple sites at oriC

• Hydrolyzes ATP to unwind DNA

• Opens A–T-rich DUE regions

Why are A–T pairs favored at oriC? (Slide 9)

• Only 2 hydrogen bonds → easier to separate

What happens during elongation? (Slide 10)

• Replisome moves along DNA to copy each strand

• Unwinds and separates the helix

• Synthesizes new DNA using base pair rules

• Produces two dsDNA molecules each with one parent and one new strand → semi-conservative

Is replication unidirectional or bidirectional? (Slide 11)

• Bidirectional from oriC

• Two replisomes → opposite directions

• Each creates a replication fork

• Stop at terminator sequence

What are the main enzymes in the replisome? (Slides 12–13)

• Helicase: unwinds and separates DNA

• DNA polymerase III: synthesizes new DNA

• Clamp proteins: keep polymerase attached

• Clamp loader: loads clamps and holds complex together

Which polymerase performs main replication in bacteria? (Slide 14)

• DNA polymerase III – major enzyme of replisome

• Performs nucleophilic attack from 3′ OH on incoming nucleotide triphosphate

• Releases two phosphates → forms new covalent bond

• Explains why DNA can grow only 5′ → 3′

How does RNA polymerase differ? (Slide 14)

• Can begin RNA synthesis without free 3′ OH

• DNA polymerase cannot → requires primer

What issue arises due to antiparallel strands? (Slide 15)

• Only one template offers a free 3′ OH for continuous synthesis

• The other strand is oriented opposite → requires special handling

How are the two strands synthesized? (Slide 16)

• Leading strand: continuous 5′ → 3′ synthesis

• Lagging strand: discontinuous in small segments (Okazaki fragments)

• Continuous = fast; discontinuous = slow

What is the trombone loop? (Slide 17)

• Loop formed in lagging strand so it enters polymerase in proper orientation

• Allows 5′ → 3′ synthesis while polymerase moves forward

• Loop protected by single-stranded binding proteins (SSB)

What are the roles of SSB proteins? (Slide 17)

• Bind and stabilize exposed single DNA

• Prevent degradation or secondary structure formation

How does synthesis start on the lagging strand? (Slide 18)

• Primase (an RNA polymerase) synthesizes short RNA primers

• Primers provide the needed 3′ OH for DNA polymerase III

Describe Okazaki fragment formation. (Slide 19)

• Primase adds RNA primers

• DNA polymerase III extends DNA between primers

• DNA polymerase I removes RNA primers and replaces with DNA

• DNA ligase joins fragments into one strand

What does DNA ligase do? (Slide 20)

• Seals DNA nicks between Okazaki fragments

• Forms covalent phosphodiester bond using ATP

List all major components of the replisome.

• Two DNA polymerase III enzymes (one per strand)

• Clamp proteins – hold polymerases on DNA

• Helicase – unwinds DNA and breaks H-bonds

• SSB proteins – protect single-strand loops

• Clamp loader – assembles and stabilizes replisome

Why are mutations important in microbial populations? (Slide 1)

Because a protein’s function depends on its amino acid sequence, mutations in genes can alter both protein sequence and function. Mutations are a major source of genetic diversity in microbes, especially in bacteria and archaea, which lack meiosis and sexual reproduction.

How do prokaryotes generate genetic diversity? (Slide 1)

Through mutation and horizontal gene transfer — the transfer of DNA between individuals of the same or different species.

What are the two main categories of mutations? (Slide 2)

1. Spontaneous mutations – occur naturally due to replication or repair errors, base instability, or damage by reactive molecules (e.g., ROS).

2. Induced mutations – caused by exposure to external mutagens such as UV radiation, X-rays, or chemicals that damage DNA.

What causes spontaneous mutations? (Slide 2)

• DNA replication/repair errors

• Natural chemical instability of bases

• Reactive oxygen species (ROS) formed during aerobic metabolism

What causes induced mutations? (Slide 2)

• Intense radiation (e.g., UV, X-rays)

• Mutagenic or carcinogenic chemicals that alter DNA structure

What are the three major types of mutations? (Slide 3)

1. Substitution – one base is replaced by another; sequence length stays the same.

2. Deletion – one or more bases permanently removed; sequence becomes shorter.

3. Insertion – one or more bases permanently added; sequence becomes longer.

What are the four main effects mutations can have on proteins? (Slides 4–6)

• Silent mutations – substitution changes codon but not amino acid; no effect on protein.

