Question 1

In bacterial DNA replication, which DNA polymerase is the primary replicative enzyme responsible for leading and lagging strand synthesis at the replication fork?

Accepted Answer

Introduction / Context: Bacterial cells replicate their genomes rapidly and accurately. Multiple DNA polymerases exist, but only one serves as the main replicative enzyme at the fork. Identifying DNA polymerase III as the principal enzyme is a foundational concept in molecular genetics and microbiology. Given Data / Assumptions: The organism category is bacteria (for example, Escherichia coli). We consider the replication fork with leading and lagging strands. DNA polymerases differ in roles: replication, repair, primer removal. Concept / Approach: In E. coli, DNA polymerase III holoenzyme is a multi-subunit complex (including core polymerase, beta clamp, clamp loader) that performs high-processivity DNA synthesis on both strands. DNA polymerase I has 5′→3′ exonuclease activity to remove RNA primers and fills short gaps, while DNA polymerase II functions mainly in DNA repair and restart. Therefore, Pol III is the correct answer. Step-by-Step Solution: List functions: Pol III (replication), Pol I (primer removal and gap filling), Pol II (repair).Identify the enzyme with highest processivity at forks: Pol III with beta clamp.Recognize that both leading and lagging strand synthesis are coordinated by Pol III dimers.Conclude Pol III is the primary replicative polymerase. Verification / Alternative check: Genetic and biochemical experiments show that mutations in Pol III subunits are lethal or strongly replication-defective, while Pol I mutants specifically impair primer removal, confirming functional distinctions. Why Other Options Are Wrong: DNA polymerase I: essential for primer removal and short-patch synthesis, not the main processive replicase.DNA polymerase II: repair-associated; not the fork replicase.All of these: overbroad; only Pol III is the primary replicative enzyme. Common Pitfalls: Assuming all polymerases replicate chromosomal DNA equally. Processivity and accessory factors (sliding clamp) define the true replicase. Final Answer: DNA polymerase III

Question 2

During prokaryotic DNA replication, what are the short DNA segments synthesized opposite to the direction of fork movement on the lagging strand called?

Accepted Answer

Introduction / Context: DNA replication is semi-discontinuous. The antiparallel nature of DNA and the 5′→3′ synthesis constraint force the cell to create short DNA pieces on one strand. Recognizing these as Okazaki fragments is central to understanding lagging-strand synthesis. Given Data / Assumptions: Synthesis proceeds only 5′→3′. The replication fork moves in one direction, creating a leading and a lagging strand. RNA primers initiate each short segment on the lagging strand. Concept / Approach: The leading strand is synthesized continuously by the replicative polymerase. The lagging strand is synthesized discontinuously as short segments called Okazaki fragments, each initiated by an RNA primer. These fragments are later processed: primers removed (Pol I), DNA filled in, and nicks sealed (ligase). Step-by-Step Solution: Identify continuous versus discontinuous synthesis.Name the short discontinuous DNA segments: Okazaki fragments.Differentiate primers (short RNA initiators) from the DNA fragments themselves.Confirm that “lagging strand” is the template/strand, not the fragment name. Verification / Alternative check: Pulse-chase labeling experiments produced short labeled DNA pieces that matured into longer DNA, validating the Okazaki fragment model. Why Other Options Are Wrong: Leading strands: continuous synthesis; not short pieces.Primers: RNA oligonucleotides that start fragments but are not the DNA fragments themselves.Lagging strand: refers to the discontinuously synthesized strand overall. Common Pitfalls: Confusing RNA primers with Okazaki fragments, or thinking both strands are made continuously. Final Answer: Okazaki fragments

Question 3

What is the molecular outcome of semi-conservative DNA replication in cells?

