Lecture 14: DNA Replication

What are parental vs. daughter strands?

Parental = the original template strands. Daughter = the newly made strands.

Three proposed models of DNA replication?

Conservative, semiconservative (correct), dispersive.

Describe the semiconservative model.

Each new double helix has one parental strand and one daughter strand.

Describe the conservative and dispersive models.

Conservative: both parental strands stay together. Dispersive: parental and daughter segments are mixed within both strands.

How did Meselson & Stahl (1958) tell strands apart?

Grew E. coli in heavy ¹⁵N, then switched to light ¹⁴N, and separated DNA by density on a CsCl gradient.

Meselson–Stahl: result after one generation?

All DNA was half-heavy. Consistent with semiconservative and dispersive (rules out conservative).

Meselson–Stahl: result after two generations?

Equal amounts of light and half-heavy DNA. Only fits semiconservative.

How many origins of replication does a bacterial chromosome have?

One. Replication is bidirectional and the two forks meet on the opposite side.

What is oriC, and is it DNA, RNA or protein?

The origin of chromosomal replication in E. coli. It is a DNA sequence.

Three key sequences in oriC?

DnaA boxes (DnaA binding), AT-rich region (strands separate), GATC methylation sites (regulation).

Why do strands separate at the AT-rich region?

A–T pairs have only 2 H bonds, so they come apart more easily.

What do DnaA proteins do at oriC?

Bind DnaA boxes and each other, bend the DNA, and cause strand separation at the AT-rich region.

Features of DnaB (DNA helicase)?

Breaks H bonds between strands; six subunits; moves 5′→3′; uses ATP.

What does topoisomerase II (DNA gyrase) do?

Travels ahead of helicase and relieves the positive supercoiling it creates.

What do single-strand binding proteins do?

Keep the separated parental strands apart so bases are exposed.

What does primase make, and how long?

Short RNA primers, about 10–12 nucleotides.

Primers needed on the leading vs. lagging strand?

Leading: one primer. Lagging: many primers.

Which polymerase makes the daughter strands in E. coli?

DNA polymerase III.

What does DNA polymerase I do?

Removes RNA primers (5′→3′ exonuclease) and fills the gap with DNA (5′→3′ polymerase).

What does DNA ligase do?

Forms the covalent (ester) bond that joins Okazaki fragments’ backbones.

Which E. coli DNA polymerases do replication vs. repair?

Pol I and III: normal replication. Pol II, IV, V: repair and replicating damaged DNA.

Two limitations of DNA polymerase?

It can’t start a strand on bare DNA (needs a primer), and it only adds nucleotides 5′→3′.

Leading vs. lagging strand synthesis?

Leading: continuous, toward the replication fork. Lagging: discontinuous, away from the fork, in Okazaki fragments.

How long are Okazaki fragments?

1000–2000 nt in bacteria; 100–200 nt in eukaryotes.

What is the primosome?

DNA helicase + primase bound together.

What is the replisome?

The primosome plus two DNA polymerase III holoenzymes.

What is the looping (trombone) model of lagging-strand synthesis?

The lagging strand loops out so its polymerase can synthesize 5′→3′ while still moving toward the fork with the leading-strand polymerase. The clamp loader reloads it at each new primer.

Overall error rate of DNA pol III?

About 1 mistake per 10⁸ bases.

Three reasons replication fidelity is high?

Stability of correct base pairs, induced fit in the polymerase active site, and proofreading.

Error rates from each fidelity step?

Base pairing alone: 1/1,000. With active site fit: 1/100,000–1,000,000. With proofreading: ~1/10⁸.

Which activity does DNA polymerase use to proofread?

3′→5′ exonuclease. It digests back to remove the mismatch, then resumes 5′→3′.

Why is eukaryotic replication more complex?

Large linear chromosomes, chromatin packed in nucleosomes, more complex cell-cycle control; uses multiple origins.

What is a telomere?

The complex of telomeric DNA repeats and bound proteins at the ends of linear chromosomes.

Features of telomeric DNA?

Moderately repetitive tandem arrays rich in G and T, with a 3′ overhang of 12–16 nucleotides.

What is the end-replication problem?

The 3′ end of a linear chromosome can’t be fully replicated (no room for a primer), so the chromosome shortens each round.

What is telomerase made of and how does it work?

Protein + RNA. Its RNA is complementary to the telomeric repeat, so it binds the 3′ overhang and extends it: binding, polymerization, translocation (repeated).

Telomere length at birth vs. old age?

About 8,000 bp at birth; can shorten to about 1,500 bp in the elderly.

What happens when telomeres get too short?

Cells become senescent and lose the ability to divide.

How are telomeres linked to cancer?

Cancer cells often have mutations increasing telomerase activity, preventing shortening and senescence. Telomerase is a possible drug target.