Unit 3: Structure of Nucleic Acids; DNA Packaging and Replication - Subjective Questions

The Watson-Crick model (1953) describes DNA as a right-handed double helix. Key features include:

• Two polynucleotide strands wound around a common axis in an antiparallel fashion (one runs , the other ).
• Sugar-phosphate backbone on the outside; nitrogenous bases point inward.
• Complementary base pairing: Adenine pairs with Thymine (2 hydrogen bonds), Guanine pairs with Cytosine (3 hydrogen bonds).
• Chargaff's rules are satisfied: and .
• Helical parameters (B-DNA): diameter Å, one complete turn every Å containing about base pairs, rise per base pair Å.
• Presence of major and minor grooves which serve as protein binding sites.
• The bases are stacked, contributing to helix stability via hydrophobic stacking interactions and hydrogen bonding.

The three major conformations of DNA differ in helical geometry:

Key points:

• B-DNA is the most common biological form under physiological hydration.
• A-DNA forms under low humidity and is found in RNA duplexes.
• Z-DNA has a zig-zag backbone, is left-handed, and may play roles in gene regulation.

Denaturation is the separation of the two DNA strands due to the breaking of hydrogen bonds between complementary bases, typically caused by heat, extreme pH, or chemicals.

• It is monitored by the hyperchromic effect — an increase in UV absorbance at nm as bases become unstacked.
• The melting temperature () is the temperature at which 50% of the DNA is denatured.

Renaturation (reannealing) is the reformation of the double helix when denatured strands recombine under favorable (cooling) conditions. It depends on:

• Concentration of complementary sequences.
• Cot value (product of DNA concentration and time).
• Sequence complexity.

Factors affecting :

• GC content: higher G-C pairs (3 H-bonds) raise .
• Salt concentration: higher ionic strength stabilizes the duplex, increasing .
• pH: extreme pH destabilizes the helix.
• Presence of denaturants like urea or formamide lowers .

DNA supercoiling is the over- or under-winding of the DNA double helix, producing a higher-order twisting of the molecule upon itself. It is described by the equation:

where = linking number, = twist, and = writhe.

Types:

• Negative supercoiling: underwinding ( less than relaxed value); facilitates strand separation and is the predominant form in cells.
• Positive supercoiling: overwinding ( greater than relaxed value); makes strand separation harder.

Role of Topoisomerases:

• Type I topoisomerases cut one strand, relaxing supercoils by changing in steps of 1; they do not require ATP.
• Type II topoisomerases (e.g., DNA gyrase) cut both strands, changing in steps of 2; they require ATP and can introduce negative supercoils.

Supercoiling is essential for compaction, replication, and transcription.

RNA is a single-stranded nucleic acid containing ribose sugar and uracil instead of thymine. Major types include:

• Messenger RNA (mRNA): Carries genetic information from DNA to ribosomes; template for protein synthesis. In eukaryotes it has a cap and poly-A tail.
• Transfer RNA (tRNA): Small (~75-90 nucleotides), adopts a cloverleaf secondary structure and L-shaped tertiary structure; carries amino acids to the ribosome via an anticodon.
• Ribosomal RNA (rRNA): Structural and catalytic component of ribosomes; the most abundant RNA. Acts as a ribozyme in peptide bond formation.
• Small nuclear RNA (snRNA): Involved in splicing of pre-mRNA (spliceosome components).
• Small nucleolar RNA (snoRNA): Guides chemical modification of rRNA.
• MicroRNA (miRNA) and siRNA: Regulate gene expression by silencing mRNA.

RNA can form complex secondary structures (hairpins, loops, bulges) via intramolecular base pairing.

DNA is compacted through several levels of organization to fit within the nucleus:

1. DNA double helix (2 nm diameter): the naked DNA.
2. Nucleosome / "beads-on-a-string" (10 nm fiber): DNA wraps around histone octamers, achieving a ~6-fold compaction.
3. 30 nm fiber (solenoid/zigzag): Nucleosomes coil with the help of histone H1, giving ~40-fold compaction.
4. Looped domains (300 nm): The 30 nm fiber forms loops attached to a protein scaffold/nuclear matrix.
5. Condensed chromatid (700 nm): Loops are further coiled and compacted.
6. Metaphase chromosome (1400 nm): Maximum condensation (~10,000-fold overall).

This hierarchical folding enables an ~2 m length of DNA to fit within a nucleus of a few micrometers while allowing regulated access for transcription and replication.

The nucleosome is the fundamental repeating unit of chromatin.

Structure:

• A histone octamer core composed of two copies each of H2A, H2B, H3, and H4.
• About 147 base pairs of DNA wrapped ~1.65 times around the octamer.
• Adjacent nucleosomes are connected by linker DNA (~20-80 bp).
• Histone H1 binds the linker DNA and the entry/exit point, sealing the nucleosome and promoting higher-order folding.

Role of histones:

• Histones are small, basic (positively charged) proteins rich in lysine and arginine, which electrostatically bind the negatively charged DNA phosphate backbone.
• They contain a histone-fold domain for octamer assembly and N-terminal tails that undergo modifications (acetylation, methylation, phosphorylation).
• These modifications regulate chromatin compaction and gene expression (the histone code).

