DNA Mismatch Repair and Mutations Quiz

DNA Mutability & Repair

• DNA synthesis has a high level of accuracy, with only one mistake in 10¹⁰ base pairs.
• Base-pair geometry and complementarity reduce errors to occur in 10⁵ times.
• DNA mistakes are prevented to reach the high level of accuracy.

Topics

• Types of DNA errors:
  • SNPs (single nucleotide polymorphisms)
  • Microsatellites
  • Transposition
  • Double-strand breaks
• Sources of errors:
  • Inaccuracy of DNA replication
  • Chemical/radiation damage
  • Insertion of DNA elements (transposons)
  • Spontaneous damage
• Repair of DNA damage:
  • How DNA is repaired quickly

Mutations

• A mismatch during first replication round, will become a mutation during the second replication round in coding or non-coding DNA.

Types of DNA Errors (Nature of Mutations)

• Point Mutation (SNP)
  • Transition
  • Transversion
  • Insertion
  • Deletion
• DNA microsatellites
  • Mutation-prone sequences
  • Insertion/deletion of di-, tri-, and tetranucleotide sequences (e.g., CACACA, CGGCGGCGGCGG)
  • Hotspots for mutations
  • Used as genetic markers for mapping genetic diseases
• Transposition
  • Movement of DNA elements
• Double-strand break
  • Mostly caused by environmental factors
  • Both DNA strands break
  • Without repair, serious consequences may arise

Some Replication Errors Escape Proofreading

• Inability of DNA polymerase to add a new base pair next to a wrong one.
• Activation of exonuclease

Mismatch Repair System (MRS) Challenge

• Scans genome to detect errors.
• Fixes DNA errors accurately before second cell division.
• MRS in E. coli: Mismatch repair protein MutS (a dimer) that recognizes the DNA mismatch from distortion.

How can MRS Recognize Which Nucleotide Is Mismatched?

• E. coli tags the parental strand by transient hemimethylation.
• Dam methylase methylates A residues on both strands of the 5'GATC3' sequence.
• This sequence is widespread throughout the genome.
• All these sites are methylated by the Dam methylase.
• MutH binds to non-methylated sites.

Directionality in Mismatch Repair: Exonuclease Removal of Mismatched DNA

• Specific steps involved in removing and fixing the error.

Eukaryotic MRS

• MutS & MutL homologs (MSH, MLH, and PMS) are used.
• Multiple MutS-like proteins with different specificities.
• Lack the MutH and hemimethylation trick used by E. coli.
• During lagging strand synthesis, the Okazaki fragment separates from previously synthesized DNA via a nick, which is similar to the nick generated by MutH on the newly synthesized strand.
• Human MutS homolog (MSH) interacts with the sliding clamp component of the replisome.

Sources of Errors

• Spontaneous damage
• Environmental factors
• Inaccuracy of DNA replication
• Insertion of DNA elements (transposons)

Ames Test for Mutagens

• Detects mutagens using Salmonella bacteria.

Spontaneous Damage (A- damage caused by water (hydrolysis))

• Deamination of C → U
• Deamination of A → hypoxanthine (can H-bond to C)
• Deamination of G → xanthine (can H-bond to C (two H-bonds))
• Methylated C are hotspots for spontaneous DNA mutations in vertebrates.
• Depurination: Hydrolysis of N-glycosyl linkage → abasic site (apurinic)

Damage Caused by Environmental Factors

• Alkylation: Methyl/Ethyl group added to base or DNA backbone. Nitrosamines & N-methyl-N¹-nitrosoguanidine.
• Oxidation: O atom of guanine ç 06-alkylation, methyl oxidation of guanine.
• Radiation:
  • UV light: Two adjacent pyrimidines (T/C) join together. Photochemical fusion causes DNA polymer.
  • Gamma & X-ray radiation: Cause double-strand breaks by ionizing DNA backbone. Also forms free radicals. Cells can't divide, apoptosis. Used in chemo/radio therapy.
• Base Analogs: Structurally similar to bases. Can be incorporated into DNA, leading to mutations.

Mutations Caused by Intercalating Agents

• Intercalating agents (flat molecules) cause addition or deletion of bases during replication (e.g., proflavin, ethidium, acridine orange).

Spontaneous Damage (caused by intercalating agents)

• Flat molecules of polycyclic rings bind to DNA
• Proflavin, acridine, and ethidium
• Cause insertion/deletion of one or a few base pairs
• Skipping or addition of new base pairs during replication.
• Slipping between bases in the template strand causes DNA polymerase to insert an extra base opposite the intercalated molecule.
• In case of deletion, the distortion of the template caused by the intercalated molecule may lead to polymerase skipping a nucleotide.
• Serious consequence on translation of mRNA

DNA Repair

• G1 and G2 are check points before replication and division.
• Cells aim to repair any damage to pass to M phase.

Table of DNA Damage Repair and Tolerance Systems

• Different types of DNA repair mechanisms (Mismatch, Photoreactivation, Base Excision, Nucleotide Excision Repair, Double-Strand Break Repair, Translesion DNA Synthesis) and their associated damage, enzymes, and specificities.

Repair of DNA Damage

• Excision repair systems: Damaged nucleotide is removed, and the other undamaged strand serves as the template for the correct nucleotide insertion by DNA polymerase.
• Recombination repair: Both strands are damaged. Sequence information is retrieved from a second undamaged copy of the chromosome.

