DNA Structure and Replication

What is the clinical presentation and lab finding in a patient with Cystic Fibrosis?

Chronic respiratory infections, poor growth, and elevated sweat chloride levels.

What is the specific genetic mutation in the CFTR gene that causes Cystic Fibrosis?

Deletion of three nucleotides (the delta-F508 mutation) in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene.

What is the molecular and physiological consequence of the delta-F508 mutation in CFTR?

Causes a misfolded CFTR protein that impairs chloride (Cl-) transport across lung epithelial cells, reducing water movement into the airway and causing thick, sticky mucus buildup, decreased mucociliary clearance, inflammation, and easy pathogen access.

How many cell types are in the human body, and why do they behave differently if they contain the same DNA?

~200 cell types; every cell contains the same DNA, but gene expression is different and specific for each cell type.

Trace the pathway from an environmental signal to cancer when DNA is mutated (Slide 3 diagram).

Environment/Signal -> Cell signaling pathways -> Transcription factors/chromatin -> Gene expression (DNA -> mRNA -> protein) -> Protein function (enzymes, cyclins, receptors) -> Cell behaviors (cell cycle, division, survival) -> If disrupted by mutations -> Cancer.

What are the three processes of the Central Dogma of Molecular Biology?

Replication (DNA -> DNA), Transcription (DNA -> RNA), and Translation (RNA -> Protein).

What is bacterial transformation?

A process in which external DNA is taken up by a bacterial cell, changing its morphology and physiology (phenotype).

Contrast Smooth (S) and Rough (R) cells of Streptococcus pneumoniae.

Smooth (S) cells are pathogenic and produce a polysaccharide capsule; Rough (R) cells are nonpathogenic and do not produce a capsule because they lack the "S gene".

How did Oswald Avery set up his experiment to identify the genetic material?

He heat-killed pathogenic S cells to release cell-free S fragments, treated the extract with an enzyme that degrades one specific macromolecule, and mixed it with nonpathogenic R cells to see if R transformed into S.

In Avery's experiment, what happened when the S extract was treated with Proteinase (protease)?

Protein was degraded, and R cells still transformed into S cells (Yes).

In Avery's experiment, what happened when the S extract was treated with RNase (ribonuclease)?

RNA was degraded, and R cells still transformed into S cells (Yes).

In Avery's experiment, what happened when the S extract was treated with DNase (deoxyribonuclease), and why?

DNA was degraded, and R cells did NOT transform into S cells (No), because destroying the DNA destroyed the "S gene" required for transformation.

How are nucleotides joined in a polynucleotide chain, and how are sequences written?

Joined by phosphodiester bonds connecting the 3'-OH of one nucleotide to the 5'-phosphate of the next; sequences are always written 5' to 3'.

What is Chargaff's first rule of DNA base composition?

In DNA, the concentration of A = T and G = C (total purines A + G = total pyrimidines T + C).

According to Chargaff's rules, how does base composition vary across species vs. within an individual organism?

Base composition varies between species (e.g., human genome is ~59% AT / 41% GC; yeast is 64% AT / 36% GC), but is the same across different cells within an organism and does NOT change with age, nutrition, or environment.

What technique did Rosalind Franklin use to produce Photo 51, and how does it work?

X-ray diffraction; X-rays bend (diffract) in different directions based on the location of atoms in a macromolecule.

What three structural parameters of DNA did Rosalind Franklin's Photo 51 reveal?

1. DNA is helical with a consistent diameter (shown by the Helical X); 2. Each turn of the helix is 34 Angstroms long; 3. Each turn contains ~10 base pairs separated by 3.4 Angstroms each.

What is the repeating monomeric unit of DNA?

2'-deoxyribonucleoside 5'-monophosphate (dNMP).

Describe the Watson-Crick-Franklin model of DNA structure.

Two unbranched polynucleotide chains in an antiparallel orientation, with the sugar-phosphate backbone on the outside and bases interacting via hydrogen bonds on the inside.