Semiconservative replication means each daughter DNA molecule consists of one parental (old) strand and one newly synthesized strand.

Meselson-Stahl Experiment (1958):

• E. coli was grown for several generations in a medium containing heavy nitrogen (), labeling all DNA as heavy.
• Cells were then shifted to a medium with light nitrogen ().
• DNA was extracted at successive generations and analyzed by cesium chloride (CsCl) density gradient centrifugation.

Results:

• Generation 0: single heavy band ().
• Generation 1: single band of intermediate density () — ruling out conservative replication.
• Generation 2: two bands — one intermediate and one light () — ruling out dispersive replication.

This pattern confirmed the semiconservative mode of DNA replication.

DNA replication requires a coordinated set of enzymes and proteins:

• Helicase: Unwinds the double helix at the replication fork by breaking hydrogen bonds.
• Single-strand binding proteins (SSBs): Stabilize the separated single strands and prevent reannealing.
• Topoisomerase / DNA gyrase: Relieves the torsional strain ahead of the fork.
• Primase: Synthesizes short RNA primers to provide a free -OH group.
• DNA polymerase III (prokaryotes): Main enzyme that synthesizes new DNA in the direction with proofreading ( exonuclease) activity.
• DNA polymerase I (prokaryotes): Removes RNA primers and fills gaps.
• DNA ligase: Joins Okazaki fragments by forming phosphodiester bonds.
• Sliding clamp (β-clamp) and clamp loader: Enhance polymerase processivity.

Together these components form the replisome.

Prokaryotic DNA replication occurs in three phases:

1. Initiation:

• Begins at a single origin, oriC.
• DnaA proteins bind to oriC and cause local unwinding of AT-rich regions.
• DnaB (helicase) is loaded with the help of DnaC, unwinding DNA bidirectionally.
• SSBs stabilize the single strands and gyrase relieves supercoiling.

2. Elongation:

• Primase (DnaG) lays down RNA primers.
• DNA polymerase III extends primers synthesizing the leading strand continuously and the lagging strand discontinuously as Okazaki fragments.
• DNA polymerase I removes primers and fills gaps.
• DNA ligase seals the nicks.

3. Termination:

• The two replication forks meet at the ter region, where Tus proteins halt the forks.
• Topoisomerase IV separates the interlinked daughter chromosomes (decatenation).

Replication is bidirectional, semiconservative, and highly processive (~1000 nucleotides/sec).

Eukaryotic replication is more complex due to larger, linear genomes packaged in chromatin.

Key differences:

• Origins: Eukaryotes have multiple origins of replication per chromosome; prokaryotes have a single origin.
• Speed: Eukaryotic forks are slower (~50 nt/sec) vs prokaryotic (~1000 nt/sec).
• Okazaki fragments: Shorter in eukaryotes (~100-200 nt) than prokaryotes (~1000-2000 nt).
• Polymerases: Eukaryotes use Pol α (primase), Pol δ (lagging strand), Pol ε (leading strand); prokaryotes use Pol III and Pol I.
• Chromatin: Nucleosomes must be disassembled and reassembled during eukaryotic replication.
• Cell cycle control: Eukaryotic replication is restricted to S phase and licensed by the pre-replication complex (ORC, Cdc6, Cdt1, MCM helicase).
• Telomeres: Ends of linear chromosomes are replicated by telomerase, which is not needed in circular prokaryotic DNA.

Fidelity refers to the accuracy with which DNA is replicated; the overall error rate is extremely low (~1 error per - nucleotides).

Mechanisms ensuring fidelity:

1. Base selection (nucleotide selectivity): DNA polymerase's active site favors correct Watson-Crick base pairing; incorrect nucleotides fit poorly. Error rate ~.

2. Proofreading (3' \rightarrow 5' exonuclease activity): DNA polymerase detects a mismatched nucleotide, removes it, and resynthesizes. This improves accuracy ~100-fold.

3. Mismatch repair (MMR): After replication, mismatches escaping proofreading are recognized and corrected. In E. coli, the MutS-MutL-MutH system distinguishes the new strand by its lack of methylation ( methylation of GATC). This improves fidelity another ~100-1000 fold.

Combined, these layers yield the overall very low mutation rate essential for genome stability.

Rolling circle replication (RCR) is a mode of unidirectional replication used to rapidly produce multiple copies of circular DNA/RNA.

Mechanism:

• An initiator protein nicks one strand of the circular double-stranded DNA at the origin, generating a free -OH end.
• The intact circular strand serves as the template.
• DNA polymerase extends the end, displacing the nicked () strand as a growing single-stranded tail.
• The displaced strand is coated with SSBs.
• Continuous rounds produce a long concatemer (multiple genome copies in tandem).
• The concatemer is later cleaved into unit lengths and circularized/converted to double-stranded form.

Biological significance:

• Replication of many bacteriophages (e.g., λ, φX174, M13).
• Bacterial conjugation (F-plasmid transfer).
• Amplification of rRNA genes in amphibian oocytes.
• Basis of laboratory techniques like rolling circle amplification (RCA).

