Meselson–Stahl & Replication Origins
What question did Meselson and Stahl set out to answer in 1958?
How DNA is replicated — whether it’s conservative, semi-conservative, or dispersive.
Replication forks are asymmetric because DNA polymerase can only add nucleotides to which end of a growing strand?
The 3′ end.
What is the main enzyme that adds nucleotides to a new DNA strand?
DNA polymerase (5′→3′ polymerase activity).
What is the “end replication problem”?
After RNA primer removal on the lagging strand, the final gap cannot be filled — chromosome ends shorten each cycle.
What type of repair corrects errors made during replication?
Mismatch repair.
What isotope was used to label “heavy” DNA in their experiment?
¹⁵N (nitrogen-15).
What is the difference between the leading and lagging strands?
Leading strand is continuous (5′→3′ toward the fork); lagging strand is discontinuous, forming Okazaki fragments.
What activity gives polymerase its proofreading ability?
3′→5′ exonuclease activity (removes mis-paired bases).
What is telomerase and what does it carry?
A reverse transcriptase enzyme that carries its own RNA template to extend chromosome ends.
Which enzyme detects and removes incorrectly inserted bases as they are added?
DNA polymerase via its 3′→5′ exonuclease (proofreading).
Why do eukaryotic chromosomes need many origins of replication but bacteria only one?
Eukaryotic DNA is much longer; multiple origins ensure replication finishes in a reasonable time.
What enzyme creates short RNA primers on the lagging strand?
DNA primase.
What does DNA helicase do at the replication fork?
Unwinds the DNA double helix using ATP energy.
Why is telomerase highly active in stem cells and cancer cells?
Because they divide frequently and need to maintain telomere length for continued replication.
List the three general steps in nucleotide excision repair.
1️⃣ Excision of damage → 2️⃣ Resynthesis by repair polymerase → 3️⃣ Ligation by DNA ligase.
Why are origins of replication critical for chromosome duplication?
They’re specific sequences where DNA unwinds and replication bubbles form, allowing polymerases to start synthesis.
Which enzyme seals the nicks between Okazaki fragments after RNA primers are replaced?
DNA ligase (seals phosphodiester bonds using ATP).
What is the function of single-strand binding proteins (SSBPs)?
Stabilize unwound DNA and prevent strands from re-annealing or forming hairpins.
What happens to somatic cells when telomerase is inactive and telomeres shorten too much?
Cells enter senescence or apoptosis (programmed cell death).
What type of damage is caused by UV radiation and how is it repaired?
Thymine dimers; removed by nucleotide excision repair (enzymes cut out and replace damaged DNA).
After one generation in ¹⁴N medium, what band pattern was seen in the CsCl gradient?
A single intermediate band (¹⁵N/¹⁴N hybrid) → proof of semi-conservative replication.
Why does the replication fork move bidirectionally from each origin?
Because two helicases move in opposite directions, creating two replication forks that double efficiency.
Topoisomerase prevents supercoiling ahead of the fork by doing what?
Introducing temporary nicks to relieve torsional strain, then resealing the DNA.
How does the RNA component of telomerase guide DNA synthesis at chromosome ends?
It base-pairs with the 3′ overhang and serves as a template to add repetitive DNA sequences (e.g., TTAGGG).
Why is DNA repair vital for cell survival and genetic stability?
It prevents mutations that can lead to cancer, aging, and cell death by maintaining DNA integrity.