DNA Replication, Repair, and Mutations

DNA Replication Mechanisms

• Semi-conservative replication occurs via DNA polymerase III (main enzyme) synthesizing leading strand continuously and lagging strand discontinuously as Okazaki fragments (1000-2000 nucleotides in eukaryotes); primase creates RNA primers every 100-200 bp requiring removal and replacement
• Semi-discontinuous synthesis creates inherent asymmetry: leading strand (3'→5' direction) replicates processively while lagging strand requires multiple primers and DNA ligase sealing; this asymmetry is the basis of the end-replication problem
• Replication fork movement at ~1000 nucleotides/second requires topoisomerases (Type I and II) to relieve tension from DNA unwinding by DNA helicase; failure causes replication stress and fork collapse

The End-Replication Problem and Telomeres

• Telomerase deficiency causes progressive telomere shortening (50-200 bp per division) because DNA polymerase cannot replicate the 5' end of the lagging strand; after 50-70 divisions (Hayflick limit), critically short telomeres trigger senescence or apoptosis
• Telomerase (reverse transcriptase in germline/stem cells) adds TTAGGG repeats using its RNA template; somatic cells lack sufficient telomerase, explaining replicative senescence as aging mechanism
• Cancer escape mechanism: telomerase reactivation (85-95% of cancers) or ALT pathway (5-15% of cancers) allows unlimited replication

DNA Repair Pathways - Critical for Maintaining Genomic Stability

• Nucleotide Excision Repair (NER) removes bulky lesions (UV-induced pyrimidine dimers, benzo[a]pyrene adducts): XPA-XPG proteins form excision complex removing 24-32 nucleotide segment; defects cause xeroderma pigmentosum (XP) with 1000-fold increased skin cancer risk
• Base Excision Repair (BER) corrects small, non-distorting lesions (oxidative damage, alkylation, deamination): DNA glycosylase recognizes abnormal base creating AP site, endonuclease removes sugar-phosphate, polymerase fills gap, ligase seals; handles ~10,000 lesions/cell/day
• Mismatch Repair (MMR) recognizes errors post-replication (mismatched bases, insertions/deletions in repetitive sequences): MutS homologs (MSH2-MSH6, MSH2-MSH3) identify error, MutL complex removes newly synthesized strand; defects cause Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC) with 70% lifetime colorectal cancer risk
• Double-Strand Break (DSB) Repair via two pathways: Non-Homologous End Joining (NHEJ) quickly ligates broken ends (error-prone, active throughout cell cycle, preferred in G1); Homologous Recombination (HR) uses sister chromatid as template for error-free repair (S/G2 phase only, requires BRCA1/BRCA2 and RAD51)
• ATM and ATR checkpoint kinases detect DNA damage and halt cell cycle via p53 activation ("guardian of the genome"), allowing repair before S phase or triggering apoptosis if damage is irreparable

Mutation Types and Molecular Consequences

• Point mutations (substitutions): missense (altered amino acid), nonsense (premature stop codon), silent; transition mutations (purine↔purine or pyrimidine↔pyrimidine) occur more frequently than transversions due to spontaneous deamination of methylated cytosine→thymine
• Insertion/deletion mutations cause frameshift if number not divisible by 3, catastrophically altering downstream sequence; microsatellite instability from MMR defects causes repetitive sequence expansions
• Chromosomal rearrangements: translocations (balanced vs. unbalanced), inversions, deletions cause gene dosage imbalance or oncogenic fusion proteins (Philadelphia chromosome t(9;22) BCR-ABL in CML)

Hereditary Cancer Syndromes from Repair Defects

• Lynch Syndrome (HNPCC) - mucinous colorectal cancer typically presenting before age 50, increased endometrial/ovarian/gastric cancer risk; patients often asymptomatic until advanced disease with hematochezia or weight loss; autosomal dominant inheritance
• Hereditary Breast/Ovarian Cancer (HBOC) - BRCA1/BRCA2 mutations cause 45-87% lifetime breast cancer risk and 40-46% ovarian cancer risk; younger age at diagnosis (30s-50s vs. 60s-70s in sporadic); classic presentation includes premenopausal bilateral breast cancer or early-onset triple-negative breast cancer

