Lecture 10: DNA Replication (chad)

What is semiconservative replication

Each replicated DNA molecule consists of one original (old) strand and one newly synthesized strand.

What are the three proposed models of DNA replication

Conservative, semiconservative, and dispersive.

What did the Meselson–Stahl experiment (1958) demonstrate

DNA replication in E. coli is semiconservative; each new molecule has one old and one new strand.

How does DNA replication occur in bacteria

It begins at a single origin of replication and proceeds bidirectionally.

What enzyme catalyzes DNA synthesis

DNA polymerase, which requires a DNA template and all four dNTPs.

In which direction does chain elongation occur

In the 5′ to 3′ direction by adding nucleotides to the 3′ hydroxyl end.

What happens to the terminal phosphates during elongation

Two terminal phosphates are cleaved off as each nucleotide is added.

Which enzyme is primarily responsible for DNA synthesis in E. coli

DNA polymerase III.

What is the function of DNA polymerase III

Performs 5′→3′ polymerization and 3′→5′ exonuclease proofreading.

What is the function of DNA polymerase I

Removes RNA primers (5′→3′ exonuclease activity) and fills gaps with DNA.

What are the seven key issues that must be resolved during DNA replication

1. Unwinding of the helix, 2. Reducing supercoiling, 3. Synthesizing a primer, 4. Discontinuous synthesis of the lagging strand, 5. Removal of RNA primers, 6. Joining DNA fragments, 7. Proofreading.

What initiates unwinding of the bacterial DNA helix

The protein DnaA binds to the origin of replication.

What is the function of DnaB and DnaC

Further open and destabilize the DNA helix during unwinding.

What are helicases

Enzymes that use ATP to break hydrogen bonds and unwind the DNA double helix.

What enzyme relieves supercoiling during replication

DNA gyrase, a type of topoisomerase.

What enzyme synthesizes the RNA primer

Primase.

Why is a primer needed for DNA replication

DNA polymerase III can only add nucleotides to a preexisting 3′ hydroxyl group.

What is the leading strand

The DNA strand synthesized continuously in the 5′→3′ direction toward the replication fork.

What is the lagging strand

The strand synthesized discontinuously away from the fork in short Okazaki fragments.

What are Okazaki fragments

Short DNA fragments synthesized on the lagging strand, each beginning with an RNA primer.

How are Okazaki fragments joined

DNA polymerase I removes RNA primers and DNA ligase seals the fragments.

What does DNA ligase do

Joins Okazaki fragments by forming phosphodiester bonds.

What does looping of the lagging strand accomplish

Allows both DNA strands to be synthesized simultaneously in the 5′→3′ direction.

What proofreading mechanism exists during replication

3′→5′ exonuclease activity that removes mispaired nucleotides.

Which enzymes are required at a replication fork

DNA polymerase III, DNA gyrase, helicases, primase, DNA ligase, and single-stranded binding proteins.

Why is eukaryotic DNA replication more complex than prokaryotic

Eukaryotes have more DNA, linear chromosomes, and DNA complexed with proteins.

How do eukaryotic cells replicate large genomes efficiently

Multiple origins of replication on each chromosome.

Which polymerases replicate nuclear DNA in eukaryotes

DNA polymerases α, δ, and ε.

Which polymerase replicates mitochondrial DNA

DNA polymerase γ.

Which polymerases are involved in DNA repair

DNA polymerases β and ζ.

What is the role of DNA polymerase α

Synthesizes RNA primers on both leading and lagging strands during initiation.

What is polymerase switching

The replacement of Pol α with Pol δ for elongation during replication.

Why are the ends of linear chromosomes a problem for replication

The lagging strand lacks a free 3′ hydroxyl after primer removal, creating incomplete ends.

What are telomeres

Repetitive DNA sequences at chromosome ends that preserve stability.

What is telomerase

A ribonucleoprotein enzyme that extends chromosome ends using its own RNA as a template.

Who discovered telomerase

Elizabeth Blackburn and Carol Greider.

What is the function of telomerase

Synthesizes telomeric repeats to fill the 3′ overhang gap via reverse transcription.

Is telomerase active in all cells

No, it is inactive in most somatic cells but active in germline and cancer cells.

What happens when telomerase is inactive

Telomeres shorten with each cell division, leading to cellular aging and division arrest.

Why are malignant cells considered “immortalized”

They maintain telomerase activity, preventing telomere shortening.

What is homologous recombination

Genetic exchange at equivalent positions between two homologous DNA molecules.

What steps are involved in genetic recombination

Endonuclease nicking, strand displacement, ligation, branch migration, and duplex separation (Holliday structure).

What is a Holliday junction

A cross-shaped structure formed during homologous recombination where DNA strands exchange segments.

Which proteins promote recombination in E. coli

RecA promotes strand exchange; RecB, RecC, and RecD cleave and unwind DNA.

Which other enzymes assist in recombination and repair

DNA polymerases, DNA ligase, gyrases, and single-stranded binding proteins.