How many hydrogen bonds form in an Adenine-Thymine (A-T) pair vs. a Guanine-Cytosine (G-C) pair, and what are their C-1' to C-1' distances?

A-T forms 2 hydrogen bonds (11.1 Angstrom C-1' to C-1' distance); G-C forms 3 hydrogen bonds (10.8 Angstrom C-1' to C-1' distance).

What percentage of cellular DNA is in the B-DNA conformation, and what are its main structural features?

>99% of cellular DNA; a right-handed helix (~20 Angstrom diameter, 36 Angstroms per turn) with 10.5 bp/turn, 3.4 Angstrom rise per bp, base pairs flat and perpendicular to the backbone (6 degree tilt), and exposed major and minor grooves.

Where are the hydrophobic portions of bases located in B-DNA, and what interactions stabilize them?

Located on the interior of the helix, stabilized by hydrophobic and base-stacking interactions.

What is A-DNA, and when does it occur?

The conformation of dehydrated DNA; also serves as a structural model for double-stranded RNA and RNA-DNA hybrid molecules.

What is Z-DNA, and where is it found?

A left-handed helix accounting for < 1% of cellular DNA, seen in regions with high G-C content.

Compare the helical sense, diameter, and base pairs per turn of A-DNA, B-DNA, and Z-DNA (Slide 15 table).

A-DNA: Right-handed, ~26 Angstroms wide, 11 bp/turn (2.6 Angstrom rise); B-DNA: Right-handed, ~20 Angstroms wide, 10.5 bp/turn (3.4 Angstrom rise); Z-DNA: Left-handed, ~18 Angstroms wide, 12 bp/turn (3.7 Angstrom rise).

Compare the sugar pucker and glycosyl bond conformations of A-DNA, B-DNA, and Z-DNA (Slide 15 table).

A-DNA: C-3' endo, Anti; B-DNA: C-2' endo, Anti; Z-DNA: C-2' endo for pyrimidines / C-3' endo for purines, and Anti for pyrimidines / Syn for purines.

Define DNA denaturing (melting) and annealing (hybridization).

Denaturing (melting) is separating two DNA strands; annealing (hybridization) is when complementary single strands of DNA rejoin by base pairing.

What is the melting temperature (Tm) of DNA, and how does G-C content affect it?

The temperature at which a solution of DNA is 50% denatured; more G-C base pairs results in a higher Tm.

In the delta-F508 allele-specific probe experiment (Slide 16), why does the delta-F508 duplex have a lower Tm than the normal CFTR allele?

The probe is designed to match the normal 15-nt CFTR sequence (15/15 bases paired -> higher Tm); the delta-F508 allele has a 3-nt deletion (CTT), leaving only 12/15 bases paired with a 3-nt bulge (GAA) in the probe, creating a less stable duplex with a lower Tm.

What are the primary structural features of RNA?

Typically a linear, single-stranded polynucleotide with a ribose sugar-phosphate backbone (has a 2'-OH), uracil (U) in place of thymine (T), sequence read 5' to 3', and base-stacking interactions that give it a helical shape.

What conformation does base-paired RNA resemble, and what special base pair is allowed in RNA-RNA pairing?

Resembles the A-DNA conformation (with antiparallel strands); G-U base pairing is allowed when RNA base pairs with itself or another RNA.

How do RNA molecules form secondary and tertiary structures?

Internal base pairing between complementary regions within a single RNA strand folds it into secondary structures (stems, hairpin loops, internal loops, stem-loops) and complex 3D tertiary structures that determine function.

What two components make up a gene, and what is a genome?

A gene contains the DNA encoding a functional RNA or protein PLUS the regulatory elements controlling its expression; a genome is the complete set of genetic material in a cell or organism.

Compare the genome size, chromosome structure, and number of genes in E. coli K12 vs. Homo sapiens (Slide 22 table).