Accepted Answer

Introduction / Context: Since the Meselson–Stahl experiment, it is established that DNA replication is semi-conservative. Each daughter duplex contains one parental strand and one nascent strand. This question confirms that core principle of molecular biology. Given Data / Assumptions: DNA double helix consists of two antiparallel strands. Replication duplicates the genome prior to cell division. Base pairing rules guide accurate copying. Concept / Approach: Helicases unwind DNA; primase makes RNA primers; DNA polymerases extend in the 5′→3′ direction; and ligase seals nicks. Template-directed synthesis means each parental strand serves as a template for a new complementary strand, yielding two hybrid molecules that each contain one old and one new strand. Step-by-Step Solution: Parental duplex separates into two templates.DNA polymerase synthesizes complementary strands on both templates.Leading strand is continuous; lagging strand is discontinuous (Okazaki fragments).RNA primers are removed and gaps filled; ligase seals remaining nicks.End result: two daughter DNA molecules, each hybrid old/new. Verification / Alternative check: Meselson–Stahl used density-labeled nitrogen (15N to 14N shift) and CsCl gradients to show hybrid densities after one replication round, consistent only with semi-conservative replication. Why Other Options Are Wrong: Two completely new DNA molecules: would imply conservative replication, which is false.One new DNA and one old unchanged: also conservative model, disproven.One new RNA molecule: RNA is not the replication product of DNA; that is transcription. Common Pitfalls: Confusing conservative, semi-conservative, and dispersive models; assuming both strands are new after replication. Final Answer: Two DNA molecules such that each contains one original (parental) and one newly synthesized strand

Question 4

Which antibiotic binds to DNA and induces strand breaks by generating hydroxyl radicals, thereby cutting the DNA?

Accepted Answer

Introduction / Context: Some chemotherapeutic antibiotics act directly on DNA structure, producing breaks that halt replication and transcription. Bleomycin is a classic example used in oncology. Distinguishing it from antibiotics that target ribosomes or glycosylation highlights different mechanisms of action. Given Data / Assumptions: We are comparing mechanisms: DNA cleavage vs. protein synthesis inhibition vs. glycosylation interference. Hydroxyl radical formation is a key clue. Agents listed: bleomycin (antitumor), erythromycin (macrolide), tunicamycin (glycosylation inhibitor). Concept / Approach: Bleomycin chelates metal ions (often Fe2+) and, in the presence of oxygen, generates reactive oxygen species including hydroxyl radicals. These radicals induce single- and double-strand DNA breaks. By contrast, erythromycin binds the 50S ribosomal subunit to inhibit peptide elongation, and tunicamycin blocks N-linked glycosylation in the endoplasmic reticulum by inhibiting UDP-N-acetylglucosamine: dolichol phosphate N-acetylglucosamine-1-phosphate transferase. Neither erythromycin nor tunicamycin directly cleaves DNA via hydroxyl radicals. Step-by-Step Solution: Identify the mechanism described: DNA binding and radical-mediated cleavage.Match to drug known for ROS-dependent DNA strand scission: bleomycin.Exclude drugs with ribosomal or glycosylation targets.Select bleomycin as the correct answer. Verification / Alternative check: Clinical pharmacology references report pulmonary toxicity as a notable bleomycin adverse effect, consistent with oxidative mechanisms, and its inclusion in combination cancer regimens (e.g., ABVD for Hodgkin lymphoma). Why Other Options Are Wrong: Erythromycin: protein synthesis inhibitor; does not cleave DNA.Tunicamycin: disrupts glycoprotein biosynthesis; no DNA cutting activity.All of these: incorrect because only bleomycin matches the described mechanism. Common Pitfalls: Assuming all “antibiotics” target DNA. Many act on ribosomes, cell walls, or metabolic enzymes. Final Answer: Bleomycin

Question 5

In Escherichia coli DNA replication, what happens to the RNA primers used to initiate synthesis on the lagging strand?