• Missense mutations – substitution changes one amino acid to another.

• Nonsense mutations – substitution changes codon to a stop codon, producing truncated protein.

• Frameshift mutations – insertion or deletion shifts the reading frame, altering downstream amino acids.

What is an example of a silent mutation? (Slide 5)

A change from CAA → CAG still codes for glutamine, producing no change in the amino acid sequence.

What is a missense mutation? (Slide 5)

A single-base substitution that changes an amino acid in the protein sequence (e.g., CAA → GAA changes glutamine to glutamate).

What is a nonsense mutation and what are its types? (Slide 6)

A substitution that changes an amino acid codon to a STOP codon, prematurely terminating translation.

Types include:

• UAG (amber)

• UAA (ochre)

• UGA (opal)

What is a frameshift mutation and how does it occur? (Slide 8)

Frameshifts result from insertions or deletions that change how mRNA codons are grouped into triplets. This shifts the reading frame and drastically alters downstream amino acids, often producing nonfunctional proteins.

How does DNA polymerase III correct replication errors? (Slide 10)

• Performs DNA proofreading during synthesis.

• Detects mismatched bases because they distort the DNA backbone.

• Stops elongation (“stalling”) and uses its exonuclease activity to remove the incorrect base.

• Resumes synthesis with the correct nucleotide.

What happens if DNA polymerase III fails to proofread correctly? (Slide 11)

The cell uses a backup repair system called methyl-directed mismatch repair to fix replication errors.

What is methyl-directed mismatch repair? (Slide 11)

A repair system (in E. coli and similar bacteria) that identifies and corrects mismatched bases after replication using Mut proteins.

How does methyl-directed mismatch repair identify the newly synthesized DNA strand? (Slide 11)

• After replication, enzymes add methyl (-CH₃) groups to A and C bases.

• Old (parental) strand = methylated

• New strand = temporarily unmethylated

• The system repairs mismatches on the unmethylated (new) strand.

How does methyl-directed mismatch repair fix the error? (Slide 12)

1. Mut proteins form a complex that scans DNA for distortions.

2. When mismatch found → DNA unwound and cut on the unmethylated strand.

3. UvrD helicase removes the cut region, creating a gap.

4. SSB proteins protect the gap.

5. DNA polymerase I fills in new nucleotides.

6. DNA ligase seals the nick.

What happens if DNA damage is too extensive for proofreading or mismatch repair? (Slide 13)

The recombination repair system is activated, led by the RecA protein.

What is RecA and what does it do? (Slide 13)

RecA binds to the replication fork and cuts both DNA backbones, swapping damaged DNA with undamaged DNA from the other strand. This allows DNA polymerase I to fill in the gap using the intact strand as a template.

What are the two major DNA repair systems for damage occurring outside replication? (Slide 14)

1. Nucleotide excision repair

2. Base excision repair

What is nucleotide excision repair (NER) and when is it used? (Slide 14–15)

• Repairs DNA regions with backbone distortions (e.g., UV-induced thymine dimers).

• Uses proteins of the uvr operon.

• Works outside replication to remove bulky lesions.

Describe the steps of nucleotide excision repair. (Slides 15–16)

1. UvrA scans DNA for distortions and stops when one is found.

2. UvrB binds and marks the damage site.

3. UvrC cuts DNA backbone on both sides of the damage.

4. UvrD helicase removes the damaged fragment.

5. DNA polymerase I fills in the gap.

6. DNA ligase seals the strand.

What is base excision repair (BER) and when is it needed? (Slides 17–18)

• Fixes non-distorting base damage (e.g., oxidation, deamination).

• Removes damaged bases that don’t alter DNA shape.

