DNA Repair

What is the primary DNA repair mechanism for double-strand breaks (DSBs) in mammalian cells?

Non-homologous end joining (NHEJ), as homologous recombination is limited to S/G2 phases due to its reliance on a sister chromatid.

What are the three main NHEJ pathways?

Classical NHEJ (c-NHEJ), Microhomology-Mediated End Joining (MMEJ), and Alternative End Joining (Alt-NHEJ or a-EJ).

What protein first binds DSBs in classical NHEJ?

The Ku70/80 heterodimer (Ku complex).

What is the role of DNA-PKcs in c-NHEJ?

It forms the DNA-PK complex with Ku, autophosphorylates to change conformation, and aligns DNA ends for repair.

Which enzyme processes DNA ends that prevent ligation in c-NHEJ?

Artemis nuclease, which trims incompatible DNA ends.

What complex is responsible for ligating DNA ends in classical NHEJ?

XRCC4-DNA ligase IV complex, with polynucleotide kinase and DNA polymerase µ or λ for gap-filling.

How accurate is classical NHEJ?

It is moderately accurate but can result in minor sequence loss; it avoids larger chromosomal aberrations.

What defines MMEJ compared to c-NHEJ?

MMEJ requires short microhomology sequences (5–25 bp) to align DNA ends before ligation.

What initiates MMEJ?

Resection of DNA ends by MRN complex (Mre11-Rad50-Nbs1) and CtIP to expose microhomologies.

Which polymerase is essential in MMEJ?

DNA polymerase theta (Polθ).

What ligase is used in MMEJ?

DNA ligase III (often in complex with XRCC1).

How does MMEJ affect genome stability?

It is more mutagenic than c-NHEJ, often causing deletions around the resected microhomology regions.

What distinguishes Alt-NHEJ from the other pathways?

It does not require Ku, relies on extensive end resection, and is highly error-prone—often overlapping mechanistically with MMEJ.

What type of mutations are common in Alt-NHEJ?

Large deletions, insertions, and translocations due to poor alignment and end processing.

Which enzyme is commonly used in both MMEJ and Alt-NHEJ?

DNA polymerase theta (Polθ).

When is Alt-NHEJ or MMEJ more likely to occur?

When classical NHEJ is suppressed or Ku is absent (e.g., in deficient cells or stressed conditions).

What is the advantage of NHEJ despite its mutagenicity?

It prevents lethal chromosomal breaks and maintains genome integrity when homologous recombination is not possible.

How does classical NHEJ prevent resection of DNA ends?

Rapid Ku binding protects ends from exonucleases and commits the repair to c-NHEJ.

How can NHEJ mechanisms be exploited in cancer therapy?

Inhibiting DNA-PKcs or Polθ can induce synthetic lethality in tumors with HR deficiencies, enhancing sensitivity to DNA-damaging agents.

What role does mutagenic NHEJ play in immunity?

It contributes to antibody diversity via programmed DSB repair during V(D)J recombination.

What types of DNA errors does mismatch repair (MMR) correct?

Base–base mismatches and small insertion/deletion loops, often from replication errors or microsatellite instability.

What is the overall sequence of events in both prokaryotic and eukaryotic MMR?

Mismatch recognition → strand discrimination → excision of the error-containing strand → resynthesis → ligation.

Which protein in E. coli recognizes mismatches?

MutS.

What is the role of MutL in E. coli MMR?

MutL stabilizes the MutS–DNA complex and facilitates recruitment of MutH for strand discrimination.

How does E. coli distinguish the newly synthesized strand from the template strand?

By methylation: MutH nicks the unmethylated daughter strand at GATC sites.

Which enzyme unwinds the DNA in E. coliMMR after nicking?

UvrD helicase.

What enzymes perform exonucleolytic degradation in E. coli MMR?

Various exonucleases depending on the direction (Exo I, Exo VII, RecJ, etc.).

Which polymerase resynthesizes DNA in E. coli MMR?

DNA polymerase III.

What is the ligase’s role in E. coli MMR?

Seals the nicked backbone after resynthesis.

What are the homologs of MutS and MutL in eukaryotes (e.g., yeast)?

MutS homologs: MSH2-MSH6 (for base mismatches) and MSH2-MSH3 (for IDLs); MutL homologs: MLH1-PMS2 (major), MLH1-MLH3 (minor).

What is a major difference in the strand discrimination mechanism between E. coliand yeast?

E. coli uses methylation; in yeast/eukaryotes, the mechanism is unclear but likely involves nicks in the daughter strand or replication machinery cues.

What factor in yeast provides exonuclease activity for mismatch removal?

Exonuclease 1 (Exo1).

What is the role of PCNA in eukaryotic MMR?

Acts as a sliding clamp to recruit and orient MutL homologs; may help identify the daughter strand.

What is RFC’s role in eukaryotic MMR?

RFC loads PCNA onto DNA, facilitating mismatch repair.

What is RPA and what is its function during eukaryotic MMR?

