DNA Replication, Repair, and Transcription: Key Concepts for Biology

Why nucleosides/nucleotides matter

Nucleotides (base, sugar, phosphate) are DNA/RNA monomers; nucleosides (base + sugar) are precursors and drug scaffolds (e.g., antivirals/anticancer).

DNA directionality

Polynucleotides have distinct ends: 5′ phosphate and 3′ hydroxyl; sequences written 5′→3′.

DNA double helix essentials

Right-handed, ~10-10.5 bp/turn; antiparallel, complementary strands; stabilized by H-bonds and base stacking (hydrophobic).

Chargaff's rules

A=T, G=C; purines=pyrimidines. A-T has 2 H-bonds; G-C has 3 H-bonds (stronger).

Glycosidic bond vs phosphodiester bond

Glycosidic: base (N9 purines / N1 pyrimidines) to sugar C1′. Phosphodiester: 3′-OH of one sugar to 5′-phosphate of next. Targets: glycosylases (DNA repair) vs nucleases.

Intercalating agents (EtBr, acridine orange, actinomycin D, SYBR/Cyber Green)

Flat aromatic molecules insert between base pairs (often minor groove), distort helix, mutagenic/carcinogenic; used as DNA stains or drugs.

Key replication enzymes (E. coli)

DnaA (origin recognition), helicase (unwinds, ATP), SSB (protects ssDNA), primase (RNA primers), DNA Pol III (5′→3′ polymerase; 3′→5′ exonuclease proofreading), DNA Pol I (5′→3′ exonuclease removes primers, also polymerase and 3′→5′ exonuclease), ligase (seals nicks), topoisomerases I/II (relieve supercoils).

Replication fork problems & solutions

Open helix (helicase), protect ssDNA (SSB), prime synthesis (primase), extend (Pol III), remove primers/fill gaps (Pol I), seal nicks (ligase), relieve supercoils (topoisomerases).

DNA polymerase requirements

Needs template, primer with 3′-OH; synthesizes 5′→3′; adds dNTPs; key is 3′-OH for nucleophilic attack.

Proofreading

DNA Pol III and Pol I possess 3′→5′ exonuclease to remove misincorporated nucleotides, lowering error rate.

Topoisomerases and drugs

Topo I: single-strand nicks; Topo II (DNA gyrase in bacteria): double-strand breaks. Quinolones (ciprofloxacin) target gyrase; euk drugs: etoposide (Topo II), camptothecin/indenoisoquinolines (Topo I).

Eukaryotic replication differences

Multiple origins; S-phase only; polymerases: Pol ε (leading), Pol δ & α (lagging/priming with primase), RNase H removes RNA primers; ligase; topoisomerases.

Chromatin compaction levels

Naked DNA → nucleosomes (histone octamer) → 30Inm fiber (nucleofilament) → higher-order loops/scaffold → chromosome. DNA wraps around (H3-H4)2 tetramer + two H2A-H2B dimers.

Telomerase & telomeres

Telomerase (reverse transcriptase) extends 3′ ends using its RNA template (adds TTAGGG repeats; telomerase RNA carries AAUCCC). Maintains linear chromosome ends.

Reverse transcriptase (RT) activities

a) RNA-dependent DNA polymerase (5′→3′), b) RNase H (5′→3′ exonuclease degrades RNA in RNA:DNA hybrid), c) DNA-dependent DNA Pol (5′→3′). Basis for cDNA synthesis.

Inhibiting DNA synthesis: nucleoside analogs

Modify 3′-OH (e.g., Ara-C cytarabine; Ara-A vidarabine; ddA dideoxyadenosine) to terminate DNA synthesis. HIV antivirals exploit high RT affinity & poor repair removal.

Cisplatin and DNA crosslinks (chemo)

Cisplatin crosslinks adjacent purines (e.g., guanines), distorting DNA; blocks replication/transcription → apoptosis; used in cancer therapy.

Replication error rates with/without proofreading

Without: ~10I³ (1/1,000 bp). With proofreading: ~10II (1/1,000,000 bp).

Common DNA damage types

Mismatch; base alterations (deamination/methylation, oxidation to 8IoxoG, alkylation), depurination/depyrimidination (AP sites), thymine dimers (UV), double-strand breaks (ionizing radiation).

Deamination examples

C→U (then repaired), 5ImC→T (C:G to T:A transition; most common point mutation in cancer); A→hypoxanthine (pairs with C).

Oxidation: 8IoxoG

ROS convert G to 8IoxoG, which pairs with A → G:C→T:A transversion.

Alkylation and cytochrome P450

P450 activates procarcinogens (e.g., vinyl chloride, styrene) to DNA-alkylating agents → adducts and mutations.

Thymine dimers & DSBs

UV causes pyrimidine dimers; ionizing radiation causes double-strand breaks and ROS damage.