Accepted Answer

Introduction / Context: Lagging-strand synthesis requires repeated priming. Understanding primer removal and fragment maturation clarifies roles of individual enzymes at the replication fork and during post-replicative processing in bacteria. Given Data / Assumptions: Organism: E. coli (model bacterium). Lagging strand forms Okazaki fragments initiated by RNA primers. Enzymes available: DNA polymerase I, DNA ligase, helicase, DNA polymerase III. Concept / Approach: DNA polymerase I has a unique 5′→3′ exonuclease activity that removes RNA primers and simultaneously fills the resulting gaps with DNA (nick translation). DNA ligase then seals the remaining nicks between adjacent DNA fragments. Helicase unwinds DNA ahead of the fork but does not remove primers, and the leading strand does not form Okazaki fragments. Step-by-Step Solution: Recognize that RNA primers are necessary for Pol III to start synthesis.After extension, primers must be removed: Pol I performs 5′→3′ exonucleolytic removal.Pol I replaces RNA with DNA to create a continuous DNA stretch.DNA ligase seals the final phosphodiester bond between fragments. Verification / Alternative check: Mutations in Pol I impair primer removal and nick translation; ligase-deficient mutants accumulate nicks, confirming the division of labor. Why Other Options Are Wrong: They result in Okazaki fragments on the leading strand: Okazaki fragments occur on the lagging strand only.They are joined together by DNA ligase: ligase seals DNA–DNA nicks, not RNA primers.They are removed by helicase + ATP: helicase unwinds duplex DNA; it does not excise RNA primers. Common Pitfalls: Confusing the roles of Pol I and ligase; ligase cannot replace RNA with DNA or remove primers. Final Answer: They are removed by DNA polymerase I

Question 6

In IUPAC nucleotide ambiguity codes, which letter represents a position that can be A, C, or T (but not G)?

Accepted Answer

Introduction / Context: IUPAC ambiguity codes are used in consensus sequences and degenerate primers to indicate multiple possible nucleotides at a single position. Knowing each code facilitates reading and designing sequences. Given Data / Assumptions: We need the code for A, C, or T. G must be excluded. Common codes: R, Y, S, W, K, M, B, D, H, V, N. Concept / Approach: The mnemonic approach: H stands for “not G” (H = A, C, T), D stands for “not C” (D = A, G, T), B stands for “not A” (B = C, G, T), and V stands for “not T” (V = A, C, G). S represents strong (G or C), W represents weak (A or T), R is purines (A or G), Y is pyrimidines (C or T), K is keto (G or T), M is amino (A or C), and N is any base. Step-by-Step Solution: List candidate codes that include three bases.Match A/C/T to code H (excludes G).Verify other options: B = C/G/T (excludes A); S = C/G (two bases only).Confirm H is correct for A, C, or T. Verification / Alternative check: IUPAC tables consistently define H as A or C or T; this is standard across sequencing and primer design tools. Why Other Options Are Wrong: B: represents C, G, T (excludes A), not the requested set.S: represents G or C only, not three bases.None of these: incorrect because H is a valid choice. Common Pitfalls: Confusing B, D, H, V sets; the “not X” mnemonic helps remember which base is excluded. Final Answer: H

Question 7

Replication origins: compared with bacteria that have a single origin, how many origins of replication are typically found across the much larger genomes of mammals and other eukaryotes?

Accepted Answer

Introduction / Context: Genome size and chromatin organization influence how replication is initiated and completed within the S phase of the cell cycle. Eukaryotic genomes are large and linear, necessitating many initiation sites to finish replication efficiently and reliably. Given Data / Assumptions: Bacteria such as E. coli have one circular chromosome with a single well-defined origin (oriC). Eukaryotes have multiple linear chromosomes and far larger genomes. S phase timing constraints require many replication origins firing in regulated patterns. Concept / Approach: Eukaryotic chromosomes contain numerous potential origins, only a subset of which fire in any given S phase according to replication timing programs. Mammalian genomes typically use thousands to tens of thousands of origins, ensuring complete duplication within hours. Origin licensing involves ORC, Cdc6, Cdt1, and loading of MCM helicases, which later activate to form replication forks. Step-by-Step Solution: Contrast a single bacterial origin with the complexity of eukaryotic chromatin.Consider the scale: billions of base pairs vs. millions in bacteria.Infer the need for numerous origins to complete replication on time.Select the option indicating thousands to tens of thousands of origins. Verification / Alternative check: DNA combing, nascent strand mapping, and replication timing analyses demonstrate dense origin usage across mammalian chromosomes. Why Other Options Are Wrong: One: true for many bacteria, not for mammals.About 100: too few for mammalian genome sizes and S phase duration.None of these: incorrect since a correct quantitative range is provided. Common Pitfalls: Assuming each gene needs its own origin; origins are independent of gene boundaries and are regulated by chromatin state and replication programs. Final Answer: Thousands and tens of thousands

Question 8

In molecular biology, what is the enzyme called that recognizes specific foreign DNA sequences and cleaves the DNA at or near those sites?