1- Direct Reversal of DNA Damage

• Repair of damage caused by UV radiation (photoreactivation).
• 0⁶-methylguanine is repaired directly by methyl transferase.

2- Repair of Hydrolysis Damage (Base Excision Repair System)

• Glycosylase enzyme recognizes damaged base, hydrolyzes glycosidic bond.
• Repair is completed by polymerase and ligase.
• Different specificities (11 in humans).
• Base excision pathway: the uracil glycosylase reaction

Base Excision Repair

• DNA glycosylases are lesion-specific, and cells have multiple DNA glycosylases.
  • Uracil glycosylase.
• Another specific glycosylase removes oxoG.

How can Glycosylase Complex Recognize Abnormal Base?

• Glycosylases diffuse laterally along the minor groove of DNA until a specific lesion is detected.
• Damaged base is flipped out

Fail-safe Glycosylase (oxoG: A repair)

• Works if glycosylase did not recognize error before replication.
• Fail-safe system that removes T opposite G (from deamination of 5-methylcytosine).

3- Nucleotide Excision Repair (NER)

• Recognizes DNA distortion (T dimers or bulky adducts on a base)
• Short single-stranded segment is removed.
• 4 proteins involved in E. coli (UvrA, UvrB, UvrC, UvrD)

Principle of NER in Higher Cells

• Similar to E. coli but more complicated, involving 25 or more polypeptides.
• XPC detects DNA distortion like UvrA.
• Bubble formation involves helicases XPA and XPD (equivalent to UvrB in E. coli) and the SSB protein RPA.
• Cleavage sites on the 5' side of the lesion for nuclease ERCC1-XPF on the 3' side.

Nucleotide Excision Repair Enzyme

• UVR proteins recognize a broad variety of changes (including dimers) caused by UV.
• Mutants to UVR proteins are sensitive to UV light.
• Xeroderma pigmentosum (7 XP genes)

Nucleotide Excision Repair

• The principles of nucleotide excision repair in higher cells are similar to those in E. coli, but more complicated, involving 25 or more polypeptides.
• The UVR proteins are needed to mend damage from UV light.
• Mutants of uvr genes are sensitive to UV light, and lack the capacity to remove T-T or T-C adducts.
• In humans, xeroderma pigmentosum patients have mutations in seven genes (XP genes).
• These XP proteins correspond to proteins involved in nucleotide excision repair.

4 - Transcription-coupled Repair

• RNA polymerase serves as damage sensing protein.
• Stopt RNA polymerase and call nucleotide excision repair proteins
• Has the advantage of focusing on genes being transcribed.

Transcription - coupled repair in Eukaryotes

• TFIIH includes XPA & XPD subunits.
• Unwinds DNA template during transcription.
• Melts DNA around the lesion during nucleotide excision repair/transcription-coupled repair.

5- Double-strand Break (DSB) Repair (Recombination repair)

• Both strands are broken (no template).
• Sister chromosome is a template.
• Helps repair errors in DNA replication.

What Happens if the Break Took Place Before Replication (no sister chromosome)?

• Fail-safe method nonhomologous end joining (NHEJ), a backup system in yeast.
• Two broken DNA ends joined together.
• Because the sequence information is lost from the broken ends, the original sequence is not restored (its mutagenic but less hazardous to the cells than unrepaired broken ends).
• 7 components found in mammalian cells (Ku70, Ku80, DNA-PKcs, Artemis, XRCC4, Cernunnos-XLF, and DNA ligase IV).

6 - Translesional DNA Synthesis

• Translesion synthesis is catalyzed by specialized class of DNA polymerases that synthesize DNA directly across the site of the damage.

• In E. coli, DNA Pol IV (DinB) or DNA Pol V (a complex of the proteins UmuC and UmuD`) performs translesion synthesis.

• DinB and UmuC are members of the Y family of DNA polymerases.

• There are five translesion polymerases known in humans, four of which belong to the Y family.

• Y-Family DNA polymerases specialize in translesion synthesis, bypassing damaged bases to block the normal progression of replication forks.

• Y-family polymerases are characterized by low catalytic efficiency, low processivity, and low fidelity on normal DNA.

Translesion DNA Synthesis

• Translesion DNA synthesis proceeds in a template-dependent manner, which is independent of base pairing.
• DNA Polymerase III
• Obstacles to progression of DNA polymerase (or AP site).
• Complex of proteins UmuC and D´ (Y-family of DNA polymerase)

Y-Family DNA Polymerases

• List of Y-family DNA polymerases in E. coli, mammalian cells and additional information

How a translesion polymerase gains access to the stalled replication machinery at the site of DNA damage?

• In mammalian cells, entry into the translesion repair pathway is triggered by chemical modification of the sliding clamp (attachment of ubiquitin).
• Once ubiquitinated, the sliding clamp recruits translesion polymerase through ubiquitin.
• The translesion polymerase somehow displaces the replicative polymerase from the 3' end of the growing strand and extends it across the site of damage.

Alternative models for translesion synthesis

• Polymerase-switching model
• Gap-filling model

Test your knowledge on DNA mismatch repair mechanisms and the consequences of mutations. This quiz covers key proteins involved in the process, types of DNA errors, and their implications on genetic integrity. Perfect for students studying molecular biology or genetics.