Lecture 1 - DNA replication, mutation and repair

DNA replication

• The duplication of chromosomal DNA before cell division
• Ensures both daughter cells receive complete genomes
• Occurs mainly in proliferative cells (e.g., skin, bone marrow, mucosa)
• Mitochondrial DNA replication is independent of cell division

Cell cycle and DNA replication

• Cells alternate between interphase and mitotic (M) phase
• Interphase includes G1, S, and G2 phases
• M phase = mitosis + cytokinesis
• DNA replication occurs during the S phase

Semiconservative replication

• Each new DNA double helix contains one original and one new strand
• Original strands act as templates for new complementary strands
• Occurs at the replication fork where strands separate

Phases of DNA replication

• Initiation – origin recognition and strand separation
• Elongation – synthesis of new strands
• Termination – replication completion at chromosomal ends

Initiation: overview

• Origins of replication are defined after cytokinesis (in G1)
• Origin Recognition Complex (ORC) binds to DNA
• Pre-replicative complex (Pre-RC) forms with MCM helicase loading
• Pre-initiation complex (Pre-IC) activates in S phase via CDK and DDK

Origin Recognition Complex (ORC)

• Made of six proteins (ORC1–ORC6)
• Binds to DNA at replication origins
• Anchors other replication proteins
• Cdc6 and Cdt1 help load MCM2–7 helicase

Activation of replication origins

• S-phase kinases (CDK and DDK) phosphorylate Pre-RC components
• Phosphorylation triggers binding of GINS and Cdc45
• Pre-IC forms, enabling DNA unwinding and synthesis start
• Geminin prevents reactivation of origins within one cell cycle

Direction of DNA synthesis

• Always occurs in the 5′ → 3′ direction
• Requires a primer to provide a free 3′-OH group
• Template is read in the 3′ → 5′ direction

Primase and primer formation

• Primase synthesizes short RNA primers (7–12 nucleotides)
• Primase = RNA polymerase
• DNA polymerase α extends primer with ~20 nucleotides of DNA
• Main synthesis continues with polymerases δ and ε

Leading and lagging strands

• Leading strand: synthesized continuously (polymerase ε)
• Lagging strand: synthesized discontinuously in Okazaki fragments (polymerase δ)
• Lagging strand requires multiple primers
• Fragments later joined by DNA ligase

Okazaki fragments

• Short DNA fragments (~150–200 bp) on lagging strand
• Each begins with an RNA primer
• RNA primer later removed and replaced with DNA
• Joined by DNA ligase to form continuous strand

PCNA function

• Acts as a sliding clamp for DNA polymerases
• Prevents polymerase dissociation from the template
• Increases processivity during synthesis

Topoisomerases

• Relieve DNA supercoiling ahead of replication fork
• Type I cuts one strand, allows rotation, then reseals
• Type II cuts both strands, passes one through, then reseals

Termination of replication

• Neighboring replication bubbles meet and fuse
• Leading and lagging strands from adjacent regions join
• Requires DNA ligase to complete continuity

Telomeres

• Chromosome ends composed of TTAGGG repeats
• Protect coding DNA from loss during replication
• Contain a 3′ single-stranded overhang

Telomerase complex

• Enzyme adds DNA repeats to chromosome ends
• Has its own RNA template
• Acts as reverse transcriptase (RNA → DNA)
• Extends the 3′ overhang so polymerase can complete the strand

Telomerase activity

• High during embryonic development
• Retained in stem cells, skin, mucosa, bone marrow, and germ cells
• Normally inhibited in somatic cells by telomerase inhibitors
• Reactivated in cancer cells, enabling unlimited division

Telomere shortening and ageing

• Shortening triggers “DNA damage” response
• Leads to cell cycle arrest and senescence
• Impairs tissue regeneration and promotes inflammation
• Contributes to ageing and degenerative diseases

Mutation definition

• Change in DNA sequence affecting a few nucleotides
• Can be spontaneous or induced by external agents
• Usually occurs in somatic cells
• Frequency <1% in population

Types of DNA changes

• Small: point mutations or indels
• Large: chromosomal deletions, inversions, translocations, amplifications
• Example: Philadelphia chromosome (BCR-ABL fusion) → leukemia

Point mutation types

• Transition: purine ↔ purine or pyrimidine ↔ pyrimidine
• Transversion: purine ↔ pyrimidine

Indel mutations

• Insertion or deletion of 1–few nucleotides
• Can cause frameshift or codon changes