E. coli K12: 4,639,675 bp (~4.6 x 10^6 bp), 1 circular chromosome, ~4,435 genes; Homo sapiens: 3,070,128,600 bp (diploid), 46 linear chromosomes (diploid), ~29,000 genes (haploid).

What proteins are most commonly bound to eukaryotic chromosomal DNA, and what structures do they form?

Histone proteins; DNA wraps around histones to form nucleosomes, which pack into chromatin and chromosomes.

What key structural regions are found on a linear eukaryotic chromosome, and what is the chromosomal location of the human CFTR gene?

Telomeres (at both ends), a centromere, unique sequences (genes), dispersed repeats, and multiple replication origins; the CFTR gene is fixed at 7q31.2.

Define the three proposed models of DNA replication: Conservative, Semiconservative, and Dispersive.

Conservative: yields one original intact parental DNA duplex and one entirely new DNA duplex; Semiconservative: yields two DNA molecules, each with one parental and one newly synthesized strand; Dispersive: yields two DNA molecules that are mixtures/hybrids of parental and new DNA throughout both strands.

How did Meselson and Stahl label and separate DNA in E. coli?

They grew E. coli in heavy nitrogen (15N) medium so all parental DNA was 15N/15N, moved the cells to light nitrogen (14N) medium for replication, and isolated and centrifuged the DNA in a CsCl density gradient.

In the Meselson and Stahl experiment, what was observed after Round 1 of replication, and which model did it rule out?

All DNA formed a single intermediate hybrid band (14N/15N); this ruled out the conservative model (which predicted one heavy 15N/15N band and one light 14N/14N band).

In the Meselson and Stahl experiment, what was observed after Round 2 of replication, and which model did it rule out?

DNA formed one hybrid band (14N/15N) and one light band (14N/14N); this ruled out the dispersive model (which predicted a single band of intermediate density) and proved semiconservative replication.

What are the substrates and cofactor requirements of DNA Polymerase?

Substrates are dNTPs; requires a single-stranded DNA template, a DNA or RNA primer with a free 3'-OH (made by primase, an RNA polymerase), and two Mg2+ ions coordinated by Asp residues at the active site.

Describe the chemical reaction catalyzed by DNA Polymerase.

A phosphoryl group transfer reaction in which the 3'-OH of the 3' nucleotide on the growing strand (primer) nucleophilically attacks the alpha-phosphate of the incoming dNTP, releasing pyrophosphate (PPi) and synthesizing DNA strictly 5' to 3'.

What are the three structural domains of DNA Polymerase, and what happens at the insertion and postinsertion sites?

Palm, Thumb, and Fingers domains; the incoming dNTP base-pairs with the template at the insertion site, is joined to the 3' primer terminus, and the polymerase translocates one base pair so the new pair moves to the postinsertion site.

If a template DNA strand has the sequence 3'-CTTAGCAGTGCAAT-5', what is the sequence and polarity of the newly synthesized daughter strand?

5'-GAATCGTCACGTTA-3' (synthesized antiparallel in the 5' to 3' direction).

What disease does AZT treat, when was it approved, and what drug class does it belong to?

Treats HIV (first FDA-approved HIV drug in 1987) and prevents mother-to-child transmission; it is a nucleoside reverse transcriptase inhibitor (NRTI).

Why is AZT classified as a prodrug, and how does it stop DNA synthesis?

AZT is inactive until the patient's own cellular kinases add three phosphates to form AZT-triphosphate, which mimics dTTP and pairs with A; once incorporated, its 3'-azido (-N3) group lacks a 3'-OH, causing chain termination.

What explains AZT's selective toxicity versus its clinical side effects (anemia, bone marrow suppression, myopathy)?

Selective toxicity: HIV reverse transcriptase takes up AZT-triphosphate far more readily than human nuclear DNA polymerases; Side effects: Human mitochondrial DNA polymerase (Pol gamma) incorporates AZT, causing mitochondrial toxicity.

