4540 section 2

What are the three components of a DNA nucleotide?

1. A nitrogenous base (purines A/G or pyrimidines C/T)

2. A deoxyribose sugar

3. A phosphate group

What is the covalent linkage between nucleotides in DNA?

A phosphodiester bond, which connects the 5' phosphate group of one nucleotide to the 3' hydroxyl (OH) group of the next.

What are the base-pairing rules and their hydrogen bond counts?

• Adenine (A) pairs with Thymine (T) (2 hydrogen bonds)

• Guanine (G) pairs with Cytosine (C) (3 hydrogen bonds)

How does RNA differ from DNA?

• Usually single-stranded

• Contains a ribose sugar (with a 2' hydroxyl)

• Uses Uracil (U) instead of Thymine

• Often forms complex secondary structures like hairpins

What are the three phases of DNA replication?

1. Initiation: At the origin (oriC)

2. Elongation: At the replication forks

3. Termination: At the terminus (ter)

What does semiconservative replication mean?

Each new DNA double helix contains one original parental strand and one newly synthesized daughter strand.

How is bacterial replication initiated?

It begins at a single origin (oriC) where a replication bubble forms with two forks moving in opposite directions (bidirectional).

What are the roles of DnaA, DnaB, and DnaC in initiation?

• DnaA: The initiator protein that melts the DNA at the origin.

• DnaB: The helicase that unwinds the DNA strands.

• DnaC: The helicase loader that places DnaB onto the template.

What is the function of DnaG (Primase)?

It synthesizes short RNA primers that provide the starting point for DNA synthesis.

What is the function of DNA Gyrase in replication?

A type 2 topoisomerase that relieves positive DNA supercoiling (torsional stress) ahead of the replication fork.

What is the role of DNA Polymerase III?

The primary replication enzyme responsible for adding nucleotides to the growing DNA chain.

Leading vs. Lagging Strand Synthesis

• Leading strand: Continuous synthesis toward the replication fork.

• Lagging strand: Discontinuous synthesis away from the fork in short Okazaki fragments.

How are RNA primers removed and replaced?

1. RNase H cleaves the RNA primer.

2. DNA Polymerase I fills the resulting gap with DNA.

3. DNA Ligase seals the remaining nick (using NAD in bacteria or ATP in eukaryotes).

What is the function of the Tus protein?

It binds to terminator sequences (ter) to stop movement of the polymerase and ensure termination.

What is a catenane and how is it resolved?

A catenane is a pair of interlocked daughter chromosomes; they are separated by Topoisomerase IV.

What are the 5 steps of replication Initiation?

1. DnaA-ATP binds to 9-mer sequences at oriC.

2. DNA melts at 13-mer repeats, forming a bubble.

3. DnaC loads DnaB (helicase) onto the strands.

4. DnaB recruits DnaG (primase) to make RNA primers.

5. DNA Pol III is loaded with a sliding clamp to complete the replisome.

What are the key steps of Elongation?

• Unwinding/Protection: DnaB unwinds strands; SSB proteins stabilize them.

• Tension Relief: DNA Gyrase reduces stress ahead of the fork.

• Synthesis: Leading and lagging strands are synthesized; the lagging strand loops to keep both Pol III enzymes together.

• Processing: RNA primers are removed by RNase H/Pol I, gaps are filled by Pol I, and fragments are joined by DNA Ligase.

What are the steps of Termination?

1. Replication forks meet at ter sites.

2. Tus protein blocks the forks.

3. Daughter chromosomes (catenanes) are unlinked by Topoisomerase IV.

4. Proteins like FtsK, XerC, and XerD segregate chromosomes to opposite cell ends.

How is DnaA regulated based on nutrition?

• Rate-limiting: DnaA accumulation is required for initiation; its focus increases with growth rate.

• Starvation: Amino acid lack induces (p)ppGpp, which inhibits DnaA transcription.

• SeqA: Sequesters the origin to prevent premature re-initiation.

What are the functions of Hda and YabA?

• Hda: Dephosphorylates DnaA.

• YabA: Locks DnaA to the polymerase to regulate the process.

What is the difference between Type I and Type II Topoisomerases?

• Type I: Single proteins; cleave one strand to relieve supercoiling; no ATP required.

• Type II: Multiple subunits; cleave both strands to introduce supercoils; requires ATP.

How do Quinolone antibiotics work?

They bind to the DNA cleavage complex of DNA Gyrase and Topoisomerase IV, blocking ligation and causing lethal double-strand breaks.

What are the mechanisms of Quinolone resistance?

1. Target-mediated: Mutations in Gyrase/Topo IV.

2. Plasmid-mediated: Qnr proteins block quinolones from the cleavage complex.

3. Chromosome-mediated: Reduced porin expression or increased efflux pumps.

How does Gepotidacin differ from Quinolones?

It stabilizes the cleavage complex but causes single-strand breaks instead of double-strand breaks; it is effective against quinolone-resistant strains.

Match the Nucleoid Associated Proteins (NAPs) to their functions

• H-NS: Bridges DNA loci together.

• IHF: Causes dramatic bending to facilitate loop formation.

• HU: Wraps/bends DNA by inserting proline into the minor groove.

• Fis: Bends DNA; most abundant during rapid growth.

• Dps: Protects DNA during starvation.

What is the SMC complex?

A large ring-like protein complex that encircles DNA to assist in chromosome condensation and organization.