RPA is a single-stranded DNA-binding protein that stabilizes unwound DNA during repair.

What experimental evidence supports how strand discrimination works in eukaryotic MMR?

Modrich et al. showed that in vitro, the strand with a pre-existing nick is selectively repaired, suggesting nick-directed discrimination.

Which MSH complex is typically used for simple mismatches in eukaryotes?

MSH2–MSH6.

Which MSH complex is typically used for insertion/deletion loops (IDLs)?

MSH2–MSH3.

How is the diversity of MutS/MutL homologs in eukaryotes advantageous?

It allows specialized responses to different mismatch types and cellular contexts, enhancing repair fidelity and flexibility.

How does the fidelity of eukaryotic MMR compare to that of E. coli?

Both are highly accurate, but eukaryotic MMR is more complex and regulated, partly due to the larger genome and more diverse mismatch contexts.

Why is MMR important for genomic stability and disease prevention?

It corrects replication errors, and deficiencies in MMR are linked to cancers like Lynch syndrome due to microsatellite instability and mutation accumulation.

What is the main function of the MMR system?

To repair mismatched bases and small insertion-deletion loops that arise during DNA replication.

Which proteins initiate mismatch recognition in E. coli?

MutS recognizes mismatches; MutL stabilizes the MutS-DNA complex.

What enzyme introduces a nick during E. coli mismatch repair and how is the strand chosen?

MutH nicks the unmethylated (new) strand using methylation as a marker for discrimination.

How is the mismatch-containing DNA excised in E. coli?

UvrD helicase unwinds the DNA, and exonucleases degrade the strand past the mismatch in either the 5'→3' or 3'→5' direction.

What polymerase and enzyme complete E. coli MMR after excision?

DNA polymerase III synthesizes new DNA; DNA ligase seals the nick.

How does eukaryotic MMR recognize mismatches?

MSH2-MSH6 (MutSα) or MSH2-MSH3 (MutSβ) recognize mismatches; MLH1-PMS2 (MutLα) stabilizes the complex.

How is strand discrimination achieved in eukaryotic MMR?

Not fully understood; likely uses nicks in the lagging strand or termini recognized by replication proteins like PCNA.

What exonuclease is used in eukaryotic mismatch excision?

Exonuclease 1 (Exo1), with 5'→3' directionality.

Which accessory proteins assist eukaryotic MMR?

PCNA (clamp), RFC (clamp loader), RPA (ssDNA binding protein), DNA polymerase δ or ε, and ligase.

What is the role of PCNA in eukaryotic MMR?

Acts as a sliding clamp to help recruit and orient repair proteins during synthesis.

What dictates which MSH and MLH homologs are used in eukaryotic MMR?

The nature of the mismatch (e.g., base-base vs loop) and the phase of the cell cycle.

How does ATP regulate MMR in both E. coli and eukaryotes?

ATP binding and hydrolysis cause conformational changes in MutS/MSH and MutL/MLH proteins to promote downstream recruitment.

What feature makes E. coli strand discrimination possible but not directly translatable to humans?

Dam methylation at GATC sites is present in E. colibut absent in eukaryotes.

In which phase of the cell cycle is eukaryotic MMR most active?

S-phase, coordinated with replication.

How do MMR pathways contribute to genome stability?

By correcting replication errors, they prevent mutations and microsatellite instability, reducing cancer risk.

What is the primary DNA repair pathway involving TFIIH?

Nucleotide excision repair (NER).

What two types of NER exist in eukaryotic cells?

Global genomic NER (GG-NER) and transcription-coupled NER (TC-NER).

What types of DNA damage are typically repaired by NER?

Bulky adducts, UV-induced photoproducts (e.g., thymine dimers), and helix-distorting lesions.

What is TFIIH and what is its general structure?

A multi-subunit protein complex (9–10 subunits) with helicase activity; includes XPB and XPD helicase subunits.

What role does TFIIH play in transcription and how is this similar to its repair function?

In transcription, TFIIH unwinds DNA at promoters; in NER, it unwinds DNA at the damage site using similar helicase activity.

What two subunits of TFIIH have helicase activity and in which direction do they act?

XPB (3'→5') and XPD (5'→3').

How is TFIIH recruited in GG-NER?

Via damage-sensing proteins like XPC and RAD23B that recognize helix distortion.

How is TFIIH recruited in TC-NER?

Stalled RNA polymerase recruits CSA and CSB, which then recruit TFIIH.

What is the role of RPA in NER after TFIIH activity?

RPA binds to the unwound single-stranded DNA to stabilize it and prevent secondary structure formation.

What nucleases are involved in NER after unwinding by TFIIH?

XPG (3’ incision) and XPF-ERCC1 (5’ incision).

What happens after the damaged DNA is excised in NER?

DNA polymerase fills in the gap and DNA ligase seals the nick in the backbone.

What disease results from defects in XP genes, including XPD and XPB?

Xeroderma Pigmentosum (XP), characterized by light sensitivity and increased cancer risk.