What is the average mutation rate in E. coli per base pair and per genome replication?

~1 bp error per 10^9 to 10^10 bp; since the E. coli genome is ~4.6 x 10^6 bp, an error occurs only once per 1,000 to 10,000 replications.

What is presynthetic error control in DNA Polymerase, and what is its error rate?

The shape of the DNA Pol active site sterically restricts base pairing to canonical Watson-Crick-Franklin base pairs (excluding mismatched G-T, A-C, A-G geometries); with this alone, DNA Pol still adds a wrong base every 10^4 to 10^5 nucleotides.

What two active sites do high-fidelity DNA polymerases possess, and what are their directionalities?

1. A catalytic polymerase site for 5' -> 3' DNA synthesis ("typing"); 2. A proofreading 3' -> 5' exonuclease site for removing mis-incorporated nucleotides ("backspace").

Trace the 5 steps of proofreading when DNA Polymerase mispairs a nucleotide (Slide 11 diagram).

1. Polymerase mispairs dC with dT; 2. Polymerase repositions the mispaired 3' terminus into the 3' -> 5' exonuclease site; 3. Exonuclease hydrolyzes the mispaired dC (releasing dCMP); 4. The 3' terminus repositions back to the polymerase site; 5. Polymerase incorporates the correct nucleotide (dATP).

What clinical condition is caused by mutations in the proofreading domain of human DNA polymerases?

Colorectal and endometrial cancers.

Where does DNA synthesis begin, and why is it bidirectional?

Begins at an origin of replication where parental strands separate to form a replication bubble with two replication forks moving in opposite directions.

What enzyme unwinds parental DNA at the replication fork, and what is its energy source?

Helicase unwinds parental DNA using ATP hydrolysis.

Contrast synthesis of the leading strand and lagging strand at a replication fork.

Leading strand: synthesized continuously 5' -> 3' toward the replication fork; Lagging strand: synthesized discontinuously 5' -> 3' away from the fork as a series of Okazaki fragments.

How does DNA unwinding cause supercoiling ahead of and behind the replication fork?

Over-winding (

How do Type I and Type II Topoisomerases relieve supercoiling?

They cut phosphodiester bonds in one DNA strand (Type I) or both DNA strands (Type II), pass the intact strand(s) through the break to unwind the helix, and ligate (reseal) the nick.

What is bacterial DNA gyrase, and what are its two functions in bacteria?

Bacterial Topoisomerase II; it introduces negative supercoils to compact the bacterial genome and removes positive supercoils ahead of replication forks.

Clinical Connection: How do fluoroquinolone antibiotics (like ciprofloxacin) work, and what infections do they treat?

They selectively target bacterial DNA gyrase (Topoisomerase II) and block its ability to reseal cleaved DNA strands, leading to double-strand breaks and bacterial cell death while sparing human topoisomerases; used to treat UTIs, respiratory infections, and GI infections.

What are the structural genes and subunit compositions of E. coli DNA Polymerase I vs. DNA Polymerase III?

DNA Pol I is encoded by polA and consists of 1 subunit (Mr 103,000); DNA Pol III is encoded by polC (dnaE) and has 9 different subunit types (Mr 1,065,400).

Compare the exonuclease activities of E. coli DNA Polymerase I and DNA Polymerase III.

Both Pol I and Pol III have 3' -> 5' exonuclease (proofreading) activity, but ONLY DNA Pol I has 5' -> 3' exonuclease activity (used for removing RNA primers).

Compare the polymerization rate and processivity of E. coli DNA Polymerase I vs. DNA Polymerase III.

DNA Pol I has a slow polymerization rate (10-20 nt/s) and low processivity (3-200 nt added before dissociation); DNA Pol III has a very fast polymerization rate (250-1,000 nt/s) and massive processivity (>=500,000 nt).

What are the primary cellular roles of E. coli DNA Polymerase I and DNA Polymerase III?