Cell-cycle checkpoints & DNA damage

G1 & G2 arrest allow repair. ATM kinase senses DSBs/replication stress; activates p53→p21 to halt cycle.

Rb/E2F in G1→S

Hypophosphorylated Rb binds E2F to block S-phase genes; late G1 phosphorylation (Cyclin D1/CDK) releases E2F → S-phase entry.

Direct reversal repair (MGMT)

O6-methylguanine repaired by MGMT (O6-methylguanine-DNA methyltransferase) via direct removal—no backbone cutting/template needed.

Mismatch repair (MMR)

Steps: recognize mismatch (new strand often unmethylated), excise segment, fill gap with DNA Pol, seal with ligase. Defects → HNPCC (Lynch syndrome).

Base excision repair (BER)

Glycosylase removes damaged base (creates AP site) → AP endonuclease → Pol fills → ligase seals.

Nucleotide excision repair (NER)

Excinuclease removes bulky lesions (e.g., UV thymine dimers) → DNA Pol fills (5′→3′) → ligase forms phosphodiester bond. Defects → Xeroderma pigmentosum (XP).

DNA repair & cancer syndromes

HNPCC: MMR genes; XP: NER genes; AT: ATM defects; BRCA1/2: homologous recombination repair of DSBs → high breast/ovarian cancer risk.

Ames test (mutagenicity)

Uses hisI Salmonella; inclusion of rat liver S9 (P450) to activate procarcinogens; revertant colony counts indicate mutagenicity.

Proto-oncogene → oncogene mechanisms

Gene amplification; translocation (new promoter/enhancer); point mutation in control region or coding sequence → overexpression or hyperactive protein (gain-of-function).

Tumor suppressor genes

Loss-of-function in both alleles; functions: DNA repair, cell adhesion, cell-cycle inhibition (e.g., p53, Rb).

General features of transcription

Catalyzed by RNA Pol; template is DNA antisense; product matches coding strand (U for T); uses NTPs; no primer; proceeds 5′→3′; Mg²I/Mn²I required; highly selective.

Sigma (σ) factor role

Part of bacterial RNA Pol holoenzyme; lowers non-specific DNA affinity, scans for promoters, identifies −10 and −35; dissociates after initiation to recycle.

Bacterial promoter elements

−10 Pribnow box (TATAAT) and −35 element; position RNA Pol for initiation site.

Transcription termination

Rho-independent: GC-rich hairpin + U-run destabilizes RNA-DNA hybrid. Rho-dependent: Rho helicase binds C-rich RNA, uses ATP to dislodge polymerase.

Eukaryotic promoters & elements

TATA box (~−25), CAAT (~−40 to −150), GC boxes; recognized by GTFs (not Pol II directly). Enhancers: distal cis-elements acting over kb distances.

Typical human protein-coding gene map

Upstream promoter/proximal elements → TSS → 5′ UTR → start codon → exons/introns → stop codon → 3′ UTR → poly(A) signal/termination.

rRNA processing (45S precursor)

Eukaryotic 45S pre-rRNA processed to 28S, 18S, 5.8S; spacers removed. (E. coli: 30S pre-rRNA.) 'S' is sedimentation unit.

tRNA processing

Cleavage, addition (CCA by tRNA nucleotidyl transferase), base modifications (e.g., pseudouridine, methylation, dihydrouridine, inosine).

Ribozymes

Catalytic RNAs (self-splicing introns, ribonuclease-like activity); catalyze cleavage/ligation of RNA; lower k_cat vs protein enzymes; no protein required.

mRNA 5′ cap

7-methylguanosine cap via 5′-5′ linkage; promotes translation initiation (eIF4E binding), stabilizes mRNA.

Poly(A) tail

20-200 As added to 3′ end; increases stability and nuclear export.

Splicing basics (GU-AG rule)

Introns begin with GU, end with AG; spliceosome (snRNPs) recognizes donor/acceptor and catalyzes two-step transesterification; some introns self-splice (ribozymes).

miRNA/siRNA biogenesis & function

Dicer cleaves dsRNA/pre-miRNA to ~21-nt duplexes; RISC uses guide strand to silence targets. siRNA: immune/experimental; miRNA: endogenous, imperfect pairing in animals.

Transcription inhibitors

Rifampin (binds bacterial RNA Pol, blocks initiation/elongation; TB; resistance via mutation). Actinomycin D intercalates DNA template. αIAmanitin inhibits eukaryotic Pol II.

Genetic code properties

Universal (mitochondrial exceptions), specific (unambiguous), redundant (degenerate), nonoverlapping/commaless; AUG start codon.

