BCH4024 Douma Exam 4 - Lecture 4

What is primase?

An RNA polymerase that synthesizes RNA primers needed to initiate DNA synthesis.

Why is primase needed during DNA replication?

DNA polymerases cannot synthesize DNA de novo; they require a pre-existing 3' hydroxyl group to extend from.

How many primers are needed on the leading strand?

Only one primer is needed because the leading strand is synthesized continuously.

How many primers are needed on the lagging strand?

One primer is needed for each Okazaki fragment since the lagging strand is synthesized discontinuously.

What is the role of single-stranded binding protein (SSB)?

Binds and protects separated single DNA strands to prevent reannealing and degradation.

What is the function of DNA gyrase in prokaryotes?

A topoisomerase that relieves positive supercoiling ahead of the replication fork.

What structure allows DNA polymerase to stay attached to the DNA during synthesis?

The sliding clamp (Beta clamp in prokaryotes).

What loads the sliding clamp onto DNA?

The clamp loader complex.

What triggers clamp loading onto DNA?

ATP binding and hydrolysis by the clamp loader.

What happens when DNA polymerase III finishes an Okazaki fragment?

It dissociates from the DNA and is transferred to a new sliding clamp on the next primer.

What happens to old sliding clamps left behind on DNA?

They are used to recruit enzymes for lagging strand processing and DNA repair.

Why must RNA primers be removed after replication?

RNA does not belong in DNA; primers must be replaced with DNA.

What enzyme removes RNA primers in prokaryotes?

DNA polymerase I, using its 5' to 3' exonuclease activity.

What unique enzymatic activities does DNA polymerase I have?

DNA synthesis, 3' to 5' proofreading exonuclease activity, and 5' to 3' exonuclease activity.

How does DNA polymerase I process Okazaki fragments?

It removes RNA primers while simultaneously synthesizing DNA to replace them.

What enzyme seals the gaps between Okazaki fragments?

DNA ligase.

What energy molecule does DNA ligase use to form phosphodiester bonds?

AMP.

What is a catenated state?

Interlinked circular genomes formed after DNA replication in prokaryotes.

What enzyme separates catenated genomes?

Topoisomerase IV.

How does topoisomerase IV resolve catenated genomes?

It makes a double-strand break in one genome, passes the other genome through, and reseals the break.

How does eukaryotic DNA replication differ from prokaryotic DNA replication?

Eukaryotes have thousands of origins of replication, slower polymerase rates, and linear chromosomes requiring telomeres.

What is the S phase of the cell cycle?

The phase during which DNA synthesis (replication) occurs.

What ensures DNA replication happens only once per cell cycle in eukaryotes?

Licensing, which loads replication machinery only during G1 phase.

What protein complex recognizes origins of replication in eukaryotes?

The Origin Recognition Complex (ORC).

What happens at the origin during G1 phase?

ORC binds the origin and recruits proteins like Cdc6, Cdt1, and MCM helicase to form the pre-replication complex.

When does DNA replication actually begin?

In S phase, after cyclin-dependent kinases (CDKs) phosphorylate pre-replication complex proteins.

What prevents relicensing of origins during S phase?

CDK activity; phosphorylated proteins cannot reassemble a new pre-replication complex.

What is MCM in eukaryotes?

The helicase that separates DNA strands at the replication fork.

What are the main replicative DNA polymerases in eukaryotes?

DNA polymerase epsilon (leading strand) and DNA polymerase delta (lagging strand).

What complex synthesizes primers in eukaryotes?

A primase-DNA polymerase alpha complex.

What is the clamp loader for the eularyotic replisome called?

Replication factor C (RFC)

What is the sliding clamp for the eukaryotic replisome called?

Proliferating cell nuclear antigen (PCNA)

What is the single-strand DNA binding protein (SSB) for the eukaryotic replisome called?

Replication protein A (RPA)

What are eukaryotic primers made of?

A short RNA segment followed by a short DNA segment.

What unique activity does DNA polymerase delta have?

Strand displacement — it can push the RNA primer out of the way during lagging strand synthesis.

What removes the RNA primer flaps displaced by DNA polymerase delta?

Flap endonuclease 1 (FEN1).

How are Okazaki fragments joined in eukaryotes?

DNA ligase forms phosphodiester bonds between fragments.

Why do eukaryotes have telomeres?

To protect the ends of linear chromosomes from degradation and prevent loss of genetic information.

What happens to telomeres after each round of replication?

They shorten because the final RNA primer cannot be replaced with DNA.

What sequence is repeated in human telomeres?

TTAGGG repeated thousands of times.