Lecture 7 DNA Repair Slides

Base excision repair

A DNA repair pathway that fixes small, subtle damage to individual bases.

What type of damage does base excision repair fix?

Small base damage that does not strongly distort the DNA helix.

BER

Base excision repair.

Main purpose of BER

To remove and replace a damaged base in DNA.

What is the overall gist of base excision repair?

A damaged base is removed, the backbone is cut, DNA polymerase fills the gap, and ligase seals the nick.

DNA glycosylase

The enzyme that recognizes and removes a damaged base during base excision repair.

What does DNA glycosylase remove?

The damaged nitrogenous base, not the whole nucleotide.

What is left after DNA glycosylase removes a damaged base?

An abasic site, also called an AP site.

AP site

A DNA site where the base is missing but the sugar-phosphate backbone remains.

Abasic site

A DNA position where the nitrogenous base is missing.

AP endonuclease

The enzyme that cuts the DNA backbone at an abasic site.

What happens after AP endonuclease cuts the backbone?

DNA polymerase fills in the correct DNA and ligase seals the nick.

DNA ligase in BER

The enzyme that seals the remaining nick after the repair patch is filled in.

Uracil DNA glycosylase

A DNA glycosylase that removes uracil from DNA.

Why is uracil DNA glycosylase important?

Uracil in DNA usually comes from cytosine deamination, so removing it prevents mutation.

What does uracil DNA glycosylase recognize?

Uracil bases in DNA.

Why does uracil not belong in DNA?

DNA normally uses thymine instead of uracil.

What mutation could happen if uracil in DNA is not repaired?

A C=G base pair can become a T=A base pair.

Prokaryotic BER focus

The professor mainly wants the general pathway and what BER repairs, not every small eukaryotic difference.

Eukaryotic BER difference

Eukaryotes have some pathway differences, such as short-patch and long-patch repair, but the general idea is similar.

Short-patch BER

A BER pathway where usually one nucleotide is replaced.

Long-patch BER

A BER pathway where multiple nucleotides are replaced.

Nucleotide excision repair

A DNA repair pathway that removes bulky lesions by cutting out a section of DNA around the damage.

NER

Nucleotide excision repair.

What type of damage does NER fix?

Bulky DNA damage that distorts the helix.

Examples of damage repaired by NER

UV-induced pyrimidine dimers, 6-4 photoproducts, and bulky chemical adducts.

Main difference between BER and NER

BER removes a damaged base; NER removes a larger DNA segment around bulky damage.

What is the overall gist of NER?

The damage is recognized, DNA is cut on both sides of the lesion, the damaged segment is removed, DNA polymerase fills the gap, and ligase seals it.

Excinuclease

An enzyme activity that makes two cuts in one DNA strand around a lesion.

Why is NER's excinuclease activity important?

It cuts DNA on both sides of bulky damage so the damaged section can be removed.

How many nicks does NER make around the lesion?

Two nicks, one on each side of the damaged region.

How is NER different from simple nick repair?

NER cuts out a whole damaged DNA segment instead of just sealing one nick.

Uvr proteins

Bacterial proteins involved in nucleotide excision repair.

UvrA

Helps recognize DNA damage in bacterial NER.

UvrB

Helps open and stabilize DNA around the lesion in bacterial NER.

UvrC

Cuts DNA on both sides of the lesion in bacterial NER.

UvrD

A helicase that removes the damaged DNA fragment in bacterial NER.

What fills the gap after bacterial NER removes damaged DNA?

DNA polymerase I.

What seals the nick after bacterial NER?

DNA ligase.

Xeroderma pigmentosum

A human disease caused by defects in nucleotide excision repair.

XP

Xeroderma pigmentosum.

Why is XP connected to UV damage?

XP cells cannot properly repair UV-induced DNA lesions.

Main symptom/risk of XP

Extreme sensitivity to sunlight and increased risk of skin cancer.

Why do XP patients have high skin cancer risk?

UV damage is not repaired efficiently, so mutations accumulate in skin cells.

What repair pathway is defective in XP?

Nucleotide excision repair.

What does XP teach us about DNA repair?

Defects in DNA repair pathways can directly cause human disease and cancer risk.

UV-induced DNA damage

DNA damage caused by ultraviolet light, often forming pyrimidine dimers.

Pyrimidine dimer

A UV-induced lesion where adjacent pyrimidines become covalently linked.

Thymine dimer

A common UV-induced pyrimidine dimer between adjacent thymine bases.

Why are pyrimidine dimers harmful?

They distort DNA and can block replication or transcription.

What repair pathway removes pyrimidine dimers?