Coding mutations examples

Nonsense: Hb McKees Rocks (β-chain Tyr→stop) → short β-globin (↑O2 affinity, ↓delivery). Missense: Sickle cell anemia (βE6 Glu→Val) → polymerization.

Wobble hypothesis

First anticodon base determines wobble (e.g., inosine reads multiple codons); first two mRNA bases are most critical for tRNA specificity.

AminoacylItRNA synthetase (aaRS) fidelity

Two-step charging: AA + ATP → aminoacylIAMP; transfer to tRNA → aminoacylItRNA. Editing site hydrolyzes misactivated AA/tRNA; costs two highIenergy bonds (ATP→AMP+PPi).

Bacterial translation initiation

Shine-Dalgarno sequence in 5′ UTR base-pairs with 16S rRNA to position AUG in P-site; IFs assemble 30S+50S; starts ≥25 nt from 5′ end.

Elongation cycle

EFITu delivers aaItRNA to A-site (GTP); peptidyltransferase (23S/28S rRNA ribozyme) forms peptide bond; EFIG drives translocation (GTP); repeats.

Termination

Stop codon in A-site recruits RFI1 (UAA/UAG), RFI2 (UGA/UAA), RFI3IGTP; hydrolysis releases peptide; ribosome components recycled.

Eukaryotic initiation (PIC)

40S preinitiation complex (MetItRNAi + eIF2) binds 5′ cap via eIF4E, scans to AUG; 60S joins to form 80S initiation complex.

Protein targeting: SRP pathway

Signal peptide emerges → SRP binds peptide+ribosome (pauses translation) → SRP receptor docks ribosome to RER → translocation; signal peptidase cleaves; synthesis resumes into ER lumen.

Antibiotics/toxins affecting translation

Streptomycin (binds 16S, blocks fMetItRNAf binding/initiation, bacteria); Tetracyclines (block A-site entry, bacteria); Chloramphenicol (peptidyltransferase, 50S); Clindamycin/Erythromycin (block translocation, 50S); Puromycin (aaItRNA mimic, chain termination); Diphtheria toxin (inactivates eEFI2); Ricin (depurinates 28S rRNA at SRL, inactivates ribosome).

PostItranslational modifications

Phosphorylation (Ser/Thr/Tyr), hydroxylation (Pro; collagen; ER), glycosylation (OI/NIlinked), biotinylation, prenylation (farnesylation), ubiquitination, etc.

Ubiquitin-proteasome pathway

PolyIubiquitination (e.g., K48) targets proteins to 26S proteasome (19S regulatory caps + 20S core; ATPIdependent) for degradation.

lac operon regulators (negative & positive)

Repressor binds operator (blocks transcription; allolactose inducer releases it). CAP-cAMP binds CAP site to activate transcription when glucose is low (glucose inhibits adenylyl cyclase → low cAMP).

cisI vs transIacting elements

cis: DNA sequences on same molecule (promoters, enhancers, operators). trans: proteins (TFs) that bind cis elements to regulate transcription.

Nuclear hormone receptors

LipidIsoluble hormones cross membrane, bind receptors that act as TFs in nucleus; receptors bind hormone response elements (HREs) via conserved DNAIbinding and ligandIbinding domains.

CellIsurface receptors & CREB

WaterIsoluble hormones (insulin, epinephrine, glucagon) trigger second messengers (cAMP, cGMP, Ca²I); PKA phosphorylates CREB → binds CRE to activate gene transcription.

Alternative splicing impact

≥80% of human genes undergo tissue-specific splicing → >100k proteins from ~20-30k genes; mis-splicing implicated in cancer and protein disorder.

RNA editing of ApoB (C→U)

APOBEC1 deaminates C→U in apoB pre-mRNA to create stop codon: ApoB48 (intestine; chylomicrons) vs ApoB100 (liver; LDL receptor binding).

Iron regulation: TfR vs ferritin

IRPs bind IREs: at 3′ UTR (TfR) binding stabilizes mRNA → ↑translation; at 5′ UTR (ferritin) binding blocks translation. Low iron → IRP binds IRE; high iron releases IRP.

Epigenetics—histone acetylation

HAT adds acetyl to Lys → neutralizes positive charge, loosens DNA-histone interaction → open chromatin, ↑transcription; HDAC removes acetyl → closed chromatin.

DNA methylation (CpG) & disease

DNMT adds methyl to C5 of CpG; associated with transcriptional repression and long-term silencing. Hypermethylation can inactivate tumor suppressors; meCpG→TpG accounts for many p53 mutations; Fragile X: CGG expansion → promoter hypermethylation → silencing FMR1.

Drugs targeting methylation

Cytidine analogs (e.g., 5-azacytidine) incorporate into DNA; trap DNMTs → passive demethylation; used in myelodysplastic syndrome.