What causes Cockayne Syndrome (CS) and how does it affect TFIIH function?

Mutations in CSA or CSB prevent TFIIH recruitment in TC-NER; leads to developmental defects and neurodegeneration.

What causes Trichothiodystrophy (TTD) and what symptoms result?

Mutations in XPB or XPD lead to failure to unwind DNA during NER; symptoms include brittle hair, developmental delay, and premature aging.

What does the severity of XP, CS, and TTD suggest about TFIIH?

TFIIH is essential for proper DNA repair and maintaining genomic stability; its dysfunction has severe systemic consequences.

What is one proposed advanced application of TFIIH in biotechnology?

Potential alternative to CRISPR-Cas9 for genetic editing due to its precise DNA unwinding and processing capabilities.

What distinguishes TC-NER from GG-NER in terms of speed?

TC-NER is faster because it is triggered directly by the stalling of RNA polymerase at the lesion.

What does the coordinated action of TFIIH, RPA, XPG, and XPF illustrate about eukaryotic NER?

It is a tightly regulated and multi-protein complex repair process with spatial and functional coordination.

What initiates global genomic NER (GG-NER)?

Recognition of DNA helix distortion by the XPC-HR23B complex, sometimes aided by UV-DDB.

What is the specific role of XPC in GG-NER?

XPC acts as the primary damage sensor that detects helix distortions and recruits TFIIH to the site.

How does RNA polymerase contribute to initiating TC-NER?

When RNA polymerase stalls at a lesion during transcription, it signals the recruitment of CSA and CSB, which in turn recruit TFIIH.

How does TFIIH interact with RNA polymerase II during TC-NER?

TFIIH is recruited to the stalled RNA Pol II complex and helps unwind DNA to expose the lesion for repair.

How long is the DNA region unwound by TFIIH during NER?

Approximately 20–30 nucleotides.

What is the role of XPB vs. XPD in the TFIIH complex during DNA repair?

XPB (3’→5’ helicase) is involved in opening the helix and transcription initiation; XPD (5’→3’ helicase) is essential for lesion verification.

What non-helicase subunits of TFIIH are involved in regulating its function?

p44 and p62 help regulate XPD helicase activity; CDK-activating kinase (CAK) subcomplex may be involved in transcription but dissociates in repair.

What is the significance of TFIIH’s involvement in both transcription and repair?

It links gene expression to genome surveillance, coordinating cellular responses to DNA damage with transcriptional status.

Why must the CAK subcomplex (CDK7/cyclin H/MAT1) sometimes dissociate from TFIIH during NER?

To activate the helicase function of XPD for lesion scanning and allow efficient repair activity.

How do XPB and XPD mutations differentially affect transcription and repair?

XPB mutations often impair both transcription and repair; XPD mutations usually affect only repair, unless in combination with other defects.

How is TFIIH’s helicase activity verified in the NER context?

Through lesion verification by XPD, ensuring that only real DNA damage is excised.

What is the general consequence of defects in TFIIH subunits outside of XPB/XPD?

They can cause instability of the entire TFIIH complex or loss of regulation, affecting both transcription and DNA repair.

Why is TFIIH considered a master regulator of NER?

It performs damage verification, DNA unwinding, and coordinates with multiple repair and transcription factors, making it central to repair execution.

What is the role of DNA ligase I or ligase III in NER?

They seal the nick in the DNA backbone after resynthesis is complete.

What type of DNA damage does base excision repair (BER) primarily address?

Small, non-helix-distorting base lesions like oxidized, alkylated, deaminated, or spontaneously lost bases.

What is the first enzyme that acts in the BER pathway?

DNA N-glycosylase.

What is the role of DNA N-glycosylases in BER?

They recognize and remove damaged bases by cleaving the N-glycosidic bond, creating an abasic (AP) site.

How do N-glycosylases recognize damaged bases?

They bend the DNA at the lesion and flip the damaged base into the enzyme’s active site for cleavage.

What structural distortion facilitates glycosylase activity?

DNA bending and base flipping.

What additional activity can some DNA glycosylases have beyond removing the base?

AP lyase activity to cleave the DNA backbone at the 3’ side of the abasic site.

What enzyme follows DNA glycosylase if it lacks lyase activity?

AP endonuclease (e.g., Endonuclease IV in bacteria) to cleave the DNA backbone 5’ to the abasic site.

What determines whether BER proceeds via short-patch or long-patch repair?

The type of polymerase recruited after backbone cleavage.

Which polymerase is used in short-patch BER in bacteria?

DNA polymerase I (Pol I).

What two functions does Pol I perform in short-patch BER?

Incorporates a single nucleotide and removes the 5’ sugar phosphate (via DRPase or lyase activity).

Which polymerases are used in long-patch BER in eukaryotes (note for contrast)?

DNA polymerase δ or ε.

What is the role of flap endonuclease in long-patch BER?

It cleaves the displaced flap of DNA generated by strand displacement synthesis.