DNA Repair Disorder Phenotypes

• Xeroderma Pigmentosum - severe photosensitivity with erythema/blistering after minimal sun exposure, early onset (childhood) freckling and multiple skin cancers (basal cell, squamous cell, melanoma); progressive neurologic degeneration in 30-40% due to accumulated DNA damage in neurons; presents with sunburn-like reaction after 10-15 minutes outdoor exposure vs. normal 30-45 minutes
• Ataxia-Telangiectasia (A-T) - progressive cerebellar ataxia (age 2-3), oculomotor apraxia, telangiectasia (spider angiomas), immunodeficiency, 1000-fold increased lymphoid malignancy risk; ATM mutations impair DSB detection causing radiation hypersensitivity
• Werner Syndrome - premature aging phenotype with early graying/alopecia, cataracts, osteoporosis, and cancer predisposition (sarcomas, hematologic malignancies) in 3rd-4th decade; caused by RECQ helicase defect impairing DNA repair

Replication Stress and Cancer Manifestations

• BRCA-deficient tumors present with genomic instability (high mutation burden), often triple-negative breast cancer morphology; increased homologous recombination deficiency can cause sensitivity to PARP inhibitors
• Microsatellite-unstable (MSI) colorectal cancers from MMR defects often present with mucinous or poorly differentiated histology, immune infiltration, and better response to checkpoint immunotherapy despite worse conventional chemotherapy response

Recognizing Repair Pathway Defects - Diagnostic Approach

• Clinical suspicion from family history - multiple family members with early-onset cancers (especially bilateral breast cancer, colorectal cancer <50 years, ovarian cancer at any age), or multiple cancer types in single individual; Amsterdam criteria (≥3 CRC relatives, one <50 years, spanning ≥2 generations) suggests Lynch syndrome
• Genetic testing - germline mutation analysis of repair genes: BRCA1/BRCA2 (most common inherited breast/ovarian cancer predisposition), mismatch repair genes (MLH1, MSH2, MSH6, PMS2) for Lynch syndrome; testing typically offered if >10% pretest probability based on personal/family history
• Immunohistochemistry (IHC) on tumor tissue - screens for MMR protein loss (absent MLH1, PMS2, MSH2, or MSH6) suggesting Lynch syndrome; BRCA1 loss also assessable by IHC but not routinely used for screening
• Microsatellite Instability (MSI) testing - tumor DNA analysis showing expansion of microsatellite repeats indicating MMR deficiency; higher MSI (MSI-H) correlates with better checkpoint inhibitor response and Lynch syndrome

Replication fidelity — the enzymes tested

• All DNA polymerases synthesize 5'→3' while reading the template 3'→5', and all require a free 3'-OH — hence the absolute need for primase-made RNA primers.
• Proofreading is the 3'→5' exonuclease activity intrinsic to the replicative polymerase (prokaryotic pol III; eukaryotic pol δ/ε). A separate 5'→3' exonuclease (prokaryotic pol I; FEN1/RNase H in eukaryotes) excises the RNA primer. A stem describing loss of proofreading points to a hypermutator phenotype, not to a repair-pathway syndrome.
• Telomerase is a reverse transcriptase carrying its own RNA template — the classic distractor is calling it a DNA-dependent DNA polymerase.

Match the lesion to the pathway

• Bulky, helix-distorting lesions (UV pyrimidine dimers, polycyclic aromatic adducts) → nucleotide excision repair; defect = xeroderma pigmentosum. Deamination, oxidation, and alkylation of a single base → base excision repair. Post-replicative mismatches and slipped repeats → mismatch repair. Getting this triad straight is the single most tested distinction in this topic.
• Spontaneous deamination of 5-methylcytosine yields thymine, explaining why CpG dinucleotides are transition-mutation hotspots.

Best next steps

• Newly diagnosed colorectal cancer: NCCN recommends universal tumor screening with MMR immunohistochemistry and/or MSI testing regardless of age or family history; isolated MLH1 loss should prompt BRAF V600E and MLH1 promoter methylation testing to exclude a sporadic epigenetic cause before calling it Lynch syndrome. Confirmed Lynch syndrome carriers enter frequent surveillance colonoscopy beginning in the early 20s per NCCN.
• Family history suggestive of BRCA-related cancer: USPSTF advises a brief familial-risk assessment tool first, then referral for genetic counseling and testing — not reflexive population-wide sequencing.

Therapeutic associations

• Homologous recombination deficiency (BRCA1/2) → PARP inhibitor (olaparib) via synthetic lethality.
• MSI-high/dMMR tumors → PD-1 blockade (pembrolizumab), a tissue-agnostic indication driven by high neoantigen burden.
• Ataxia-telangiectasia: minimize ionizing radiation, including diagnostic CT; expect low IgA and elevated alpha-fetoprotein.