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What is a mutation?

A mutation is a change in the normal base pair sequence


If left unrepaired, mutations in the genetic code or in regulatory regions that impact gene expression, what can happen?

Cell cycle arrest, cell death (apoptosis), genomic instability and disease (like cancer)


Most instances of inherited syndromes with defects in DNA repair involve ___ mutant alleles.



What are the three classes of small-scale mutations (point mutations)? Describe them.

1. Base substitutions and modifications (replacement or modification of a single base; transitions (Pu/Pu or Py/Py) and transversions (Pu/Py); methylation; unnatural bases)
2. Deletions (one or more nucleotide is eliminated, leading to frameshift)
3. Insertions: copy/duplicative transposition (duplicated copy moved to another location); non-copy transposition (movement to a new location), leads to frameshifts)


What are the four major types of large scale mutations/changes that occur at the chromosomal level?

1. Amplification (gene duplications)
2. Deletions
3. Translocations (fusions or inversion)
4. Loss of heterozygosity


What are allelic sequence variations?

Allelic sequence variations are polymorphisms arising with a frequency >1%.


What is the average heterozygosity for human genomic DNA?

1:250-1:1000 bases are different between allelic sequences


What can be used to distinguish between individuals?

SNPs (single nucleotide polymorphisms)


Mutations that change alleles can produce new genetic traits; some are good, leading to ___, and some are deleterious, leading to ___.

Adaptation; Disease susceptibility


What are de novo mutations and where can they occur?

Naturally occurring mutations; they can occur in both somatic cells and germline cells


Are somatic mutations heritable? Why or why not?

No, they only affect those cells that result from mitotic division; mutations can accumulate in these cells, leading to disease


Are germline mutations heritable? Why or why not?

Yes, they affect all cells, thus increasing the susceptibility to disease


What are the sources of de novo mutations?

Copying errors, spontaneous chemical attack, environmental exposure


Describe how spontaneous DNA alterations occur.

Bases within DNA are altered through several types of endogenous attack, including oxidative damage, hydrolytic attack, or modifications.


What are the most frequent types of chemical attack?

1. Depurination of AG (hydrolytic cleavage of the bases from the sugar backbone)
2. Deamination (typically the removal of amino group from C, leading to conversion to U)


Replication and environmental factors can result in DNA mutations. Different types of damage can result in the activation of ___, which usually remove the mutagenic lesion.

Specific removal pathways


How do cells deal with mutated DNA that is not repaired?

If the rate of DNA damage = the rate of repair, the cell remains healthy. If the rate of DNA damage > the rate of repair, damage accumulates, which can lead to excessive proliferation (cancer), senescence (halted growth), or apoptosis (cell death)


What are the three general classes of DNA repair mechanisms?

1. Direct repair (damage reversal)
2. Excision repair (remove and replace - BER, NER, TCR, MMR)
3. Double-stranded break repair (HEJ, NHEJ)


What are the four types of excision repair?

1. Base excision repair (BER)
2. Nucleotide excision repair (NER)
3. Transcription coupled repair (TCR)
4. Mismatch repair (MMR)


True or False: Most lesions can be repaired by more than one mechanism.



What does direct repair do?

Direct repair reverses base damage in a quick, though energetically costly manner.


Describe the direct repair mechanism.

O6-meG, an abnormal base, can base pair to C or T. O6-meG can block polymerase. MGMT (O6-meg methyl transferase) removes the methyl group and reverses the base damage.


What does base excision repair (BER) do?

Uses specific glycosylase enzymes to recognize and remove abnormal bases and generate AP (abasic) sites. AP endonuclease and phosphodiesterase cut the backbone, fill the gap, and seal.


BER dysfunction has been linked to disease progression in ___.

Alzheimer's disease


What does nucleotide excision repair (NER) do?

Removes bulky lesions that distort the helix. The gap is repaired.


What are the two types of NER?

General/global NER and transcription coupled repair (TCR)


What is the difference between general NER and TCR?