Accepted Answer

Introduction / Context: Restriction endonucleases underpin recombinant DNA technology, cloning, and genomic mapping. They evolved as bacterial defense mechanisms against foreign DNA such as bacteriophages, providing sequence-specific cleavage. Given Data / Assumptions: The enzyme must recognize a DNA sequence motif. It must introduce a cut (double-strand break or nick) at or near that motif. Context implies defense or laboratory usage. Concept / Approach: Restriction enzymes (e.g., EcoRI, HindIII) recognize palindromic sequences and cleave DNA to produce sticky or blunt ends. In bacteria, methylation systems protect host DNA from self-cleavage. Other enzymes listed do not perform sequence-specific restriction: transposase moves DNA elements, helicase unwinds DNA, and reverse transcriptase copies RNA into DNA. Step-by-Step Solution: Identify the requirement: sequence-specific recognition and cleavage.Match to restriction endonucleases from bacteria.Exclude enzymes with unrelated functions (helicase, transposase, reverse transcriptase).Select restriction endonuclease. Verification / Alternative check: Restriction–modification systems pair endonucleases with methyltransferases; this pairing is well documented in microbial genetics. Why Other Options Are Wrong: Transposase: catalyzes transposition; not a defense nuclease against foreign DNA sequences.Helicase: unwinds duplex DNA; no site-specific cutting.Reverse transcriptase: synthesizes DNA from RNA; no sequence-specific DNA cutting. Common Pitfalls: Confusing general nucleases with sequence-specific restriction enzymes; only the latter recognize defined motifs. Final Answer: Restriction endonuclease

Question 9

In gel electrophoresis of linear DNA, how can the fragment size be estimated accurately during analysis?

Accepted Answer

Introduction / Context: Agarose gel electrophoresis separates linear DNA fragments primarily by size, with mobility inversely related to the logarithm of fragment length. Accurate size estimation requires internal or parallel standards known as DNA ladders. Given Data / Assumptions: We are analyzing linear DNA bands on an agarose gel. Staining (e.g., ethidium bromide, SYBR dyes) provides fluorescence but not absolute size. Standards such as HindIII-digested lambda DNA or commercially prepared ladders have known fragment sizes. Concept / Approach: To estimate fragment size, run a DNA ladder in a lane beside samples. Plot distance migrated versus log10(size) for standard bands to create a calibration curve, then read sample sizes by interpolation. Relying solely on fluorescence brightness, sedimentation coefficients, or run time is unreliable because they are influenced by concentration, gel composition, voltage, buffer, and conformation. Step-by-Step Solution: Load molecular weight standard (e.g., HindIII-digested lambda) in one lane.Run samples under the same conditions.Measure migration distances of ladder bands and construct a standard curve: distance vs. log(size).Measure migration of sample band; interpolate size from the standard curve.Report size with an appropriate error margin. Verification / Alternative check: Using different ladders (100 bp, 1 kb) tailored to expected size ranges cross-validates estimates. Including an internal standard band within the same sample lane further improves accuracy. Why Other Options Are Wrong: Fluorescence intensity: reflects DNA quantity and dye binding, not size.Sedimentation coefficient: pertains to ultracentrifugation, not agarose mobility.Run time: affects overall migration but cannot directly translate to size without standards. Common Pitfalls: Misreading bands due to gel smearing, running gels too hot (band smiling), or using an inappropriate ladder range for the fragment sizes of interest. Final Answer: By comparison with a molecular weight standard such as HindIII-digested phage lambda DNA

Question 10

In molecular biology, which catalytic activities are present as distinct active sites within the single polypeptide of E. coli DNA polymerase I?

