Molecular Biology: DNA Structure, Replication, and PCR

Comprehensive practice flashcards reviewing the nature of genetic material, DNA structure, prokaryotic and eukaryotic replication mechanisms, and PCR.

What did Frederick Griffith's 1928 transformation experiment demonstrate about genetic material?

Heat-killed virulent $S$ strain cells transfer genetic information to live nonvirulent $R$ strain cells of $S.\,\text{pneumoniae}$, permanently converting them into live, capsule-producing virulent cells.

How did Avery, MacLeod, and McCarty (1944) prove that DNA is the transforming principle?

They treated heat-killed virulent extracts with specific enzymes and showed that only DNA-digesting enzymes (DNase)—and not proteases or RNases—abolished the ability of the extract to transform $R$ cells.

How did the 1952 Hershey-Chase blender experiment confirm that DNA is the viral genetic material?

$^{32}\text{P}$-labeled viral DNA entered the bacterial pellet upon centrifugation, whereas $^{35}\text{S}$-labeled viral protein coats remained in the supernatant, confirming that bacteriophage T2 injects DNA into host cells.

What are the structural components of a nucleotide, and how are they joined to form a polynucleotide chain?

A deoxyribose pentose sugar, a $5'$ phosphate group, and a nitrogenous base, linked into chains with $5'\text{-to-}3'$ polarity via covalent phosphodiester bonds between the $5'$ phosphate of one nucleotide and the $3'\text{-OH}$ of the next.

What structural differences distinguish purines from pyrimidines, and which nitrogenous bases belong to each class?

Purines are double-ringed molecules consisting of adenine ($\text{A}$) and guanine ($\text{G}$), while pyrimidines are single-ringed molecules consisting of cytosine ($\text{C}$), thymine ($\text{T}$), and uracil ($\text{U}$).

What mathematical base ratios in double-stranded DNA are described by Chargaff's rules?

The ratio of purines to pyrimidines is equal ($1:1$), where the amount of adenine equals thymine ($\text{A} = \text{T}$) and the amount of guanine equals cytosine ($\text{G} = \text{C}$).

What structural dimensions and pairing properties define the Franklin-Watson-Crick double helix model?

Two antiparallel polynucleotide chains with a $2\,\text{nm}$ diameter and a $3.4\,\text{nm}$ helical turn, held together by complementary base pairs ($\text{A-T}$ via two hydrogen bonds, $\text{G-C}$ via three).

How do the conservative, semiconservative, and dispersive models of DNA replication distribute parental DNA?

Semiconservative replication produces daughter duplexes with one parental and one new strand; conservative replication keeps the parental duplex intact while forming an entirely new duplex; dispersive replication fragments parental DNA across daughter strands.

How did the 1958 Meselson-Stahl experiment disprove the conservative and dispersive replication models?

Centrifugation using $^{15}\text{N}$ and $^{14}\text{N}$ showed a single intermediate hybrid band after round 1 (disproving conservative replication) and distinct light and hybrid bands after round 2 (disproving dispersive replication).

In what direction does DNA polymerase synthesize DNA, and what chemical group is required to add incoming dNTPs?

It synthesizes exclusively in the $5'\text{-to-}3'$ direction and requires an existing free $3'\text{-OH}$ group paired to a template strand, releasing pyrophosphate ($\text{PP}_\text{i}$) to drive nucleotide addition.

What is an origin of replication, and what defines a replicon?

An origin of replication is a specific sequence where replication initiates bidirectionally via two replication forks; a replicon is the entire chromosomal DNA segment replicated under the control of that single origin.

What roles do DNA helicase, single-strand-binding proteins (SSBs), and topoisomerase perform at the replication fork?

Helicase unwinds the double helix using ATP, SSBs coat single strands to prevent reannealing, and topoisomerase (DNA gyrase) relieves upstream torsional strain and supercoiling.

Why is DNA primase necessary during replication initiation?

Because DNA polymerases cannot synthesize DNA de novo; primase synthesizes short complementary RNA primers that provide the free $3'\text{-OH}$ group required for DNA polymerases to elongate.

What are the primary replication and repair functions of DNA Polymerases I, II, and III in E. coli?

Pol III conducts bulk leading- and lagging-strand synthesis; Pol I removes RNA primers via $5'\text{-to-}3'$ exonuclease activity and replaces them with DNA; Pol II handles repair. All three exhibit $3'\text{-to-}5'$ exonuclease proofreading.

How do synthesis mechanisms differ between the leading strand and the lagging strand?

The leading strand is synthesized continuously toward the replication fork from one primer; the lagging strand is synthesized discontinuously away from the fork as Okazaki fragments, each requiring an RNA primer before being processed by Pol I and ligase.

What multi-protein assemblies comprise the replisome architecture at the replication fork?

The primosome (primase, helicase, and accessory proteins) and two DNA Polymerase III holoenzymes anchored to DNA strands by $\beta$ sliding clamps that are loaded by clamp loaders.

How do eukaryotic replication forks differ from prokaryotic replication forks in replicon count and polymerases?

Eukaryotes use multiple replicons across linear chromosomes, initiate priming via a Pol $\alpha$-primase complex, synthesize the leading strand using Pol $\epsilon$, and synthesize lagging Okazaki fragments using Pol $\delta$.

What causes the end-replication problem in eukaryotic chromosomes, and how is chromosome integrity preserved?

Terminal lagging-strand RNA primers cannot be replaced after removal, causing linear chromosomes to shorten with each division; telomeres buffer this loss with noncoding repetitive DNA sequences.

How does telomerase elongate chromosome ends, and what is its role in cellular senescence and cancer?

It uses an internal RNA template to extend the $3'$ ends of telomeres; lack of telomerase in somatic cells causes progressive shortening and senescence, whereas telomerase reactivation allows cancer cells to avoid senescence.

What are the three temperature stages of a Polymerase Chain Reaction (PCR) cycle and their functions?

Denaturation ($94\,^\circ\text{C}$) separates the double-stranded DNA template, annealing ($54\,^\circ\text{C}$) binds flanking DNA primers, and extension ($72\,^\circ\text{C}$) allows thermostable Taq polymerase to synthesize complementary DNA.