Both function in genomic replication and DNA repair: Pol III synthesizes the bulk of the leading and lagging strands, while Pol I removes RNA primers (5' -> 3' exonuclease) and fills the resulting gaps on the lagging strand.

What are the functions of the three components of the E. coli DNA Pol III Holoenzyme (Core polymerase, Clamp loader, and Beta sliding clamp)?

1. Core polymerase (Pol III): catalyzes DNA synthesis at ~1,000 nt/s; 2. Clamp loader: uses ATP hydrolysis (3 ATP -> 3 ADP + 3 Pi) to assemble the beta clamp onto DNA and coordinates the fork via tau subunits interacting with DnaB helicase and Pol III cores; 3. Beta sliding clamp: ring-shaped dimer that tethers core Pol III to DNA, decreasing dissociation and increasing processivity.

How often does bacterial replication initiation occur, and what are the sequence elements of E. coli oriC (245 bp)?

Occurs only once per cell division (most regulated step); oriC contains: DUE (DNA Unwinding Element: tandem array of three 13-bp AT-rich repeats, consensus GATCTNTTNTTTT), five R sites (R1-R5, 9-bp repeats, consensus TT(A/T)TNCACC) and three I sites (I1-I3) that bind DnaA, plus IHF and FIS binding sites for initiation factors.

List the sequential steps of E. coli DNA replication initiation at oriC (Slide 22).

1. DnaA binds ATP to become active; 2. ATP-DnaA binds oriC (with IHF), causing positive supercoiling that denatures the AT-rich DUE region; 3. DnaC binds ATP and loads a hexameric DnaB helicase at both ends of the replication bubble; 4. DnaB helicase unwinds DNA 5' -> 3' using ATP; 5. DnaC dissociates; 6. DNA Pol III and replisome proteins assemble on both DnaB helicases; 7. ATP bound to DnaA is hydrolyzed to ADP and DnaA dissociates (slow ADP release prevents immediate re-initiation).

Describe the coordinated action of proteins in the prokaryotic replisome at the replication fork (Slide 23).

DNA gyrase removes positive supercoils ahead of the fork; DnaB helicase unwinds DNA 5' -> 3'; Primase synthesizes RNA primers; Single-stranded DNA binding protein (SSB) coats ssDNA to protect from nucleases, reannealing, and secondary structures; the lagging strand loops around so the Pol III holoenzyme synthesizes both leading and lagging strands simultaneously.

How are RNA primers removed and Okazaki fragments joined on the prokaryotic lagging strand (Slide 24)?

1. DNA Pol I (or RNase H1) removes the RNA primer using Pol I's 5' -> 3' exonuclease activity while Pol I simultaneously fills the gap with dNTPs (5' -> 3' synthesis); 2. Bacterial DNA ligase repairs the remaining nick using NAD+ (releasing AMP + NMN).

What is the state of two circular bacterial chromosomes immediately after replication completes, and how are they separated (Slide 25)?

They are interlinked like links in a chain in a "catenated" state; DNA Topoisomerase IV separates (decatenates) the circular chromosomes.

What fundamental rules are shared between prokaryotic and eukaryotic DNA replication?

Both are semiconservative, bidirectional, template- and primer-dependent, synthesize DNA strictly 5' -> 3' with a continuous leading strand and discontinuous lagging strand, and use high-fidelity polymerases with 3' -> 5' proofreading.

How many DNA polymerases and replication origins do human/eukaryotic cells have, and how fast is eukaryotic synthesis?

Eukaryotes have ~15 specialized DNA polymerases and ~30,000-50,000 AT-rich origins of replication (~25,000 bp apart, associated with actively transcribed genes); synthesis rate is slower (max ~50 nt/sec) and Okazaki fragments are shorter (100-200 nt; ~50 million made per round in humans).

How does replication "licensing" regulate eukaryotic origins across G1 and S phases (Slide 30)?