Accepted Answer

Introduction / Context: DNA polymerase I from Escherichia coli is a single polypeptide enzyme that performs multiple catalytic functions during DNA repair and processing. This question tests recognition of the distinct active sites carried on that one polypeptide chain and how they contribute to fidelity and repair. Given Data / Assumptions: Organism: E. coli. Enzyme: DNA polymerase I (Pol I). Activities to evaluate: 5' → 3' polymerase, 3' → 5' exonuclease, 5' → 3' exonuclease. Concept / Approach: Pol I possesses three separable catalytic activities located at distinct active sites on the same polypeptide. The polymerase adds nucleotides. The 3' → 5' exonuclease proofreads newly added bases. The 5' → 3' exonuclease removes RNA primers or damaged DNA ahead of a nick (nick translation). Step-by-Step Solution: 1) Identify polymerase site: adds dNTPs in the 5' → 3' direction.2) Identify proofreading site: 3' → 5' exonuclease removes misincorporated nucleotides, increasing fidelity.3) Identify primer-removal site: 5' → 3' exonuclease cleaves nucleotides ahead of a nick, enabling primer removal and gap filling.4) All three sites are present on the single Pol I polypeptide; thus the inclusive option applies. Verification / Alternative check: Biochemical domain mapping shows the Klenow fragment (polymerase + 3' → 5' exonuclease) after limited proteolysis, while the small fragment contains the 5' → 3' exonuclease. This confirms distinct active sites on one polypeptide. Why Other Options Are Wrong: 5' → 3' polymerase alone: incomplete; Pol I also has both exonucleases.3' → 5' exonuclease alone: incomplete; lacks polymerase and 5' → 3' exonuclease.5' → 3' exonuclease alone: incomplete; lacks other functions. Common Pitfalls: Confusing 5' → 3' exonuclease with 3' → 5' proofreading; assuming separate subunits rather than distinct sites within one polypeptide; mixing up Pol I with Pol III core enzyme. Final Answer: All of the above

Question 11

In nucleic acid hybridization, which Wallace rule expression correctly estimates the melting temperature (Tm) of a short oligonucleotide under standard conditions?

Accepted Answer

Introduction / Context: The Wallace rule provides a quick mental estimate of the melting temperature (Tm) for short DNA oligonucleotides in typical salt conditions. It helps select hybridization stringency in PCR, cloning, and probe design without complex calculations. Given Data / Assumptions: Short oligonucleotides (about 14–20 bases). Standard monovalent salt conditions. We only compare formulae; no sequence is given. Concept / Approach: G:C base pairs contribute more to duplex stability than A:T because G:C forms three hydrogen bonds and stronger stacking. The Wallace approximation weights G:C pairs by 4 and A:T pairs by 2 to reflect this increased stability. Step-by-Step Solution: 1) Identify correct weighting: G:C should contribute more to Tm than A:T.2) Examine options: only one assigns 4 per G:C and 2 per A:T.3) Select that expression as the Wallace rule suitable for quick estimates. Verification / Alternative check: For longer oligonucleotides, nearest-neighbor models or empirical equations (e.g., Tm = 69.3 + 0.41 * %GC - 650 / length) provide better accuracy, but the Wallace rule remains appropriate for short primers. Why Other Options Are Wrong: Option A swaps weights and underestimates G:C stability.Option B greatly overemphasizes A:T contributions.Option D is a different empirical formula for longer sequences, not the Wallace rule. Common Pitfalls: Applying the Wallace rule to very long primers, ignoring salt/mismatch effects, or confusing %GC equations with the short-oligo heuristic. Final Answer: Tm = 4 * (number of G:C pairs) + 2 * (number of A:T pairs)

Question 12

Automated DNA sequencing based on Sanger chain termination typically uses which primer/dye strategy?

Accepted Answer

Introduction / Context: Sanger sequencing (chain termination) underpins most automated capillary sequencing systems. The method requires a defined primer to initiate DNA synthesis and fluorescent labeling for automated base calling. Given Data / Assumptions: Sequencing is based on chain termination chemistry. Automation implies fluorescence detection. We compare primer and labeling strategies. Concept / Approach: Two fluorescence strategies exist: dye-primer (fluorescent label on the primer) and dye-terminator (fluorescent ddNTPs). Both require an oligonucleotide primer. A polynucleotide primer is not used, and sequencing cannot proceed without a primer. Step-by-Step Solution: 1) Chain termination needs a 3'-hydroxyl provided by a primer annealed to template.2) Automation relies on fluorescent signals to identify terminated fragments.3) In dye-primer chemistry, the primer itself carries the fluorophore; in dye-terminator, ddNTPs are labeled, but an unlabeled oligonucleotide primer is still used.4) Therefore, the only correct statement among the options is that an oligonucleotide primer labeled with a fluorescent dye is used (captures dye-primer usage and affirms primer necessity). Verification / Alternative check: Even when dye-terminator chemistry is chosen, an oligonucleotide primer is required. Options claiming “no primer” are incompatible with DNA polymerase mechanics. Why Other Options Are Wrong: Polynucleotide primer: not the standard practice for Sanger sequencing.No primer: impossible for polymerase initiation on single-stranded template.Either polynucleotide or no primer: both contradict core requirements. Common Pitfalls: Confusing dye-primer vs dye-terminator and concluding that a primer is unnecessary; using the term “polynucleotide primer,” which is non-standard in this context. Final Answer: An oligonucleotide primer labeled with a fluorescent dye