Restriction endonucleases (EcoRI)

Bacterial enzymes cut specific palindromic DNA sites; awarded 1978 Nobel (Arber, Nathans, Smith). Naming: genus, species/strain, order of discovery.

Recombinant DNA & ligase

Cut vector and insert with same enzyme to create compatible sticky ends; DNA ligase forms phosphodiester bonds; foundational to cloning (1980 Nobel to Paul Berg et al.).

Reverse transcriptase & cDNA libraries

cDNA made from mRNA by RT → lacks introns and upstream regulatory regions; represents expressed genes.

Cloning workflow

1) Cut plasmid and insert with same restriction enzyme. 2) Ligate to make recombinant plasmid. 3) Transform host. 4) Select on antibiotic. 5) Screen colonies (e.g., PCR).

PCR essentials

Template, primers, dNTPs, thermostable DNA Pol (Taq). Steps: denature, anneal primers to flanking regions, extend. Exponential amplification in 20-30 cycles.

Gel electrophoresis basics

DNA is negatively charged; shorter fragments migrate farther. Visualize with intercalating dyes (e.g., ethidium bromide) under UV.

Southern/Northern/Western blots

Southern: DNA; Northern: RNA; Western: Protein (SDS-PAGE → transfer → antibody detection via fluorescence or HRP chemiluminescence).

qPCR (real-time PCR)

Fluorescent reporters (e.g., SYBR Green) enable real-time quantification of DNA/cDNA/RNA during amplification.

SDS-PAGE

SDS denatures proteins and imparts uniform negative charge; PAGE separates by size (mass).

Western blot detection

Transfer proteins to membrane; detect with labeled antibodies (fluorophore or HRP + chemiluminescent substrate).

ELISA

Plate-based immunoassay to detect/quantify native antigens (e.g., SARS-CoV-2 Spike) using enzyme-linked antibodies.

SARS-CoV-2 diagnostics overview

TaqPath RT-PCR kit targets ORF1ab, N, S genes. Antigen rapid tests use antibody-antigen binding.

COVID therapeutics

Remdesivir: nucleoside analog targeting viral RNA polymerase. Dexamethasone: anti-inflammatory corticosteroid. Monoclonal antibodies: anti-Spike (e.g., casirivimab+imdevimab). Paxlovid: nirmatrelvir (Mpro inhibitor) + ritonavir.

Vaccine types (COVID)

mRNA (Pfizer, Moderna), viral vector-based, protein-based. mRNA encodes Spike → translated in cells → immune response.

Genome editing & CRISPR-Cas9

Cas9 is RNA-guided endonuclease; requires PAM adjacent to target; bacteria origin (antiviral defense). Enables targeted additions/deletions/modifications.

DNA helix: bp/turn & handedness

B-DNA is right-handed with ~10-10.5 base pairs per turn under physiological conditions.

Antiparallel orientation

One strand runs 5′→3′ while the complementary strand runs 3′→5′.

Base stacking stabilization

Hydrophobic interactions and van der Waals forces between adjacent aromatic bases stabilize the helix.

Phosphodiester linkage direction

The backbone connects 3′-OH of one nucleotide to the 5′-phosphate of the next.

Glycosidic bond specificity

N9 of purines (A,G) or N1 of pyrimidines (C,T) to sugar C1′; target of depurination/depyrimidination.

Intercalators—examples & risks

Ethidium bromide, acridine orange, actinomycin D, SYBR/Cyber Green; distort DNA, frameshift mutations, carcinogenic potential.

Helicase function & energy

Unwinds duplex DNA at forks using ATP hydrolysis; creates ssDNA templates.

SSB protein roles

Binds ssDNA to prevent reannealing, protects from nucleases, and removes secondary structure.

Primase role

Synthesizes short RNA primers to provide 3′-OH for DNA polymerase initiation.

Okazaki fragments

Short DNA fragments synthesized discontinuously on the lagging strand, later joined by ligase.

DNA ligase mechanism

Seals nicks by forming phosphodiester bonds; uses ATP (or NADI in bacteria).

DNA Pol I—three activities

5′→3′ exonuclease (primer removal), 5′→3′ polymerase (fill-in), 3′→5′ exonuclease (proofreading).

DNA Pol III core functions

Primary replicative polymerase in E. coli; high processivity; 5′→3′ synthesis and 3′→5′ proofreading.

Topo I vs Topo II covalent intermediates

Both form transient covalent bonds with DNA phosphates; Topo I cuts 1 strand, Topo II cuts both strands; energy stored to reseal breaks.

Quinolones (ciprofloxacin) target

Inhibit bacterial DNA gyrase (Topo II), blocking negative supercoiling and leading to DNA breaks.

Eukaryotic topo inhibitors

Etoposide inhibits Topo II; Camptothecin and indenoisoquinolines inhibit Topo I to treat cancers.