Describe the steps of base excision repair. (Slide 18)

1. DNA glycosylase recognizes and removes the damaged base (backbone intact).

2. Site left behind = AP site (apurinic/apyrimidinic site). AP endonuclease cuts the DNA backbone at the

3. AP site → creates a gap.

4. DNA polymerase I fills the gap with correct nucleotides.

5. DNA ligase seals the DNA backbone.

What is an AP site? (Slide 18)

A site in DNA where the base has been removed, but the sugar-phosphate backbone remains intact; a key intermediate in base excision repai

List the major DNA repair mechanisms and the main enzymes involved. (Slides 10–18)

Repair Type	Key Enzymes / Proteins	Function
Proofreading	DNA polymerase III	Exonuclease removes mispaired bases
Mismatch repair	Mut proteins, UvrD, SSB, Pol I, Ligase	Removes mismatched bases post-replication
Recombination repair	RecA, Pol I	Swaps damaged DNA for undamaged template
Nucleotide excision repair	UvrA, UvrB, UvrC, UvrD, Pol I, Ligase	Removes bulky, backbone-distorting lesions
Base excision repair	Glycosylase, AP endonuclease, Pol I, Ligase	Removes non-distorting damaged bases

Why are frameshift mutations often more severe than point mutations? (Slide 8)

Because they alter the entire downstream reading frame, potentially changing every amino acid and often introducing premature stop codons, resulting in nonfunctional proteins.

What are the four types of point mutation effects? (Slides 4–6)

• Silent – no amino acid change

• Missense – one amino acid replaced by another

• Nonsense – converts codon to stop codon

• Frameshift – alters codon grouping due to insertion/deletion

What balance must microbes maintain between mutation and stability? (Slide 1)

Microbes must balance genomic stability (accurate inheritance of DNA and proteins) with genomic plasticity (ability to evolve). Preventing all mutation would eliminate the ability to adapt, while excessive mutation threatens cell function.

Define genomic stability. (Slide 1)

The ability of a cell to faithfully and completely transfer genetic information, proteins, and cellular systems to the next generation.

Define genomic plasticity. (Slide 1)

The capacity of a cell to adapt and evolve, allowing changes in DNA, proteins, and cell systems to respond to environmental changes.

What are the two major groups of processes that create genomic plasticity in microbes? (Slide 2)

1. Genetic rescue systems – allow replication of heavily damaged DNA.

2. Genetic transfer systems – allow acquisition of DNA from other cells or the environment.

What is the main genetic rescue system in bacteria? (Slide 3)

The SOS repair system, composed of about 40 genes called the SOS regulon.

How is the SOS regulon controlled? (Slide 3)

• Each gene has an operator called the SOS box.

• A repressor protein, LexA, binds as a dimer to the SOS box and prevents transcription of SOS genes.

How is the SOS response activated when DNA is damaged? (Slide 4)

• DNA damage causes accumulation of single-stranded DNA (ssDNA) during replication.

• RecA binds to ssDNA and becomes activated (RecA*), gaining protease activity.

• RecA* cleaves LexA, disabling repression and allowing SOS gene transcription.

What are the two phases of the SOS response? (Slide 5)

1. Early response: Increases transcription of genes for recombination repair and nucleotide excision repair.

2. Late response: Activates transcription of polymerases IV (dinB) and V (umuC, umuD) to replicate through DNA damage.

What is the function of DNA polymerases IV and V? (Slide 6)

• DNA polymerase V inserts random bases across damaged DNA, allowing replication to continue.

• DNA polymerase IV bypasses replication stalls by “looping out” damaged regions.

• Both lack proofreading ability and together perform translesion DNA synthesis.

What is translesion DNA synthesis? (Slide 6)

A process where specialized polymerases replicate over damaged DNA (lesions) without repairing them, allowing survival but introducing errors.

Why does translesion DNA synthesis cause mutations? (Slide 7)

Because polymerases IV and V lack proofreading and insert random bases, creating replication errors that can become permanent mutations.

How does the SOS response contribute to evolution and antibiotic resistance? (Slide 7)

Though error-prone, SOS-induced mutations can provide adaptive advantages (e.g., antibiotic resistance) by increasing genetic diversity under stress.

What is horizontal gene transfer (HGT)? (Slide 8)

The transfer of genetic material between organisms (not parent to offspring), contributing to microbial evolution and plasticity.

What process does most horizontal gene transfer depend on? (Slide 8)

Homologous recombination, which integrates transferred DNA into the chromosome using matching sequences.

Describe the steps of homologous recombination. (Slide 9)

1. Donor DNA is nicked and unwound to form ssDNA.

2. ssDNA is protected by SSB proteins.