Question 13

In circular DNA topology, the number of times the two strands are intertwined is called the linking number. What is this quantity?

Accepted Answer

Introduction / Context: DNA topology in covalently closed circular DNA is described by geometric quantities that remain conserved unless strands are broken. Recognizing these terms is essential to understanding supercoiling and enzymatic action by topoisomerases. Given Data / Assumptions: DNA is circular and covalently closed. We need the term that counts how many times the two strands wind around each other. Concept / Approach: The linking number (Lk) is an integer describing total interlinking of the two strands. It is decomposed as Lk = Tw + Wr, where Tw is twist (local helical turns) and Wr is writhe (supercoiling). Lk changes only when a strand is cut and rejoined. Step-by-Step Solution: 1) Identify quantity tied to interlinking of both strands: linking number.2) Distinguish from twist (twisting number), which measures helical turns along the axis.3) Note writhe (not listed) measures supercoiling of the axis in space.4) Therefore, the correct term is linking number. Verification / Alternative check: In relaxed B-DNA of N base pairs, Lk ≈ N / 10.5 when writhe is zero; experimental manipulation with topoisomerases demonstrates Lk changes only via strand passage. Why Other Options Are Wrong: Helix frequency: vague and not a standard conserved topological invariant.Twisting number: refers to Tw component, not total interlinking.None of these: incorrect because “linking number” is the accepted term. Common Pitfalls: Equating twist with linking number, ignoring writhe, or assuming Lk can change without strand breakage. Final Answer: Linking number

Question 14

Chromatin structure: what is the correct composition of the nucleosome core particle with respect to histones and H1?

Accepted Answer

Introduction / Context: Nucleosomes are the fundamental repeating units of eukaryotic chromatin. Understanding their precise composition is critical to interpreting chromatin packaging, accessibility, and epigenetic regulation. Given Data / Assumptions: Focus on the nucleosome core particle. Consider whether linker histone H1 is part of the core. Concept / Approach: The nucleosome core particle is an octamer comprising two copies each of H2A, H2B, H3, and H4. About 146 base pairs of DNA wrap around this core. H1 is a linker histone that binds at the DNA entry/exit site to stabilize higher-order structure but is not part of the core octamer. Step-by-Step Solution: 1) Enumerate histones in the core: H2A, H2B, H3, H4 (two of each).2) Confirm H1 function: binds linker DNA, not within the octameric core.3) Therefore, the correct composition is an octamer of four histones without H1. Verification / Alternative check: Biochemical reconstitution experiments form stable core particles with H2A/H2B/H3/H4 alone; addition of H1 shifts to chromatosome (~166 bp DNA), evidencing H1 as linker-associated. Why Other Options Are Wrong: Octamer with H1: incorrectly includes H1 in the core.Hexamer options: do not match established octameric stoichiometry. Common Pitfalls: Confusing the chromatosome (core + H1) with the nucleosome core particle, or miscounting histone copies. Final Answer: An octamer of four histones without H1 protein

Question 15

Which additional nucleolytic activities are exhibited by E. coli DNA polymerase I besides its polymerase function?