Nucleotide excision repair.

Double-strand break

A DNA lesion where both strands of the DNA double helix are broken.

DSB

Double-strand break.

Why are double-strand breaks dangerous?

Both DNA strands are broken, so the cell may lose genetic information and replication can collapse.

Why is a DSB worse than a single-strand nick?

A single-strand nick still has the opposite strand as a template, but a DSB breaks both strands.

What can cause double-strand breaks?

Ionizing radiation, oxidative damage, UV damage, and replication forks encountering DNA breaks.

Replication fork collapse

A dangerous event where a replication fork breaks down, often creating a double-strand break.

How can a single-strand break lead to a double-strand break?

If a replication fork reaches a nick in the template strand, the fork can collapse and create a DSB.

Why are DSBs important for this lecture?

They are repaired by homologous recombination or nonhomologous end joining.

Homologous recombination repair

A repair pathway that fixes double-strand breaks using a homologous DNA sequence as a template.

HR

Homologous recombination.

What type of damage does homologous recombination repair?

Double-strand breaks and collapsed replication forks.

Why is homologous recombination accurate?

It uses an undamaged homologous DNA molecule, often a sister chromatid, as a template.

When is homologous recombination most useful?

During S phase and G2, when sister chromatids are available.

Why is HR favored during the replicative phase?

Because replicated sister chromatids are present and can be used as accurate templates.

What is needed for homologous recombination?

A homologous DNA template.

Basic purpose of homologous recombination

To repair broken DNA accurately using a matching DNA sequence.

General first step of HR

The broken DNA ends are processed to create 3' single-stranded overhangs.

3' overhang

A single-stranded DNA end with a free 3' end that can invade a homologous DNA molecule.

Strand invasion

The step in HR where a 3' overhang pairs with a homologous DNA template.

D-loop

A DNA displacement loop formed when an invading strand pairs with a homologous template.

Why is strand invasion important in HR?

It allows the broken DNA end to use the homologous template for repair synthesis.

What happens after strand invasion in HR?

DNA polymerase extends the invading strand using the intact template.

How does HR restore lost DNA information?

DNA polymerase copies information from the undamaged homologous template.

Nonhomologous end joining

A double-strand break repair pathway that directly joins broken DNA ends without a homologous template.

NHEJ

Nonhomologous end joining.

What type of damage does NHEJ repair?

Double-strand breaks.

Does NHEJ need a homologous template?

No.

Why is NHEJ useful?

It can repair DSBs when no sister chromatid or homologous template is available.

When is NHEJ most important?

During G1 or G0, when sister chromatids are not available.

Why is NHEJ considered mutagenic?

It can lose or add nucleotides when joining broken DNA ends.

Main difference between HR and NHEJ

HR uses a homologous template and is accurate; NHEJ directly joins ends and is more error-prone.

Which repair pathway is more accurate, HR or NHEJ?

Homologous recombination.

Which repair pathway is more mutagenic, HR or NHEJ?

Nonhomologous end joining.

Why does NHEJ not conserve the original DNA sequence?

The broken ends may be trimmed or filled before ligation, changing the sequence.

Why is NHEJ used when HR is not feasible?

NHEJ does not require a sister chromatid or homologous template.

What does Ku70-Ku80 do in NHEJ?

It binds to broken DNA ends.

Ku70-Ku80

A protein complex that recognizes and binds DNA ends during NHEJ.

DNA-PKcs

A protein kinase involved in NHEJ after Ku binds DNA ends.

Artemis

A nuclease involved in processing DNA ends during NHEJ.

DNA ligase IV

The ligase that seals DNA ends during NHEJ.

XRCC4 and XLF

Proteins that help DNA ligase IV seal breaks during NHEJ.

Basic NHEJ steps

Ku binds DNA ends, repair proteins process the ends, gaps may be filled, and ligase seals the break.

Why might NHEJ create small insertions or deletions?

The DNA ends may be trimmed or filled in before being joined.

Compare HR and NHEJ by template use

HR uses a homologous template; NHEJ does not.

Compare HR and NHEJ by accuracy

HR is usually accurate; NHEJ is error-prone.

Compare HR and NHEJ by cell cycle timing

HR is favored in S/G2; NHEJ is important in G1/G0.

Compare HR and NHEJ by mutation risk

HR has lower mutation risk; NHEJ has higher mutation risk.

Compare HR and NHEJ by speed/simplicity

NHEJ is simpler and faster; HR is more complex but more accurate.

Why will HR vs NHEJ probably be on the exam?

The professor specifically said comparing and contrasting them is important and will appear on the exam.