DNA Replication & Repair

Origins, strands, and the replication fork

Semiconservative and bidirectional. Each daughter double helix keeps one parental strand and gains one newly made strand. Replication moves outward from each origin in both directions, forming two replication forks. Prokaryotes use a single origin (oriC); eukaryotes use multiple origins per chromosome.

Why origins are AT-rich. An A–T pair is held by 2 hydrogen bonds and a G–C pair by 3, so AT-rich consensus sequences are the easiest place to pull the strands apart. Memory hook: "AT = Attach Two."

Opening and protecting the template. Helicase uses ATP to unwind the double helix at the fork, and single-stranded binding (SSB) proteins coat the exposed strands so they cannot reanneal. Unwinding overwinds the DNA ahead of the fork; topoisomerases relieve these supercoils by nicking and resealing the backbone (topo I cuts one strand, topo II — called DNA gyrase in bacteria — cuts both). A deficient helicase (BLM) causes Bloom syndrome.

Primase supplies the starting point. No DNA polymerase can start a chain de novo; each needs a free 3'-OH to extend. Primase lays down a short RNA primer to provide one, so every new strand begins as RNA — no primer, no polymerase. All DNA synthesis then proceeds 5'→3', adding nucleotides to the growing 3' end.

Leading vs lagging strand. The template strands are antiparallel and every polymerase works only 5'→3', so only one new strand can grow continuously. The leading strand is made continuously toward the fork from a single RNA primer. The lagging strand is made away from the fork in short Okazaki fragments, each needing its own primer, which are later stitched together.

Polymerases and finishing the strand

DNA polymerase III — the replicase. In prokaryotes, pol III does the bulk of synthesis on both strands. It pairs 5'→3' polymerase activity with a 3'→5' exonuclease that backs up and removes a mismatched base it has just added (proofreading), giving it the highest fidelity of the polymerases.

DNA polymerase I — primer replacement. Pol I carries all three activities: 5'→3' polymerase, 3'→5' proofreading exonuclease, and a 5'→3' exonuclease. That last activity lets it excise the RNA primer ahead of it while its polymerase fills the gap with DNA.

DNA ligase — the last step. Ligase forms the final phosphodiester bond, joining adjacent Okazaki fragments and sealing the remaining nick in the backbone.

Eukaryotic counterparts. Eukaryotes use a different set: pol α (bound to primase; starts each strand), pol δ (lagging strand), pol ε (leading strand), pol β (gap filling in base excision repair), and pol γ (mitochondrial DNA).

Telomerase. The last lagging-strand primer at a chromosome end cannot be replaced, so linear chromosomes shorten each cycle. Telomerase, a reverse transcriptase with its own RNA template, adds TTAGGG repeats to 3' ends; it is active in germ and stem cells and often reactivated in cancer.

Replication enzymes as drug targets

Fluoroquinolones. Ciprofloxacin and related drugs inhibit bacterial topoisomerase II (DNA gyrase) and topoisomerase IV, halting replication — the basis for their use in infections such as UTI and pyelonephritis. Toxicities to know: tendon rupture and QT prolongation; they are generally avoided in pregnancy and children because of cartilage damage.

Anticancer topoisomerase inhibitors. Human topo I is blocked by the "-tecans" and human topo II by etoposide/teniposide; the trapped enzyme leaves strand breaks that kill dividing cells.

Chain terminators. Nucleoside analogs that lack a usable 3'-OH (e.g., activated acyclovir, zidovudine and other NRTIs) are incorporated into the growing strand and stop elongation, because the next phosphodiester bond cannot form.

DNA repair pathways

Nucleotide excision repair (NER). Removes bulky, helix-distorting lesions, classically UV-induced pyrimidine dimers. Endonucleases cut out the damaged oligonucleotide, DNA polymerase fills the gap, and ligase seals it. NER operates in G1.

Base excision repair (BER). Fixes a single damaged base, typically from spontaneous deamination (cytosine → uracil). It is active throughout the cell cycle.

Mismatch repair (MMR). Corrects replication errors that escaped polymerase proofreading, predominantly in S phase (post-replication): the mismatched stretch of the new strand is removed, refilled, and resealed. Without MMR, short repeat sequences expand and contract — microsatellite instability.

Double-strand break repair. Nonhomologous end joining (NHEJ) ligates the two broken ends directly without any homology; it is fast but error-prone, and the same machinery joins gene segments in V(D)J recombination. Homologous recombination instead copies the lost sequence from an intact sister chromatid, so it is accurate but limited to S/G2; it depends on BRCA1/BRCA2 and is defective in hereditary breast/ovarian cancer and Fanconi anemia.

Repair-defect syndromes

Xeroderma pigmentosum. Autosomal recessive NER defect: UV pyrimidine dimers cannot be excised. Children burn severely after brief sun exposure and show dry skin, facial freckling, corneal damage, and skin cancers (basal cell, squamous cell, melanoma) in the first decade. Strict sun avoidance is essential.

Lynch syndrome (HNPCC). Autosomal dominant germline MMR mutation, most often MSH2 or MLH1. Tumors show microsatellite instability; patients develop early colorectal cancer (often right-sided) and endometrial cancer, the most common extracolonic tumor. Contrast familial adenomatous polyposis, which is driven by APC/β-catenin signaling rather than a repair defect.

Ataxia-telangiectasia. Autosomal recessive loss of ATM, a kinase that senses double-strand breaks and triggers cell-cycle arrest; review tables often list it beside NHEJ, but ATM signals the break rather than joining it. Features: cerebellar ataxia in early childhood, telangiectasias, IgA deficiency with recurrent sinopulmonary infections, radiosensitivity, and increased lymphoma/leukemia risk.

Pattern recognition: sun-triggered childhood skin cancer → NER; colon plus endometrium with microsatellite instability → MMR; ataxia, telangiectasias, and IgA deficiency → ATM.