Lecture 2 - DNA Replication

How fast can E. coli replicate DNA per strand?

60,000 nucleotides per minute.

How large is the E. coli genome?

4.6 million base pairs.

How long does E. coli take to replicate and divide?

20 minutes.

How fast is human DNA replicated?

3,000 nucleotides per minute.

Why can humans still replicate their genome quickly?

Multiple origins of replication.

Why is there a reduction in speed for eukaryotic organisms?

We have to be able to correct mistakes that are being made.

How large is the human genome?

3.3 billion base pairs.

Typical DNA replication error rate?

1 error per 10,000,000 nucleotides.

Where do mutations come from?

DNA polimerase

What enzyme activity allows proofreading?

3′ → 5′ exonuclease activity.

Final error rate after proofreading and repair?

1 error per 1,000,000,000 nucleotides.

How many de novo mutations in a child on average?

30–35.

What is conservative replication?

Original duplex remains intact; new duplex entirely new.

these strands are the same as parent

What is dispersive replication?

DNA fragments mix old and new in every strand.

these strands are intermixed with the new one

What is semi-conservative replication?

Each daughter DNA has one old strand and one new strand.

one strand is old and the other is new

Which isotopes were used by Meselson and Stahl’s?

N14 and N15

Why grow bacteria in ¹⁵N first?

To make heavy DNA.

What technique separated heavy and light DNA?

Density gradient centrifugation.

After two rounds, what result supported semi-conservative replication?

Some hybrid DNA and some light DNA.

What would dispersive replication show after two rounds?

All intermediate density DNA.

What is theta replication?

Circular DNA replication forming a bubble.

Where does theta replication occur?

E. coli and other circular genomes.

What happens first in theta replication?

The two strands melt (slowly separate) or unwind the two strands.

What is the origin of replication?

Site where polymerase binds and starts replicating.

What is the replication bubble?

site where proteins enter and do its job (unidirectional or bidirectional)

What direction does polymerase synthesize DNA in?

5’ to 3’ Direction

DNA is antiparallel, so what direction is the template?

5’ to 3’ facing opposite way

What is a replication fork?

Site of unwinding.

What is bidirectional replication?

Forks move both directions.

What is rolling-circle replication?

One strand displaced while new strand synthesized.

What critical set of elements are required for DNA polymerase?

3’ OH group

How do you access a plasmid that’s in a circle (rolling circle replication)?

Create a nic in the DNA (break the DNA) - exposing the 3’ OH group

What happens to the original strands when polymerase is synthesizing DNA in rolling circle replication?

As the new strands are being made, original strands get displaced.

Why does displacement happen in rolling circle replication?

The old strand can be used as a template strand to make new strands. (continuous cycle)

Where does rolling-circle occur?

Some viruses and F-factor plasmid.

What direction does rolling-circle go?

Unidirectional

Why do linear chromosomes in eukaryotes need multiple origins?

Chromosomes are large. they are too large to have a single replication origin.

What forms at each origin in eukaryotic DNA replication?

Replication bubbles.

What direction does linear go in?

Bidirectional

Four stages of replication?

Initiation, unwinding, elongation, termination.

What binds oriC (initiation)?

DnaA protein

DNAa box binds to protein in order to what?

Causes DNA To twist (unwind) and separate the AT-rich region

Helicase function? (unwinding)

Break hydrogen bonds.

SSB (single stranded binding protein) function? (unwinding)

Stabilize single strands.

Gyrase/topoisomerase function? (unwinding)

Relieve supercoiling.

Topoisomerase I breaks? (unwinding)

clips and twist One strand onto the other one (undo a turn)

Topoisomerase II breaks? (unwinding)

clips and twist Both strands (to undo a single turn)

Why is a primer needed in elongation?

Polymerase requires 3′-OH group to anchor

What makes the primer?

Primase.

DNA polymerase III function?

Main DNA synthesis.

DNA Pol III activities?

5′→3′ polymerase, 3′→5′ proofreading.

DNA Pol I activities?

5′→3′ polymerase, 3′→5′ proofreading, 5′→3′ exonuclease.

What else can DNA Pol I do?

Replace RNA with DNA

What seals nicks?

Ligase.

How does replication end?

replication forks meet or Tus–Ter system (protein) blocks fork.

Direction DNA is synthesized?

5′ → 3′.

Leading strand synthesis?

Continuous.

Lagging strand synthesis?

Discontinuous.

What are Okazaki fragments?

Short lagging-strand fragments (~1500 nt).

difference in Origins of prokaryotes and eukaryotes?

Prokaryotes: one. Eukaryotes: many.

ARS sequences found where?

Yeast.

When does eukaryotic replication occur?

Specific cell-cycle phase (S phase).

When does prokaryote replication occur?

always replicating

What is licensing?

Licensing takes place to ensure that replication forks initiate at the same time and doesn’t initiate more than once per cycle

When is licensing factors laid down?

Before replication.

What prevents re-initiation?

Removal of licensing factors.

What does Geminin do?

Removes licensing factor, so replication can continue

what does DNA Pol α (alpha) do?

Makes primer.

What does DNA Pol δ (delta) do?

Lagging strand synthesis.

What does DNA Pol ε (sigma) do?

Leading strand synthesis.

What is a nucleosome?

DNA wrapped around 8 histone proteins.

What happens to the histones during replication?

Nucleosome is removed —> some old histones find their way to their location on the new strand + new histones are made and attach to the old histones.

What is a telomere?

Repetitive DNA at chromosome ends.

Human telomere repeat?

TTAAGGG.

What is the Purpose of telomeres?

Protect genes from loss (buffer the DNA)

Short telomeres cause?

Apoptosis.

The older you get the ____ the telomeres are.

The more stress, the ____ the telomeres are.

shorter

What cells experience shorter telomere growth?

Cells that aren’t critical (not sperm and egg)

What enzyme extends telomeres?

Telomerase.

Telomerase contains what?

RNA template + protein reverse transcriptase.

Where is telomerase active?

Germ cells & rapidly dividing cells.

Telomerase in cancer cells leads to?

Immortalization in cancer.

Does telomerase extend the longer or shorter strand?

Longer strand