In G1 phase (CDKs OFF; licensing allowed, origin firing prevented): Origin Recognition Complex (ORC) binds origins, and Cdc6 and Cdt1 join to load the MCM helicase to form the Pre-RC; In S phase (CDKs ON; licensing prevented, origin firing allowed): Cyclin-CDKs phosphorylate ORC/MCM (and Cdc6/Cdt1 leave), activating the replisome and initiating bidirectional synthesis.

Identify the function of each protein in the eukaryotic replisome: MCM, Pol epsilon, Pol delta, Pol alpha-primase, RFC, PCNA, and RPA (Slide 31).

MCM: helicase; DNA Pol epsilon: synthesizes leading strand (high processivity, proofreading); DNA Pol delta: synthesizes lagging strand (proofreading, strand-displacing); DNA Pol alpha-primase: synthesizes the initial RNA primer plus a short DNA stretch; RFC (Replication Factor C): clamp loader; PCNA (Proliferating Cell Nuclear Antigen): trimeric sliding clamp (increases processivity); RPA (Replication Protein A): single-strand DNA binding protein (SSB).

Trace the steps of eukaryotic lagging strand processing (Slide 32).

1. DNA Pol delta synthesizes an Okazaki fragment and performs "strand displacement synthesis," pushing the downstream RNA-DNA primer into a single-stranded overhang flap; 2. Flap endonuclease-1 (FEN1) cleaves off the overhang flap; 3. DNA ligase uses ATP to seal the nick in the sugar-phosphate backbone.

Why do linear eukaryotic chromosomes shorten with every round of DNA replication (the end-replication issue)?

Once the terminal RNA primer on the lagging strand is removed from the extreme 5' end of the daughter strand, DNA polymerase cannot fill the gap because there is no upstream 3'-OH primer available at the chromosome end.

What is the structure and repeat sequence of human telomeres?

Non-coding repetitive sequences at the ends of linear chromosomes; in humans, the repeat is 5'-TTAGGG-3' on the TG strand (which overhangs and is longer at the 3' end than the complementary 3'-AATCCC-5' CA strand) so that shortening does not lose genes.

What is a T-loop in telomeres, and what proteins form it (Slide 35)?

A protective structure where the single-stranded 3' TG overhang loops back and base-pairs within the double-stranded telomere duplex, bound by telomere duplex DNA-binding proteins and TRF1 and TRF2; it sequesters and protects the 3' chromosome ends from nucleases and double-strand break repair enzymes.

How does telomere length change over time in Germ/Stem cells, Primary Somatic cells, and DKC/Progeria cells (Slide 37 graph)?

Germ and stem cells maintain constant telomere length (due to active telomerase); Primary somatic cells shorten each division until reaching critical length, triggering Replicative Senescence (permanent G0 arrest) or Crisis/Apoptosis (preventing gene loss and cancer); DKC (Dyskeratosis Congenita) and Progeria cells have abnormally rapid telomere shortening associated with premature aging.

What is telomerase, and what are its components?

A specialized ribonucleoprotein (composed of both RNA and protein) that acts as a reverse transcriptase, carrying its own internal RNA molecule with a CA-rich repeat (3'-AACCCCAAC-5') that serves as the template for synthesizing the TG telomere strand.

Trace the step-by-step mechanism of telomere synthesis by telomerase (Slide 38 diagram).

1. Telomerase's internal 3'-AACCCCAAC-5' RNA template anneals to the overhanging 3' TG primer strand; 2. Telomerase catalyzes 5' -> 3' DNA synthesis using dGTP and dTTP (releasing PPi) to add TTAGGG repeats to the 3' end; 3. Telomerase translocates along the newly extended 3' end and repeats synthesis; 4. Telomerase dissociates, and DNA Pol alpha-primase synthesizes an RNA primer on the new TG strand; 5. DNA polymerase fills in the complementary CA strand, DNA ligase seals the nick, RNase removes the primer, and telomere-binding proteins (TRF1/TRF2) bind the remaining 3' overhang to form a T-loop.