Replication fork

• Helicase is the enzyme that unzips the two DNA strands.
• Each old strand acts as a template to make a new matching strand.
• DNA polymerase builds new DNA, but it can add nucleotides only in one direction: 5′ → 3′.

               Helicase opens DNA
                       ↓
Old DNA strand  ========\ 
                         \   ← Replication fork
                          \
Old DNA strand  ===========

Leading strand

Lagging strand and Okazaki fragments

1. Primase lays down a short RNA primer for each fragment.
2. DNA polymerase extends it to make DNA.
3. The RNA primers are removed and replaced with DNA.
4. DNA ligase acts like glue, joining all the fragments into one continuous lagging strand.

Leading strand:   ---------------------------->  continuous

Lagging strand:   <---  <---  <---  <---         short pieces
                  Okazaki fragments
                         ↓
                  DNA ligase joins them

Simple comparison

Transcription: easy explanation

Basic steps

1. Initiation
   RNA polymerase binds the promoter, a DNA sequence before the gene, and locally unwinds DNA.
2. Elongation
   It reads the DNA template strand in the 3′ → 5′ direction and builds complementary RNA 5′ → 3′.
   DNA template: 3′-TACG-5′
   RNA made: 5′-AUGC-3′
   The RNA sequence is nearly the same as the DNA coding strand, except RNA has U (uracil) instead of T (thymine).
3. Termination
   RNA polymerase reaches a stop signal, releases the RNA, and DNA rewinds.

Prokaryotic transcription

RNA polymerase

• Bacteria have one RNA polymerase.
• It needs a sigma (σ) factor to recognize and bind the promoter.
• After initiation, sigma usually detaches and the core polymerase continues RNA synthesis.

Promoter sequences

• -35 region
• -10 region, also called the Pribnow box: TATAAT

Important feature

Termination

• Newly made RNA forms a hairpin loop.
• This is often followed by many U residues.
• RNA polymerase stops and the RNA is released.

• Rho (ρ) is an ATP-dependent helicase protein.
• It moves along RNA, catches the paused RNA polymerase, and releases the RNA transcript.

Eukaryotic transcription

RNA polymerases

• Pol I makes rRNA, except 5S
• Pol II makes mRNA
• Pol III makes tRNA and 5S rRNA

Initiation

• TATA box may be present in the promoter.
• TBP (TATA-binding protein), part of TFIID, binds it.
• Other transcription factors assemble.
• Then RNA polymerase II begins transcription.

RNA processing of eukaryotic mRNA

1. 5′ cap added
2. Splicing removes introns and joins exons
3. 3′ poly-A tail added

Prokaryotes vs eukaryotes

Inhibitors of transcription

High-yield memory lines

• Rifampicin: bacteria, RNA polymerase β subunit, tuberculosis.
• Actinomycin D: binds DNA by intercalation.
• α-Amanitin: mushroom toxin, blocks RNA polymerase II, so mRNA formation falls.

Post-transcriptional modifications

The three main modifications of eukaryotic mRNA

Pre-mRNA
5′ ─ exon ─ intron ─ exon ─ intron ─ exon ─ 3′

   ↓ 5′ capping + splicing + 3′ polyadenylation

Mature mRNA
5′ cap ─ exon ─ exon ─ exon ─ poly-A tail

1. 5′ cap

• It has an unusual 5′-to-5′ triphosphate linkage.
• It protects mRNA from degradation by exonucleases.
• It helps the mRNA leave the nucleus.
• It helps the ribosome recognize the mRNA and start translation.

2. 3′ poly-A tail

• This tail is not directly encoded by the DNA template.
• It binds poly-A-binding proteins.
• It increases mRNA stability.
• It promotes export from the nucleus and efficient translation.

3. RNA splicing

• Exons: sequences retained in mature mRNA
• Introns: intervening sequences removed from pre-mRNA

High-yield splice-site rule

5′ GU .......... AG 3′

• Intron begins with GU
• Intron ends with AG
• An internal branch-point adenine (A) helps form a lariat-shaped intron during removal.

Alternative splicing

Same gene → different exon combinations → different proteins

Other post-transcriptional RNA changes

RNA editing

• C → U editing in apolipoprotein B mRNA can produce apoB-48 instead of apoB-100.
• A → I editing occurs in some RNAs; inosine is read similarly to guanosine during translation.

Processing of tRNA and rRNA

• tRNA: trimming of ends, addition of CCA at the 3′ end, base modifications, and sometimes intron removal.
• rRNA: cleavage of precursor rRNA and chemical modifications, then assembly with proteins into ribosomal subunits.

Regulation by non-coding RNAs

• Blocking translation
• Promoting mRNA degradation

Prokaryotes versus eukaryotes

One-line memory aid

• Cap at 5′ end
• Tail at 3′ end
• Splice out introns, join exons

Mutations

• DNA replication errors
• Radiation, such as UV or ionizing radiation
• Chemicals or other mutagens
• Failure of DNA repair mechanisms

• Somatic cells: affect the individual, may contribute to cancer, not usually inherited.
• Germ cells: egg or sperm cells, can be passed to offspring.