Accepted Answer

Introduction / Context: Beyond catalyzing DNA synthesis, DNA polymerase I participates in repair by excising nucleotides and correcting errors. Recognizing its exonuclease repertoire explains primer removal and proofreading. Given Data / Assumptions: Enzyme: E. coli DNA polymerase I. We evaluate exonuclease and endonuclease activities. Concept / Approach: Pol I has two exonuclease activities: a 3' → 5' exonuclease for proofreading and a 5' → 3' exonuclease that removes nucleotides ahead of a nick, including RNA primers. It does not have a 5' → 3' endonuclease; the activity is exonucleolytic and nick-dependent. Step-by-Step Solution: 1) Identify proofreading: 3' → 5' exonuclease maintains fidelity by removing incorrect bases.2) Identify nick translation: 5' → 3' exonuclease removes primers/damage ahead of a nick while polymerase fills the gap.3) Conclude both exonucleases are present; endonuclease is not. Verification / Alternative check: Proteolysis yields the Klenow fragment lacking the 5' → 3' exonuclease but retaining polymerase and 3' → 5' exonuclease; the small fragment contains the 5' → 3' exonuclease, confirming dual activities. Why Other Options Are Wrong: Only 5' → 3': incomplete; ignores proofreading.Only 3' → 5': incomplete; ignores primer removal.5' → 3' endonuclease: incorrect enzyme class. Common Pitfalls: Confusing endonuclease with exonuclease, or attributing 5' → 3' exonuclease to Pol III instead of Pol I. Final Answer: Both (a) and (b)

Question 16

In the Meselson–Stahl experiment, if E. coli grown in 15N medium is shifted to 14N and allowed exactly one round of replication, how many DNA bands appear in a CsCl density gradient?

Accepted Answer

Introduction / Context: The Meselson–Stahl experiment demonstrated semiconservative replication by monitoring DNA density changes after switching isotopic media. Understanding the banding pattern after each generation reveals the replication mechanism. Given Data / Assumptions: Cells initially labeled with heavy 15N. Shifted to light 14N medium. Exactly one round of replication has occurred. Concept / Approach: Semiconservative replication yields daughter molecules with one parental (15N) strand and one newly synthesized (14N) strand after the first generation. Both DNA duplexes have identical hybrid densities and thus co-migrate. Step-by-Step Solution: 1) Start: all DNA is heavy–heavy (15N/15N).2) After one replication: each duplex is heavy–light (15N/14N).3) Density gradient: both molecules have the same intermediate density, forming a single band. Verification / Alternative check: After two generations, bands split into one intermediate and one light band, confirming the semiconservative model rather than conservative or dispersive replication. Why Other Options Are Wrong: Two: occurs after two generations, not one.Three or smear: inconsistent with discrete semiconservative outcomes under standard conditions. Common Pitfalls: Expecting both heavy and light bands after one generation, or confusing the timeline of band formation. Final Answer: One

Question 17

Which statements correctly describe bacterial DNA replication?

Accepted Answer

Introduction / Context: Core features of bacterial chromosome replication include the number of origins, directionality of fork movement, and how parental strands are conserved. Mastery of these traits is foundational for genetics and molecular biology. Given Data / Assumptions: Organism: bacteria (e.g., E. coli). Focus: origin number, bidirectionality, conservation. Concept / Approach: E. coli initiates replication at a single origin (oriC), two replication forks proceed bidirectionally around the circular chromosome, and replication is semiconservative, preserving one parental strand in each daughter duplex. Step-by-Step Solution: 1) Origin count: one principal chromosomal origin (oriC).2) Fork movement: two forks move in opposite directions from oriC.3) Strand conservation: each product contains one old and one new strand.4) Therefore, the combination “All of the above” is correct. Verification / Alternative check: Genetic mapping and visualization of replication intermediates corroborate a single oriC and bidirectional forks; Meselson–Stahl demonstrates semiconservative inheritance. Why Other Options Are Wrong: Any single statement alone omits the other true properties; the inclusive choice best reflects bacterial replication. Common Pitfalls: Confusing bacterial single-origin replication with eukaryotic multiple origins, or assuming unidirectional fork movement. Final Answer: All of the above

Question 18

How does ethidium bromide interact with duplex DNA at the molecular level?

