#2 DNA Replication, Repair, Recombination, and Polymerase Chain Reaction

Vocabulary flashcards covering DNA replication machinery, proofreading, repair pathways, recombination mechanisms, PCR principles, and gel electrophoresis based on lecture materials.

Semiconservative Replication

The mechanism of DNA replication in which each daughter double helix consists of one intact original parental strand and one newly synthesized strand.

Meselson-Stahl Experiment

A 1958 experiment using ${}^{15}\text{N}$ and ${}^{14}\text{N}$ isotope labeling in E. coli paired with density gradient centrifugation that proved DNA replication follows a semiconservative model.

DNA Polymerase Fidelity

The accuracy of DNA synthesis, achieving 1 error per $10^4$ nucleotides via active site geometry selection, and improved to 1 mistake per $10^9$ nucleotides by $3' \rightarrow 5'$ exonuclease proofreading.

Proofreading Activity

The $3' \rightarrow 5'$ exonuclease function of DNA polymerase that detects mispaired nucleotides, reverses direction, and removes the incorrect base before continuing $5' \rightarrow 3'$ synthesis.

Leading Strand

The daughter DNA strand synthesized continuously in the $5' \rightarrow 3'$ direction toward the unwinding replication fork.

Lagging Strand

The daughter DNA strand synthesized discontinuously in the $5' \rightarrow 3'$ direction away from the replication fork as a series of Okazaki fragments.

Okazaki Fragments

Short stretches of newly synthesized DNA produced on the lagging strand that are later processed by nucleases, filled by repair DNA polymerase, and sealed by DNA ligase.

DNA Primase

A specialized RNA polymerase that synthesizes short RNA primers (~12 nucleotides) without requiring a pre-existing 3'-OH end to initiate DNA synthesis.

DNA Helicase

A hexameric rotary protein (DnaB in bacteria, CMG in eukaryotes) that hydrolyzes ATP to propel itself along single-stranded DNA and unwind the double helix ahead of the replication fork.

Single-Strand DNA-Binding Proteins (SSB / RPA)

Proteins (SSB in prokaryotes, RPA in eukaryotes) that bind cooperatively to exposed single-stranded DNA, preventing hairpin loop formation and protecting the template chain.

Sliding Clamp (PCNA / $\beta$-clamp)

A ring-shaped protein (PCNA in eukaryotes, $\beta$-clamp in bacteria) that holds DNA polymerase securely on the DNA template strand to maintain high processivity.

Clamp Loader (RFC / $\gamma$ complex)

An ATP-dependent protein complex (RFC in eukaryotes, $\gamma$ complex in bacteria) that opens and loads the sliding clamp onto DNA at primer-template junctions.

DNA Topoisomerase

An enzyme that relieves supercoiling and torsional stress ahead of the helicase by introducing transient single-strand nicks (Type I) or double-strand breaks (Type II) and resealing them.

Replisome

The multiprotein molecular machine at the replication fork that integrates DNA helicase, primase, polymerases, sliding clamps, clamp loaders, and SSBs to coordinate synthesis.

Origin Recognition Complex (ORC)

A eukaryotic protein complex bound at replication origins that loads symmetric Mcm helicase complexes onto DNA during the G1 phase of the cell cycle.

CMG Helicase

The fully active eukaryotic replicative helicase complex assembled at the onset of S phase, named for its components Cdc45, Mcm, and GINS.

Telomere

A non-coding region of repetitive DNA sequences (GGGTTA repeat in humans, repeated ~1000 times) located at eukaryotic chromosome ends to prevent gene loss and chromosome fraying.

Telomerase

A ribonucleoprotein reverse transcriptase carrying a 451-nucleotide RNA template scaffold that extends the 3' end of parental telomeric DNA to resolve the end-replication problem.

T-loop

A protective structure formed when the single-stranded 3' telomeric overhang loops back and invades upstream double-stranded telomeric repeat DNA.

Shelterin

A protein complex that caps telomeres, masking chromosome ends so they are not recognized as double-strand breaks by cellular DNA repair machinery.

Depurination

A spontaneous DNA lesion in which the N-glycosidic bond breaks, releasing a purine base (adenine or guanine) from the sugar-phosphate backbone without breaking the phosphodiester chain.

Deamination

Spontaneous loss of an amino group from a DNA base, such as converting cytosine to uracil or 5-methylcytosine to thymine.

Thymine Dimer

A bulky DNA lesion produced by UV radiation where adjacent thymine bases on the same strand become covalently cross-linked, distorting the double helix structure.

Base Excision Repair (BER)

A repair pathway for small non-bulky lesions that utilizes DNA glycosylase, APE1 endonuclease, AP lyase, DNA polymerase $\beta$, and DNA ligase to excise and replace a single damaged base.

Mismatch Repair (MMR)

A post-replication repair mechanism where MSH2/MSH6 and MLH1/PMS2 detect mispaired bases or insertion/deletion loops on newly synthesized strands and excise hundreds of surrounding nucleotides.

Nucleotide Excision Repair (NER)

A repair mechanism that senses helix distortions and excises a short oligonucleotide fragment (~24–32 nucleotides in eukaryotes) containing bulky lesions like thymine dimers.

Translesion DNA Polymerases

Low-fidelity emergency DNA polymerases (such as Pol $\eta$) lacking $3' \rightarrow 5'$ exonuclease activity that replace stalled replicative polymerases to bypass bulky lesions.

Non-Homologous End Joining (NHEJ)

An error-prone double-strand break repair pathway involving Ku70/Ku80 and DNA-PK that directly ligates broken DNA ends without a template, often resulting in small deletions.

Homologous Recombination (HR)

An error-free repair mechanism for double-strand breaks that utilizes an undamaged homologous chromosome or sister chromatid template and enzymes like RecA (prokaryotes) or Rad51 (eukaryotes).

Holliday Structure

A branched four-stranded DNA recombination intermediate formed by strand invasion during homologous recombination, whose resolution determines crossover vs. non-crossover outcomes.

Polymerase Chain Reaction (PCR)

An in vitro DNA amplification technique conceived by Kary B. Mullis in 1983 using repeated cycles of denaturation, primer annealing, and enzymatic extension.

Taq Polymerase

A thermostable DNA polymerase isolated from Thermus aquaticus that withstands $95^\circ\text{C}$ denaturation steps and functions optimally at $72^\circ\text{C}$ during PCR extension.

Short Tandem Repeats (STRs)

Polymorphic repetitive DNA loci whose length variations across individuals are amplified by PCR for human identification, such as in the 13 CODIS standard loci.

Agarose Gel Electrophoresis

A matrix-based separation technique that resolves DNA fragments by size as negatively charged DNA molecules migrate toward the positive anode under an electrical field.