Main types of mutations

Point mutation

Normal DNA:     GAA
Mutated DNA:    GTA
                   ↑
              one base changed

Types of point mutations by effect

1. Silent mutation

mRNA:  GAA  → glutamate
mRNA:  GAG  → glutamate

• Protein sequence is unchanged.
• Usually harmless, although it can occasionally affect splicing or mRNA stability.

2. Missense mutation

Normal mRNA:   GAG  → glutamate
Mutant mRNA:   GUG  → valine

• Beta-globin codon 6: GAG → GTG in DNA coding sequence
• Glutamate is replaced by valine.

3. Nonsense mutation

Normal:   UAU → tyrosine
Mutant:   UAA → STOP

• Translation stops too early.
• Produces a shortened, usually nonfunctional protein.

4. Splice-site mutation

• Normal splicing is disrupted.
• An exon may be skipped or an intron may remain in mature mRNA.
• It can lead to an abnormal protein or a frameshift.

Point mutation by chemical base change

Transition

• Purine → purine: A ↔ G
• Pyrimidine → pyrimidine: C ↔ T

Transversion

• Purine ↔ pyrimidine
• A/G ↔ C/T

Frameshift mutation

Normal:      THE CAT ATE THE RAT
Deletion:    THE ATA TET HER AT...

• Usually has a more severe effect than a single missense mutation.
• Insertions or deletions of 3, 6, 9... bases do not shift the reading frame. They add or remove whole amino acid(s).

Quick comparison

Memory trick

Proto-oncogenes and oncogenes

Proto-oncogenes

• Growth factors
• Growth-factor receptors
• Signal-transduction proteins
• Nuclear transcription factors
• Cell-cycle regulators

Oncogenes

Normal proto-oncogene
        ↓ activating alteration
Oncogene
        ↓
Continuous / excessive growth signal
        ↓
Cancer development

Mechanisms of proto-oncogene activation

1. Point mutation

Example: RAS mutation

RAS-GTP = ON
RAS-GDP = OFF

• Seen in many cancers, including colon, pancreatic, and lung cancers.

2. Gene amplification

Examples

Normal:      [MYC]
Amplified:   [MYC] [MYC] [MYC] [MYC] [MYC]
                         ↓
                  excess MYC protein

3. Chromosomal translocation

A. Promoter/enhancer swapping

t(8;14)

MYC gene from chromosome 8
     moves near
immunoglobulin heavy-chain enhancer on chromosome 14
     ↓
too much MYC expression

B. Fusion gene formation

t(9;22) = Philadelphia chromosome

BCR gene + ABL gene
        ↓
BCR-ABL fusion protein
        ↓
constitutively active tyrosine kinase
        ↓
uncontrolled myeloid-cell proliferation

4. Insertional mutagenesis

Viral promoter inserted near proto-oncogene
                    ↓
excess gene transcription
                    ↓
oncogene activation

5. Increased transcription due to regulatory mutation

Regulatory DNA mutation
        ↓
more transcription
        ↓
more normal growth-promoting protein
        ↓
excess proliferation

High-yield table

Easy mnemonic: “MAPS”

• Mutation
• Amplification
• Promoter/enhancer translocation
• Splicing/fusion? Better: Structural fusion from translocation

Proto-oncogene vs tumor suppressor gene

Tumor suppressor genes

• Oncogenes: accelerator stuck ON, gain-of-function
• Tumor suppressor genes: brakes fail, loss-of-function

1. RB gene: governor of the G1 → S checkpoint

• Gene: RB1
• Protein: Rb, retinoblastoma protein
• Main action: stops the cell from entering S phase and copying DNA.

How Rb works

When Rb is active

Active Rb (hypophosphorylated)
          ↓ binds
         E2F
          ↓
DNA-synthesis genes remain OFF
          ↓
Cell stays in G1 phase

When the cell should divide

Growth factors
      ↓
Cyclin D + CDK4/6
      ↓ phosphorylates
Rb becomes inactive
      ↓ releases
E2F
      ↓
S-phase genes ON
      ↓
G1 → S progression

Loss of RB

Rb lost/inactive → E2F always free → DNA synthesis genes ON → uncontrolled division

Associations

• Retinoblastoma
• Osteosarcoma
• Many other tumors have disruption of the Rb pathway.

2. p53: guardian of the genome

• Gene: TP53
• Protein: p53
• Main action: detects DNA damage and decides whether the cell should pause, repair DNA, or die.

Normal response to DNA damage

DNA damage
     ↓
p53 becomes stabilized and activated
     ↓
p53 acts as a transcription factor
     ↓
p21 production increases
     ↓
p21 inhibits cyclin-CDK complexes
     ↓
Rb stays active and bound to E2F
     ↓
G1 → S arrest

If the DNA damage is repaired

• p53 levels fall.
• The cell-cycle block is removed.
• The cell can proceed normally.

If DNA damage cannot be repaired

• Senescence: permanent cell-cycle arrest
• Apoptosis: programmed cell death

Severe / unrepaired DNA damage
          ↓
p53
     ↙          ↘
senescence     apoptosis

Loss of p53

• DNA damage is not properly halted.
• The cell does not reliably repair its DNA.
• Abnormal cells escape apoptosis.
• Mutations accumulate, increasing cancer risk.

