Molecular Genetics Exam 1

Recognition of the Origin Prokaryotes

1: DnaA reads a 9 base pair segment 5′-TTATCCAC-3’ called DnaA boxes and uses it as a landing pad to attach

Recognition of the Origin Eukaryotes

1: Origin Recognition Complex looks for nucleosome depleted regions, areas where DNA isn’t wrapped around histones. ORC also prefers A/T rich areas where metazoan origins are more common. ORC has 6 proteins, ORC-1 through ORC-6 which search for acetylated histone tails (weakened DNA histone binding). ORC also looks for G4 (4 stranded DNA structure) sequences based on the secondary structure of DNA,

Relieving torsional strain Prokaryotes

2: DNA gyrase introduces negative supercoils to relieve the positive supercoiling strain caused ahead of the replication fork by Helicase.

It breaks phosphodiester bonds on both DNA strands and holds onto the severed ends using active-site Tyrosine residues that covalently bond to the $5'$ phosphate groups.

It conserves the energy of the original phosphodiester bond in this tyrosine-phosphate bond, allowing it to reseal (ligate) the backbone without using ATP. (ATP is used only to power the protein's mechanical gate movement).

Relieving torsional strain eukaryotes

2: Topoisomerase II: Relieves the intense positive supercoiling build-up generated during unwinding. It creates a transient ATP-dependent double-strand break in one DNA duplex, allows an intact duplex segment to pass through, and rapidly re-ligates the phosphodiester backbone.

Preparing to separate both strands of DNA prokaryotes

3: Helicase Loader: Encoded by dnaC, it acts as an essential chaperone that holds the ring-shaped helicase in an open conformation. It escorts and loads the helicase directly onto single-stranded DNA within the open initiation bubble, hydrolyzing ATP to release the helicase so it can lock around the strand

Preparing to separate both strands of DNA eukaryotes

3: Helicase Loader: Composed of licensing factors (Cdc6 and Cdt1), this complex works alongside the Origin Recognition Complex during G1 phase. It mediates the loading of two ring-shaped inactive helicase cores onto double-stranded DNA at the origin, a process known as origin "licensing".

Separate Two Strands of DNA prokaryotes

4: Helicase: Encoded by dnaB, this hexameric ring unwinds duplex DNA at the fork. Ringing around the lagging strand template, it uses energy from ATP hydrolysis to break the interstrand hydrogen bonds between complementary nitrogenous bases in a $5' \to 3'$ direction.

Separate Two Strands of DNA eukaryotes

4: Helicase: Activated at the onset of S phase by key phosphorylation events, this active multi-protein complex surrounds the leading strand template. Driven by ATP, it moves in a 3’ - 5’ direction to melt the double helix and drive fork progression.

Prevent Rejoining of Strands prokaryotes

5: Single-stranded binding proteins: Tetrameric proteins that bind cooperatively and non-specifically to exposed single-stranded DNA (ssDNA). They physically shield the hydrophobic bases to prevent the two template strands from spontaneously re-annealing or forming secondary hairpin structures that would block polymerases.

Prevent Rejoining of Strands eukaryotes

5: Single-stranded binding proteins: Multi-subunit complexes (functionally known as RPA) that coat exposed ssDNA. Besides preventing re-hybridization and secondary structure formation, they protect the single strands from being degraded by endonucleases and act as a signaling platform for DNA damage checkpoints.

Priming Prokaryote

6: RNA primase: Encoded by dnaG, this enzyme transiently interacts with the active helicase to synthesize short RNA sequences ($\sim10\text{–}12\text{ nucleotides}$) on both templates. These RNA primers provide the free $3'\text{-OH}$ group required by DNA polymerases to initiate phosphodiester bond formation.

Priming eukaryote

6: RNA primase: Operating within a larger complex, this enzyme synthesizes a short RNA stretch ($\sim7\text{–}10\text{ nucleotides}$) on single-stranded template DNA. This provides the essential $3'\text{-OH}$ starter motif required for subsequent strand extension.

Preparing to Load the DNA Polymerase Holding Clamps prokaryote

7: Clamp loader: An ATP-dependent multi-subunit complex (gamma-tau complex) that recognizes primer-template junctions. It binds the closed ring-shaped clamp protein, forces it open using ATP, positions it precisely over the duplex DNA at the $3'$ end of the RNA primer, and hydrolyzes ATP to lock the clamp around the DNA.

Preparing to Load the DNA Polymerase Holding Clamps eukaryote

7: Clamp loader: A five-subunit AAA+ ATPase complex (Replication Factor C / RFC) that specifically binds the $3'$ end of primer-template junctions. It uses ATP energy to open the circular eukaryotic clamp protein, thread the DNA template through its center, and close the ring around the DNA strand.

Loading of DNA Polymerase on Both Strands of DNA prokaryote

8: Clamp protein: Encoded by dnaN, this homodimeric ring-shaped protein (beta-clamp) slides freely along double-stranded DNA. By binding directly to the core DNA polymerase, it prevents the enzyme from diffusing away from the template, increasing polymerase processivity from tens to hundreds of thousands of base pairs.

Loading of DNA Polymerase on Both Strands of DNA eukaryote

8: Clamp protein: A homotrimeric ring structure (PCNA) that encircles the DNA double helix. It acts as a mobile sliding platform that anchors eukaryotic DNA polymerases to the template, maintaining high speed and continuity during continuous and discontinuous strand synthesis.

Extension of RNA Primer for Synthesis of New DNA Strand prokaryote

9: DNA polymerase: The main replicative enzyme (DNA Polymerase III) synthesizes new DNA strictly in the 5’ - 3’ direction. It continuously synthesizes the leading strand toward the replication fork and discontinuously synthesizes Okazaki fragments (1000bp - 2000bp) on the lagging strand away from the fork.