DNA polymerase can only synthesize DNA in the direction, adding nucleotides to a free -OH. Because the two parental strands are antiparallel, only one strand can be copied continuously.

Leading strand:

• Synthesized continuously in the direction of fork movement.
• Requires only a single RNA primer.
• Template is read .

Lagging strand:

• Synthesized discontinuously in short fragments called Okazaki fragments.
• Each fragment needs its own RNA primer.
• Synthesis occurs in the direction opposite to fork movement.
• Primers are later removed (Pol I / RNase H) and gaps filled; DNA ligase joins the fragments.

Reason for discontinuity: As the fork opens, the lagging-strand template becomes available in the direction relative to fork movement, but polymerase must synthesize , forcing it to work backward in pieces. This mode is described as semidiscontinuous replication.

Transfer RNA (tRNA) is a small (~75-90 nucleotide) adaptor molecule that translates the genetic code into amino acids.

Secondary structure (cloverleaf):

• Acceptor stem: The end carries the conserved CCA sequence where the amino acid attaches.
• D-arm (DHU loop): Contains dihydrouridine; involved in recognition by aminoacyl-tRNA synthetases.
• Anticodon arm: Contains the anticodon that base-pairs with the mRNA codon.
• TψC arm: Contains ribothymidine, pseudouridine; interacts with the ribosome.
• Variable loop: Varies in size among tRNAs.

Tertiary structure (L-shaped):

• The cloverleaf folds into an L-shape via tertiary hydrogen bonds, placing the anticodon and the amino acid acceptor site at opposite ends (~70 Å apart).

Function: tRNA is aminoacylated (charged) by specific synthetases and delivers the correct amino acid to the ribosome during translation, ensuring accurate decoding.

Chargaff's rules (Erwin Chargaff, late 1940s) describe the base composition of DNA:

First rule (base equivalence):

• The amount of adenine equals thymine () and guanine equals cytosine ().
• Consequently, the total purines equal total pyrimidines:

Second rule:

• The base composition (%GC) varies between species but is characteristic and constant for a given species.

Significance:

• These rules provided crucial evidence for complementary base pairing (A-T and G-C), a cornerstone of the Watson-Crick model.
• They explained why the double helix has a uniform diameter (a purine always pairs with a pyrimidine).
• The species-specific %GC content underlies differences in melting temperature and DNA stability.

Note: Chargaff's rules apply to double-stranded DNA, not single-stranded DNA or RNA.

Okazaki fragments are short DNA segments synthesized discontinuously on the lagging strand. Their maturation converts them into a continuous strand:

Steps:

1. Primer synthesis: Primase lays down an RNA primer for each fragment.
2. Elongation: DNA polymerase III (prokaryotes) or Pol δ (eukaryotes) extends each fragment until it reaches the previous fragment's primer.
3. Primer removal:
   • In prokaryotes, DNA Pol I uses its exonuclease activity to remove RNA primers and simultaneously fills the gap (nick translation).
   • In eukaryotes, RNase H and FEN1 remove the primer/flap.
4. Gap filling: DNA polymerase fills the resulting gap with DNA nucleotides.
5. Ligation: DNA ligase forms the phosphodiester bond, sealing the nick.

Importance:

• Produces an intact, continuous lagging strand.
• Removes error-prone RNA and ensures genome integrity.
• Failure leads to fragmented DNA and genomic instability.

DNA and RNA are both nucleic acids but differ in several ways:

Key structural notes:

• The -OH group in RNA makes it chemically more reactive and prone to hydrolysis.
• RNA can fold into complex secondary and tertiary structures enabling catalytic (ribozyme) activity, whereas DNA typically remains a stable duplex.

DNA topology of a closed circular molecule is described by three parameters related by:

• Linking number (): The number of times one strand crosses the other; a topological invariant for a closed circle (changes only by strand breakage).
• Twist (): The number of helical turns of one strand around the other.
• Writhe (): The number of times the axis of the helix crosses itself (supercoils).

Superhelical density () measures the degree of supercoiling relative to the relaxed state:

Given: ,

Interpretation: A negative of indicates the DNA is negatively supercoiled (underwound), typical of natural DNA which favors strand separation for replication and transcription.

The end-replication problem:

• Linear eukaryotic chromosomes cannot fully replicate their ends. When the final RNA primer on the lagging strand is removed, DNA polymerase cannot fill the gap because there is no upstream -OH to extend from.
• This results in progressive shortening of chromosome ends with each round of replication.

Telomeres:

• Telomeres are repetitive, non-coding DNA sequences (e.g., in humans) at chromosome ends bound by protective proteins (shelterin complex).
• They protect coding sequences from erosion and prevent chromosome ends from being recognized as DNA damage.

Telomerase:

• Telomerase is a reverse transcriptase (ribonucleoprotein) carrying its own RNA template.
• It extends the overhang by adding telomeric repeats, providing a template for lagging-strand synthesis and counteracting shortening.

Significance:

• Telomere shortening is linked to cellular aging (senescence).
• Telomerase reactivation is a hallmark of many cancers, allowing unlimited cell division.