Bio 99 Exam 2

What is the function of DNA helicase?

separates the two DNA strands by breaking hydrogen bonds, allowing replication to occur.

How does DNA helicase work?

It binds to nucleotides when the strand opens (passive) or binds one strand and forces open the helix using ATP (active).

What does translocation mean in DNA helicase activity?

Translocation is the ATP-dependent movement of helicase along DNA to separate strands.

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

stabilizes and protects single-stranded DNA during replication.

What is the function of Primase?

synthesizes a short RNA primer to provide a starting point for DNA polymerase.

What is the function of DNA Polymerase III?

It synthesizes new DNA by adding nucleotides to a pre-existing chain in the 5’ to 3’ direction.

What is the function of the β-clamp?

holds DNA polymerase III in place on the DNA strand to ensure high processivity during replication.

What is the function of DNA Polymerase I?

It removes RNA primers and fills in the gaps with DNA.

What is the function of RNAse H?

removes RNA primers from DNA strands.

Does Pol I/RNAse H work on leading or lagging strands?

Lagging strands.

What is the function of DNA ligase?

forms phosphodiester bonds between DNA fragments to complete the strand.

Why must DNA ligase form a bond between G and C nucleotides after primer replacement?

Because a free 5’ triphosphate is not present, and DNA polymerase is no longer there to do it.

Why do we need Okazaki fragments?

Because DNA synthesis only occurs 5’ to 3’, but DNA strands are antiparallel.

What is the Trombone Model?

A model describing how the lagging strand forms loops so DNA polymerases on both strands can move together.

What is the End Replication Problem?

The inability to fully replicate the ends of linear DNA, leading to gradual shortening of chromosomes.

Why does the End Replication Problem occur?

The RNA primer at the end of the lagging strand cannot be replaced with DNA, leaving a gap.

How does the cell solve the End Replication Problem?

By using telomerase to extend the ends of DNA.

Does telomerase require a separate primer?

No.

Is telomere synthesis DNA or RNA synthesis?

DNA synthesis.

In what types of cells is telomerase active?

Germ cells and stem cells.

What happens to telomeres in differentiated cells?

They shorten with each cell division because telomerase is not active.

What conclusion was made from fibroblast experiments about telomeres?

Telomere length correlates with cellular lifespan.

What is PCR used for?

To amplify a specific region of DNA.

How do we PCR only the DNA we want?

By designing specific primers that bind only to the target sequence.

What direction must DNA replication occur in PCR?

5’ to 3’ direction.

Why does the correct annealing temperature matter in PCR?

Correct temperature ensures specific primer binding; too low allows non-specific binding.

Define DNA helicase.

An enzyme that unwinds the DNA double helix during replication.

Define SSB.

Single-stranded binding proteins that prevent DNA strands from re-annealing.

Define Primase.

An enzyme that creates RNA primers for DNA polymerase to start synthesis.

Define DNA polymerase III.

The main enzyme that extends DNA strands during replication.

Define β-clamp.

A protein that keeps DNA polymerase III attached to DNA.

Define DNA polymerase I.

An enzyme that removes RNA primers and fills gaps with DNA.

Define DNA ligase.

An enzyme that connects Okazaki fragments by forming phosphodiester bonds.

Define Okazaki fragment.

Short DNA fragments synthesized on the lagging strand during replication.

Define Telomerase.

An enzyme that extends the telomeres at chromosome ends.

Define PCR (Polymerase Chain Reaction).

A laboratory method to amplify specific DNA sequences using primers and DNA polymerase.

What is a point mutation?

A change in a single nucleotide.

What is a silent mutation?

A mutation that does not change the amino acid sequence.

What is a missense mutation?

changes one amino acid to another.

What is a nonsense mutation?

A mutation that introduces a premature stop codon.

What is an insertion mutation?

A mutation where one or more nucleotides are added to the DNA sequence.

What is a deletion mutation?

A mutation where one or more nucleotides are removed from the DNA sequence.

What is a frameshift mutation?

A mutation that changes the reading frame of the codons due to insertion or deletion.

What is a chromosome inversion, deletion, or translocation mutation?

Structural changes to chromosomes involving flipping, losing, or rearranging segments.

What causes polymerase errors?

Incorrect nucleotide incorporation during replication, about 1 error per 10⁵ nucleotides.

What is deamination of nucleotide bases?

Loss of an amino group from a nucleotide, causing mutations during replication.