Extension of RNA Primer for Synthesis of New DNA Strand eukaryote

9: DNA polymerase: Main replicative polymerases (Polymerase epsilon on the leading strand and Polymerase delta on the lagging strand) extend the primer-template junction. They catalyze 5' - 3' synthesis of complementary DNA, producing shorter Okazaki fragments (100-200bp) on the lagging strand.

Proofreading prokaryote

10: 3’ - 5’ exonuclease: Intrinsic catalytic activity (carried out by the epsilon subunit of DNA Pol III) that functions as a real-time quality control system. When a mismatched base pair disrupts the geometry of the double helix, polymerization pauses, the 3’ end shifts into the exonuclease site, the incorrect base is excised, and the strand returns to the polymerase domain.

Proofreading eukaryote

10: 3’ - 5’ exonuclease: Built-in exonuclease domains within replicative polymerases (Pol epsilon and Pol delta) They immediately recognize non-standard base geometries, pause polymerization, reverse direction to hydrolyze the erroneous $3'$ terminal nucleotide, and resume complementary synthesis, reducing error rates down to 1 in 10^7 - 10^8.

Adding Correct Nucleotide and Base Pairing prokaryote

11: DNA polymerase: Reads the parental strand template and tests incoming deoxynucleotide triphosphates (dNTPs) within its active site. Correct Watson-Crick base pairing (AT - 2 H bonds, GC - 3 H bonds) induces a conformational shift that triggers a nucleophilic attack by the template's 3’ OH group on the incoming alpha-phosphate, releasing pyrophosphate PPi.

Adding Correct Nucleotide and Base Pairing eukaryote

11: DNA polymerase: Utilizes specific spatial geometry within the active site pocket to ensure only proper dNTPs matching the template strand are incorporated. It catalyzes phosphodiester bond formation, driving chain extension step-by-step while maintaining high fidelity.

Removing the RNA Primers from Leading and Lagging Strand prokaryote

12: DNA polymerase I: A multifunctional enzyme that uses its unique 5’ - 3’ exonuclease domain (distinct from proofreading exonuclease) to hydrolyze and remove the RNA primer nucleotides located directly in front of its path on the template strand.

Removing the RNA Primers from Leading and Lagging Strand eukaryote

12: RNAsH + FEN1: RNase H specifically degrades the bulk of the RNA primer portion hybridized to DNA. As Polymerase delta extends into the region, it displaces the remaining RNA nucleotide flap, which is then specifically recognized and cleaved off by Flap Endonuclease 1 (FEN1).

Gap Filling Between Okazaki Fragments in Lagging Strand and Refilling the Primer Space in the Leading Strand prokaryote

13: DNA polymerase I: Concurrently with primer degradation, it uses its 5’ - 3’ polymerase domain to add deoxynucleotides (dNTPs) into the vacant gap, substituting RNA with DNA. This dual action (known as nick translation) proceeds until all RNA is replaced, leaving only a single backbone nick between adjacent DNA fragments.

Gap Filling Between Okazaki Fragments in Lagging Strand and Refilling the Primer Space in the Leading Strand eukaryote

13: DNA polymerase delta: Following primer removal by RNase H and FEN1, DNA Polymerase delta extends the upstream DNA fragment across the gap, synthesizing DNA until it reaches the 5’ end of the downstream Okazaki fragment, leaving behind a single nick in the sugar-phosphate backbone.

Sealing the Nick/Gap Between Two Nucleotides prokaryote

14: DNA ligase: Encoded by ligA, this enzyme seals single-stranded nicks in the phosphodiester backbone. It uses an energetic cofactor (NAD+ in bacteria) to transfer an AMP group to the 5’ phosphate group at the nick, facilitating nucleophilic attack by the adjacent 3’ OH group to form a continuous phosphodiester bond.

Sealing the Nick/Gap Between Two Nucleotides eukaryote

14: DNA ligase: Uses an ATP cofactor to covalently join the 3’ OH terminus of one DNA fragment to the 5’ - monophosphate terminus of another. This seals all phosphodiester gaps along both the leading strand and Okazaki fragments on the lagging strand, completing the continuous double helix.

Separating Two Daughter DNA Upon Completion of Replication prokaryotes

15: Topoisomerase IV: A specialized Type II topoisomerase that acts at the termination site (ter locus). Because bacterial chromosomes are circular, replication causes the two resulting circular double-stranded daughter molecules to become physically linked (catenated); Topoisomerase IV transiently cleaves both strands of one circular chromosome, passes the second chromosome through, and reseals it to un-link (decatenate) the daughter circles.

Separating Two Daughter DNA Upon Completion of Replication eukaryotes

15: Topoisomerase II: Upon termination of replication along linear chromosomes, sister chromatids remain topologically tangled and catenated at various points. Topoisomerase II introduces double-stranded cuts to untangle and resolve these interlocks, allowing sister chromatids to properly condense and separate cleanly during mitosis.

Histone

protein that dna wraps around, 8 protein histone core with 2 copies of H2A, H2B, H3, H4

nucleosome

bead made up of 8 histone molecules and 146-147 bp, connected by H1 histone protein

solenoid

30 nm fiber forcing 6 nucleosomes into scaffold loops

weight of one bp

650 daltons

Da

1.66×10^-24 g

weight of nucleotide

330 Da

Histone code Hypothesis

comboes of modifacation of methylation, acetylation, phosphorylation, ubiquitylation, sunoylation, ADP-riboslation define the function of chromatin.

weight of histone protein

11-15 kD