Accepted Answer

Introduction / Context: Ethidium bromide is a classical fluorescent dye used to visualize DNA. Understanding its interaction with DNA explains both its staining properties and its mutagenic potential. Given Data / Assumptions: Analyte: duplex DNA. Ligand: ethidium bromide (planar polycyclic compound). Concept / Approach: Ethidium bromide intercalates—slides between stacked base pairs—expanding the helical rise and unwinding DNA slightly. Intercalation enhances fluorescence by restricting rotational freedom and shielding the dye from solvent quenching. Step-by-Step Solution: 1) Consider structural complementarity: a flat aromatic dye fits between base pairs.2) Predict effects: intercalation increases contour length and reduces superhelical density.3) Conclude: the mechanism is intercalation, not specific base or phosphate binding. Verification / Alternative check: Viscosity and circular dichroism changes, along with topological experiments, confirm intercalative binding; phosphate-binding agents yield different signatures. Why Other Options Are Wrong: Adenine-only binding: inconsistent with non-specific intercalation between any adjacent base pairs.Phosphate binding: typical of cationic groove binders, not ethidium intercalation.No interaction: contradicted by strong fluorescence upon DNA addition. Common Pitfalls: Assuming base-specific hydrogen bonding or mistaking groove-binding for intercalation. Final Answer: It intercalates between bases

Question 19

During initiation of chromosomal replication in E. coli, short RNA primers are synthesized by which enzyme?

Accepted Answer

Introduction / Context: DNA replication requires short RNA primers to provide a 3'-hydroxyl for DNA polymerases. Identifying the priming enzyme clarifies the division of labor at the replication fork. Given Data / Assumptions: Organism: E. coli. Process: initiation and Okazaki fragment priming. Concept / Approach: E. coli uses DnaG primase, a specialized RNA polymerase associated with the DnaB helicase, to synthesize short RNA primers. The housekeeping DNA-dependent RNA polymerase transcribes genes and is not the primase at the fork. Reverse transcriptase is irrelevant in this context. Step-by-Step Solution: 1) Replication fork assembly recruits DnaG primase to the unwound template.2) Primase synthesizes short RNA oligonucleotides that present a 3'-OH.3) DNA polymerase III extends from these primers; Pol I later removes RNA primers and fills gaps. Verification / Alternative check: Genetic and biochemical reconstitution show DnaG activity is essential for Okazaki fragment initiation; depletion prevents lagging-strand synthesis despite the presence of transcriptional RNA polymerase. Why Other Options Are Wrong: RNA polymerase (transcriptional): does not normally synthesize replication primers in vivo.Reverse transcriptase: synthesizes DNA from RNA, absent in standard E. coli replication.Both (a) and (b): overbroad; the correct physiological primase is DnaG. Common Pitfalls: Equating any RNA-synthesizing enzyme with primase; assuming transcriptional RNA polymerase primes DNA replication. Final Answer: Primase

Question 20

In DNA agarose-gel electrophoresis, how do larger DNA fragments migrate and where do they typically appear in the gel photograph?

Accepted Answer

Introduction / Context: DNA agarose-gel electrophoresis separates fragments based on size as they migrate through a porous matrix under an electric field. This question checks understanding of how fragment length influences mobility and band position in a standard gel photo. Given Data / Assumptions: Agarose gel with wells at the top; DNA is loaded into wells. Electric field drives negatively charged DNA toward the bottom (anode). All fragments have similar charge density (phosphate backbone). Concept / Approach: Mobility in a sieving matrix is inversely related to fragment length. Larger fragments experience more frictional resistance and thus migrate more slowly than smaller fragments. Step-by-Step Solution: 1) DNA is negatively charged and moves toward the anode at the bottom of the gel.2) Agarose forms pores; migration speed depends on ability to thread through pores.3) Larger fragments are hindered more, so they travel a shorter distance in a fixed time.4) Therefore, large bands remain closer to the wells at the top in the gel photograph. Verification / Alternative check: Standard DNA ladders on gels show high-molecular-weight bands near the wells and low-molecular-weight bands farther down, confirming the rule. Why Other Options Are Wrong: They confuse speed and position: fast fragments end up near the bottom; slow fragments remain near the top. Mixing these yields incorrect pairings. Common Pitfalls: Assuming charge differences dominate; for DNA, charge per base pair is approximately constant, so size-dependent sieving is the key factor. Final Answer: Slowly, appearing nearer the top (near the wells).

DNA Structure and Replication Questions