What is oxidation of nucleotide bases?

Damage by reactive oxygen species that alters base pairing.

What is T-T dimer formation?

Structural damage where two adjacent thymine bases bond together due to UV light.

What happens in chromosome breakage?

Physical breaking of DNA strands, leading to possible deletions or rearrangements.

What is the purpose of the Ames test?

To test whether a chemical is mutagenic.

How is the Ames test performed?

Bacteria that cannot synthesize histidine are exposed to a chemical to see if mutations allow them to grow without histidine.

What does it mean if S. typhimurium cannot make their own histidine?

It likely has a mutation in a histidine synthesis gene.

How do we know if a compound is mutagenic in the Ames test?

If colonies grow without histidine, it indicates mutations and mutagenicity.

How are polymerase errors repaired?

Via proofreading (3' to 5' exonuclease activity) and mismatch repair pathway.

What is mismatch repair?

A system that identifies and repairs wrongly incorporated bases after replication.

In prokaryotes, how is the parental strand recognized during mismatch repair?

By methylation of GATC sites.

In eukaryotes, how is the parental strand recognized during mismatch repair?

By the presence of nicks in the new strand.

Where will DNA ligase act in mismatch repair?

It seals the nick after the mismatch is corrected.

What is base excision repair (BER)?

A repair pathway that fixes small, non-helix-distorting base lesions by removing the base and replacing it.

What is nucleotide excision repair (NER)?

A repair pathway that removes bulky helix-distorting lesions like T-T dimers.

What experiment revealed the NER pathway?

Adding radioactive thymidine showed DNA repair outside S-phase; absent in XP cells.

What defect do XP patients have?

An underactive nucleotide excision repair pathway.

In vitro, how is XP phenotype tested?

By measuring DNA radioactivity after UV treatment; XP cells show less repair activity.

What causes double-stranded (DS) breaks?

Irradiation, UV rays, chemicals, and collapsed replication forks.

Why are DS breaks dangerous?

They inhibit replication and transcription and can cause chromosomal rearrangements.

What is homologous recombination (HR)?

A DS break repair mechanism that uses a homologous DNA template to fix the break.

What is non-homologous end joining (NHEJ)?

A DS break repair mechanism that directly joins the broken DNA ends without a template.

Why knock out a gene?

To study gene function or create models of disease.

How do you introduce foreign DNA into cells for knockout?

By transformation using heat shock and selection media.

What is the URA3 cassette used for?

To knock out a gene by replacing it with a selectable marker (URA3).

How would you select for cells that have acquired the knockout cassette?

Use ura3Δ starting cells and media without uracil; only successfully modified cells survive.

Does this media confirm gene knockout?

No, further verification is needed.

When wild type mouse cells are transformed with the NeoR cassette, they are plated in media (LACKING/CONTAINING) neomycin. Growth in this media (DOES/DOES NOT) confirm mTR knockout

Containing, Does not

After PCR with mTR and NeoR primers, which gel lanes do you expect if mTR is knocked out?

Lanes 3 and 4

For this PCR reaction, would you use DNA or RNA primers and why?

DNA, because it is more stable.

What would happen if the annealing temperature during PCR was raised to 80°C?

No PCR products

Which is NOT a phenotype of mTR -/- knockout cells?

Slower replication compared to wild type

In wild type cells, which chromosome strand is most likely extended by telomerase?

Strand B

After adenine is deaminated to hypoxanthine and not repaired, what base pairs are found in chromatids 1A and 1B

A/T on chromosome 1A and G/C on chromosome 1B

In an in vitro DNA repair assay for hypoxanthine, which radiolabeled dNTP is necessary?

Radioactive dCTP

In the Ames test with three compounds, how many are mutagenic based on results?

2

In the Ames test, do we assume a mutagen affects only the histidine synthesis gene?

true

What is a good negative control for the Ames test?

compound that does not mutate DNA.

What is a silent mutation?

A nucleotide change that does not change the amino acid.

Why is a poly G sequence added to cDNA during library construction?

Because there is no common sequence on the 3’ end of the cDNA.

Why are sticky ends preferred in cloning with EcoRI?

Because they help make the ligation process more efficient.

Why is DNA ligase needed to join EcoRI-digested cDNA and plasmid?

Because the "G" on the plasmid side only contains an OH group.

For a shuttle vector functional in yeast, what kind of promoter must it contain?

Bacterial or Yeast promoter (either acceptable).