Lecture 8 (MCB 450)

What is the difference between a transition and a transversion mutation?

• transition: one purine replaces another purine (A—G) or one pyrimidine replaces another pyrimidine (C—T)

• Transversion: a purine is replaced by a pyrimidine or vise versa (A/G ←→ C/T)

What is the most common base substitution reported in the lecture’s cancer genome studies?

• A C→T transition at CpG sites

• Cancer genomes can contain hundreds to thousands of mutations affecting known and newly discovered cancer genes

What are the major causes of DNA damage?

• spontaneous damage: loss of bases through depurination or depyrimidination

• Endogenous chemicals: reactive oxygen species (ROS), nitrous acid, and lipid peroxidation products

• Replication errors: DNA polymerase misincorporation and mismatches

• UV radiation: pyrimidine dimers

• Ionizing radiation: single and double strand breaks

• Chemical exposure: alkylating agents and environmental mutagens

What can happen if DNA damage is not repaired?

• it can cause mutations, genomic instability, and cancer

• The lecture estimates more than 20,000 DNA-damaging events and more than 10,000 replication errors per cell per day

How do alkylating agents and intercalating agents damage DNA differently?

• Alkylating agents: covalently add alkyl groups to DNA bases, potentially causing mispairing and strand breaks

• Intercalating agents: insert between adjacent base pairs, distorting and lengthening the DNA helix and interfering with replication and transcription

Examples of intercalating agents include ethidium bromide, acridine dyes, and actinomycin D

Why do cells activate cell-cycle checkpoints after DNA damage?

Checkpoints pause the cell cycle, giving the cell time to repair damage before continuing to divide

What are the G1 and G2 checkpoints?

G1 checkpoint: can delay progression into DNA synthesis (S phase) when conditions or DNA integrity are unsuitable

G2 checkpoint: can delay entry into mitosis, allowing time to address damage after DNA replication

How does the p53-p21 pathway help respond to DNA damage?

• DNA damage activates signaling involving ATM, a kinase

• The p53 protein promotes expression of p21, which inhibits cell-cycle progression and helps delay entry into S phase

What is the role of the Rb-E2F pathway in delaying S-phase progression?

• Rb (retinoblastoma protein) restrains E2F, a transcription factor that promotes expression of genes needed for DNA synthesis

• When Rb inhibits E2F, progression into S phase is restricted

• The key idea is that Rb-E2F controls access to S phase, while p53-p21 helps impose a damage-response checkpoint

What is direct reversal repair?

It repairs certain types of DNA damage by directly reversing the chemical modification rather than removing a stretch of nucleotides

• the lecture lists it as one of the major repair pathways

What is base excision repair (BER)?

• BER repairs small, damaged or chemically altered DNA bases

• The damaged base is removed, the resulting site is processes, DNA polymerase fills the gap, and DNA ligase seals the backbone

What is nucleotide excision repair (NER)?

• NER removes a short stretch of DNA containing bulky damage that distorts the double helix

• DNA polymerase fills the gab, and DNA ligase seals it

What is mismatch repair (MMR)?

MMR corrects mismatched bases and certain replication errors that remain after DNA synthesis

How can you distinguish the four repair pathways?

• direct reversal → reverses certain chemical modifications

• BER → removes small, damaged bases

• NER → removes bulky, helix-distorting damage

• MMR → corrects mismatches and replication errors

How does UV radiation damage DNA?

• UV light can cause adjacent pyrimidines, commonly neighboring thymines, to become cross-linked, forming a pyrimidine dimer

• This disrupts normal base pairing and distorts DNA

How does NER repair a UV-induced thymine dimer?

1. The damaged DNA region is recognized

2. An excision endonuclease (exinuclease) cuts out the damaged segment

3. DNA polymerase fills the gap in the 5’→3’ direction

4. DNA ligase seals the remaining nick

What is xeroderma pigmentosum (XP), and why does it increase skin-cancer risk?

• XP is a rare, autosomal recessive disease associated with defective nucleotide excision repair

• UV damage persists, mutations accumulate, and affected individuals have extreme photosensitivity and a high risk of skin cancer at a young age

How are repair defects linked to specific cancers?

• Xeroderma pigmentosum: defects in NER; increased UV-induced skin cancers

• Hereditary nonpolyposis colorectal cancer (HNPCC/Lynch syndrome): Defects in MMR; increased colorectal cancer risk

• Familial breast and ovarian cancer: BRCA1/BRCA2 defects impair homologous recombination repair of double-strand breaks

What is the difference between homologous recombination and non-homologous end joining?

• homologous recombination (HR): uses a homologous DNA template to repair a double-strand break; generally high fidelity

• Non-homologous end joining (NHEJ): Directly joins broken DNA ends without requiring a homologous template; can be error-prone

The lecture emphasizes that defects in different repair pathways predispose people to different cancer types

What is procarcinogen, and what does cytochrome P450 do?

• a procarcinogen is a precursor that becomes carcinogenic after metabolic activation in the body

• Cytochrome P450 enzymes can convert environmental chemicals into reactive compounds that damage DNA

How can activated procarcinogens lead to cancer?

• cytochrome P450 can convert a chemical into a reactive electrophile, which may form a DNA adduct (a chemical covalently bonded to DNA)

• If the damage is not repaired correctly, mutations may accumulate and contribute to cancer

• examples include: vinyl chloride and styrene, polycyclic aromatic hydrocarbons and aflatoxin B1

What is the principle of the Ames test?

• The Ames test uses mutant salmonella bacteria that cannot grow without histidine

• if a test chemical causes reverse mutations, some bacteria regain the ability to grow without added histidine

Why is rat liver preparation sometimes added to the Ames test, and how are results interpreted?

• rat liver enzymes, including cytochrome P450, mimic metabolic activation in mammals

• this can reveal chemicals that become mutagenic only after metabolism

• a significant increase in revertant bacterial colonies suggest the chemical or its metabolites are mutagenic and potentially carcinogenic

How do alkylating agents affect DNA?

They covalently add alkyl groups to DNA bases which can alter base pairing, cause mutations, and lead to single or double strand breaks

How do intercalating agents affect DNA?

They insert between stacked base pairs without forming covalent bonds to the bases. This distorts the helix, interferes with replication and transcription, and can block topoisomerase activity

What is the key different to remember between these two classes?

Alkylating agents chemically modify DNA bases; intercalating agents physically insert between base pairs and distort DNA structure

What is the difference between a proto-oncogene and an oncogene?

• proto-oncogene: a normal gene involved in cell growth and division

• oncogene: an activated or altered form that can excessively stimulate cell growth or division

How can proto-oncogene become an oncogene?

• point mutation: can create a hyperactive or degradation-resistant protein

• gene amplification: creates multiple copies of a gene, producing excess protein

• translocation or transposition: moves a gene under new regulatory control, potentially increasing its expression

These changes are typically gain-of-function effects

What do tumor-suppressor genes do, and how can their loss contribute to cancer?

• tumor-suppressor genes help prevent uncontrolled cell growth

• their proteins may repair DNA, regulate cell adhesion, or inhibit cell-cycle progression

• loss-of-function mutations can reduce these protective activities; the lecture notes defects in both copies as a common model

• examples include p53 and Rb

How do oncogene activation and tumor-suppressor loss work together in carcinogenesis?

• Oncogene activation can push cells to divide excessively, while loss of tumor-suppressor function removes important restraints on growth and damage responses

• together, these changes can promote uncontrolled cell division and cancer development