3. RecA binds and swaps the donor strand with the recipient’s homologous region (cross-strand exchange).

4. DNA ligase seals the nicks, integrating the new DNA.

What are the three main mechanisms of horizontal gene transfer? (Slide 10)

1. Transformation – uptake of environmental DNA fragments.

2. Transduction – transfer of DNA via bacteriophages (viruses).

3. Conjugation – direct transfer of plasmid or chromosomal DNA between cells.

What is transformation? (Slide 11)

The uptake and incorporation of free DNA from the environment into a bacterial cell’s chromosome.

What is a competent cell? (Slide 11)

A bacterial cell capable of absorbing environmental DNA; can occur naturally (natural competence) or be induced in the lab (artificial competence).

How is artificial competence produced? (Slide 11)

By laboratory treatments (chemical or electrical) that make bacterial membranes permeable to DNA; used in experiments like pGLO plasmid transformation.

How does transformation work in naturally competent bacteria? (Slide 12)

• Membrane proteins bind and absorb DNA from the environment (from dead cells).

• One DNA strand is degraded by nucleases; the other integrates via RecA-mediated recombination.

How is natural competence regulated in Bacillus subtilis? (Slide 13)

• Controlled by quorum sensing using the signal peptide ComX.

• As cell density rises, ComX accumulates and activates ComP receptor, which triggers transcription of competence genes.

What is transduction? (Slide 14)

The transfer of bacterial DNA via bacteriophages (bacterial viruses).

What are the two types of bacteriophages? (Slide 14)

• Virulent phages: Immediately replicate and cause cell lysis (cytolysis).

• Temperate phages: Integrate their DNA into the host chromosome as prophages.

How does generalized transduction occur? (Slide 15)

1. During lytic infection by a virulent phage, host DNA fragments are formed.

2. Occasionally, a virus mistakenly packages bacterial DNA instead of viral DNA, creating a transducing particle.

3. When infecting another cell, it delivers bacterial DNA, which can be integrated by homologous recombination.

Why is generalized transduction “generalized”? (Slide 15)

Because any random fragment of bacterial DNA can be transferred.

How does specialized transduction occur? (Slide 16)

1. In temperate phages, prophage DNA excises from the bacterial chromosome.

2. Improper looping can include adjacent bacterial genes.

3. These genes are carried into the next host cell when the phage infects again.

Why is specialized transduction “specialized”? (Slide 16)

Because only genes near the prophage insertion site are transferred.

What is conjugation? (Slide 17)

A process of DNA transfer between bacterial cells through direct contact, typically involving plasmids.

What is a conjugative plasmid? (Slide 17)

A plasmid that carries genes required for transfer, including those for forming the conjugation bridge; often includes antibiotic resistance or metabolic genes.

What structure mediates DNA transfer during conjugation? (Slide 17)

The conjugation bridge (or F pilus) — a hollow tube connecting donor and recipient cells.

What is the F plasmid of E. coli and its role in conjugation? (Slide 18)

• The F (fertility) plasmid encodes proteins to form the F pilus.

• F⁺ cells (donors) attach to F⁻ cells (recipients) via the pilus.

• The F plasmid is replicated and transferred as single-stranded DNA, then copied into dsDNA in the recipient.

What is an Hfr strain and how does it form? (Slide 19)

• Hfr (high frequency recombination) strains form when the F plasmid integrates into the bacterial chromosome.

• During conjugation, chromosomal DNA is replicated and transferred through the F pilus.

• The transferred DNA can integrate into the recipient’s chromosome via homologous recombination.

What are the major mechanisms of genetic change in microbes summarized? (Slides 1–19)

1. Mutation – changes in base sequence.

2. Genetic rescue (SOS system) – allows repair or survival through DNA damage.

3. Horizontal gene transfer – acquires genetic material from other cells (transformation, transduction, conjugation).

What are the key points from the study guide for Part 3? (Slide 20)

• Define genomic stability and plasticity.

• Describe the SOS system, LexA, and RecA regulation.

• Differentiate early vs late SOS responses; polymerases IV & V roles.

• Define translesion DNA synthesis and why it causes mutations.

• Explain horizontal gene transfer and homologous recombination (RecA).

• Describe transformation, competence, and quorum sensing (ComX/ComP).

• Define bacteriophages, and distinguish virulent vs temperate.

• Compare generalized and specialized transduction.

• Describe conjugation, F plasmid, F pilus, and Hfr strain