Causes of mutation

• Spontaneous (replication errors, tautomeric shifts)
• Chemical mutagens (base modifications, intercalating agents)
• Physical mutagens (UV, heat, ionizing radiation)

Replication errors

• Mismatch – incorrect nucleotide incorporation
• Replication slippage – strand misalignment in repetitive sequences
• Proofreading by DNA polymerase corrects most errors

Proofreading

• DNA polymerases δ and ε have 3′–5′ exonuclease activity
• Remove incorrect nucleotides before continuation
• Ensures high-fidelity DNA replication

Replication slippage

• Template and new strand misalign during synthesis
• Can cause insertion or deletion of repeats
• Common in microsatellite regions

Huntington disease

• Caused by CAG trinucleotide repeat expansion
• Leads to neuron degeneration
• Symptoms: chorea, cognitive decline, mood changes
• Earlier onset with more repeats

Chemical mutagens: deaminating agents

• Convert bases by removing amino groups
• Cytosine → uracil (pairs with adenine)
• Adenine → hypoxanthine (pairs with cytosine)
• Guanine → xanthine (non-mutagenic but inhibits polymerase)

Chemical mutagens: alkylating agents

• Add alkyl groups to bases
• Can alter base pairing or block replication
• Examples: dimethylnitrosamine, EMS

Chemical mutagens: oxidation

• Produces 8-oxoguanine (pairs with adenine)
• Leads to G→T transversions
• Caused by reactive oxygen species or ionizing radiation

Intercalating agents

• Flat molecules inserting between base pairs
• Distort helix and block replication
• May cause insertions or deletions
• Examples: doxorubicin (antitumor), ellipticin, berberine

Physical mutagens: UV

• Induces pyrimidine dimers (especially thymine)
• Causes replication errors and deletions
• Linked to skin cancer

Physical mutagens: heat

• Breaks glycosidic bonds → apurinic/apyrimidinic (AP) sites
• Common (10,000+ per cell daily)
• Usually repaired efficiently

Effects of mutagens

• Carcinogens – initiate tumors
• Oncogens – promote tumor cell proliferation
• Mutagens – cause DNA changes
• Teratogens – cause developmental defects
• Clastogens – cause chromosomal breaks

Point mutation effects

• Synonymous – same amino acid
• Missense – different amino acid
• Nonsense – premature stop codon
• Readthrough – stop codon lost, longer protein

Indel mutation effects

• 1–2 bp change → frameshift
• 3 bp change → adds or removes one amino acid

Mutations in non-coding DNA

• Affect transcription regulation
• Can alter tissue specificity or expression level
• May cause aberrant splicing

Effects on organism

• Germline mutation → heritable
• Somatic mutation → non-heritable, can cause cancer
• Can be silent, loss-of-function, or gain-of-function

Beneficial mutations

• Source of genetic diversity and evolution
• Enable immune diversity via antibody gene hypermutation
• Somatic hypermutation in immunoglobulin genes increases antibody variability

DNA repair types

• Mismatch repair
• Base excision repair (BER)
• Nucleotide excision repair (NER)
• Repair of strand breaks (single or double)

Base excision repair (BER)

• Fixes chemically altered bases
• DNA glycosylase removes damaged base → AP site
• AP endonuclease cuts strand and removes nucleotides
• DNA polymerase β fills gap
• DNA ligase seals strand

Nucleotide excision repair (NER)

• Fixes bulky or helix-distorting damage
• Damaged strand cut on both sides
• Helicase unwinds

exonuclease removes 25–50 nucleotides
• DNA polymerase δ/ε fills gap
• DNA ligase joins ends

Mismatch repair

• Corrects replication errors (mismatches, indels)
• MSH proteins recognize mismatches
• Cleaves wrong strand 5′ to error
• Exonuclease removes segment
• DNA polymerase fills gap and ligase seals it

Single-strand break repair

• Caused by oxidative stress
• PARP1 marks breaks and recruits BER enzymes
• DNA polymerase and ligase repair the gap

Double-strand break repair

• Caused by ionizing radiation or replication errors
• Two mechanisms:
– Homologous recombination (accurate)
– Non-homologous end joining (error-prone)

Diseases caused by DNA repair defects

• Ataxia telangiectasia – ATM mutation

poor DNA repair, cancer risk
• Werner syndrome – helicase/exonuclease mutation

premature ageing
• Xeroderma pigmentosum – defective UV repair

extreme photosensitivity