Li-Fraumeni syndrome

• Sarcomas
• Breast cancer
• Brain tumors
• Leukemia
• Adrenocortical carcinoma

p53 and Rb together

DNA damage
   ↓
p53 ↑
   ↓
p21 ↑
   ↓ inhibits CDKs
Rb remains hypophosphorylated and active
   ↓ binds
E2F
   ↓
No S-phase gene transcription
   ↓
Cell-cycle arrest

• p53 detects DNA damage and makes the decision.
• Rb physically blocks the G1 → S transition by binding E2F.

HPV connection

Quick comparison

Molecular basis of cancer

Major gene groups involved

What cancer cells acquire

1. Self-sufficient growth signaling
   Example: activated RAS or HER2.
2. Resistance to growth inhibition
   Example: loss of Rb or p53.
3. Evasion of apoptosis
   Example: BCL2 overexpression or p53 loss.
4. Replicative immortality
   Cancer cells often activate telomerase, preventing progressive telomere shortening.
5. Angiogenesis
   Tumors induce blood-vessel growth, often through VEGF, to obtain oxygen and nutrients.
6. Invasion and metastasis
   Cells lose normal adhesion, degrade extracellular matrix, enter vessels, and colonize distant sites.
7. Genomic instability
   Defective DNA repair allows mutations to accumulate faster.
8. Immune evasion
   Tumors may suppress or escape immune recognition, for example through PD-L1 signaling.

Chemical carcinogens

Chemical exposure
       ↓
DNA adduct / DNA damage
       ↓
Mutation in oncogene or tumor-suppressor gene
       ↓
Abnormal cell survival and proliferation
       ↓
Cancer

Types of chemical carcinogens

1. Direct-acting carcinogens

• Often electrophilic compounds that directly damage DNA.
• Important examples include some alkylating agents used in chemotherapy.
• They can cause therapy-related cancers, especially leukemia, after a latent period.

Direct-acting chemical → directly reacts with DNA

2. Indirect-acting carcinogens, or procarcinogens

• Commonly activated by the cytochrome P450 enzyme system.
• Individual variation in these enzymes can influence susceptibility.

Procarcinogen
     ↓ CYP450 metabolism
Ultimate carcinogen
     ↓
DNA damage / mutation

Important examples and associations

Initiation, promotion, and progression

1. Initiation

• It is generally irreversible.
• The damage becomes fixed as a mutation when the cell divides.
• An initiator alone may not be enough to produce a tumor.

Initiator → DNA mutation → initiated cell

2. Promotion

• Promoters are generally not directly mutagenic.
• They stimulate cell proliferation, survival, inflammation, or altered signaling.
• Promotion usually requires repeated or sustained exposure.
• It is relatively reversible early on if exposure stops.

Initiated cell
      + repeated promoter exposure
      ↓
clonal expansion
      ↓
preneoplastic lesion

3. Progression

• Faster growth
• Resistance to apoptosis
• Angiogenesis
• Invasion
• Metastasis
• Treatment resistance

Initiation → Promotion → Progression → Malignant cancer

High-yield comparison

Easy memory line

PCR

Ingredients needed

• Template DNA containing the target sequence
• Two primers: short DNA sequences that mark the target region
• Taq polymerase: heat-stable DNA polymerase
• dNTPs: A, T, G, C building blocks
• Buffer and Mg²⁺

Three steps in each PCR cycle

Double-stranded DNA
        ↓ heat
Two single DNA strands
        ↓ cooling
Primers attach
        ↓ Taq polymerase
New DNA copies made

1 DNA target → 2 → 4 → 8 → 16 → ...

Uses of PCR

• Detecting infections with DNA-containing organisms
• Detecting mutations and inherited disorders
• Forensic testing and paternity testing
• DNA sequencing and cloning
• Cancer mutation testing

RT-PCR

Principle

RNA
 ↓ reverse transcriptase
cDNA
 ↓ PCR
Millions of DNA copies

Steps

1. Extract RNA from the sample.
2. Use reverse transcriptase to make cDNA.
3. Amplify the cDNA using standard PCR cycles:
   • Denaturation
   • Annealing
   • Extension

Uses of reverse-transcription PCR

• Detecting RNA viruses, such as influenza or SARS-CoV-2
• Measuring gene expression through mRNA detection
• Detecting RNA fusion transcripts, such as BCR-ABL in chronic myeloid leukemia

PCR vs RT-PCR

RT-PCR versus real-time PCR: common confusion

• RT-PCR = Reverse-transcription PCR: RNA → cDNA → PCR.
• Real-time PCR / qPCR = PCR product is measured during every cycle using fluorescence.
• Real-time RT-PCR / RT-qPCR = RNA is first converted to cDNA, then amplified and measured in real time. This is widely used for RNA-virus testing.

RT-PCR:     RNA → cDNA → endpoint PCR result

qPCR:       DNA → PCR + fluorescence-based quantification

RT-qPCR:    RNA → cDNA → real-time fluorescent PCR

High-yield line