TCR is specifically associated with active transcription.


What does mismatch repair (MMR) do?

Repairs mistakes AFTER DNA replciation and before mitosis


What are the four common steps of DNA excision repair?

1. Recognition of damage
2. Cleavage by endonuclease
3. Removal by nuclease or helicase activity
4. Replacement by polymerase and ligase activity


Describe the process of base excision repair.

1. Specific glycosylases recognize specific altered bases and generate abasic (AP) sites by cleaving the glycosidic bond between a base and the sugar.
2. An AP endonuclease and phosphodiesterase remove the sugar phosphate.
3. DNA polymerase adds new nucleotides.
4. DNA ligase seals the nick.


Describe the process of nucleotide excision repair.

1. Nuclease cleaves the two ends of the section of DNA including the bulky lesion.
2. DNA helicase unzips the DNA and removes the section.
3. DNA polymerase and ligase add new base pairs


How are bulky lesions generated?

By pyrmidine dimers and other chemical modifications


What type of repair is associated with actively transcribed DNA?

Transcription coupled repair (TCR)


What syndrome is caused by a defect in TCR?

Cockayne syndrome


Describe the process of transcription coupled repair (TCR).

1. TFIIH, a helicase made from 2 XP proteins, opens the double helix.
2. XPG cleaves the 3' side of the lesion.
3. XPF/ERCC1 cleaves the 5' side of the lesion


What does mismatch repair (MMR) do?

Corrects errors following DNA replication


What are the two types of double-stranded break repair and when do they occur in the cell cycle?

1. Nonhomologous end-joining (NHEJ), occurs during G0/G1
2. Homologous end-joining (HEJ), occurs during S/G2


Where do cell cycle checkpoints occur?

G1/S, G2/M


Which type of double-stranded break repair is mutagenic and why?

NHEJ, because DS ends are degraded and not replaced; typically found in non-dividing cells.


Describe the process of NHEJ.

1. Ku Heterodimers recognize the broken ends.
2. Additional proteins attach and process the DNA ends. Limited repair synthesis occurs.
3. Ligation occurs.


Describe the process of HEJ.

1. Nuclease digests 5' ends of broken strands.
2. Strand exchange occurs by complementary base-pairing with the sister chromatid.
3. Repair polymerase synthesizes DNA using undamaged DNA as a template.
4. Invading strand release; broken double helix re-formed.
5. DNA synthesis continues using strands from damaged DNA as template.
6. DNA ligation


What catalyzes strand invasion?

RecA (prokaryotes) or Rad51 (eukaryotes)


Describe the process of strand invasion by RecA/Rad51.

RecA intertwines the DNA single strand and the DNA duplex in a sequence-independent manner. The single strand searches the duplex for homologous sequences. A strand invasion occurs-the single strand displaces one strand of the duplex as it forms conventional base pairs with the other strand, creating a heteroduplex.


What is branch migration?

The process by which an unpaired region of one of the single strands displaces a paired region of the other single strand, moving the branch point without changing the total number of DNA base pairs.


What are the two types of branch migration? Describe them.

1. Spontaneous branch migration - back-and-forth, random process, makes little progress over long distances.
2. Protein-directed branch migration - requires energy and moves the branch point at a uniform rate in one direction


What are the features of general recombination?

1. 2 homologous molecules "cross-over"
2. The site of exchange can occur anywhere.
3. At the site of exchange, a strand of one DNA molecule becomes base-paired to a strand of the second molecule, creating a heteroduplex joint.
4. No nucleotide sequences are altered at the exchange site.
5. Frequency is not constant along the chromosome.


What initiates genetic recombination during meiosis?

Pairing between the non-sister chromatids of the homologous chromosome pairs.


What happens if the point of crossing over is not a perfect match?

MMR is utilized


When does recombination occur in meiosis?

During the first meiotic prophase


Describe the process of general recombination in meiosis.

1. Begins with a double stranded break - Spo11 breaks the DS DNA to start the process.
2. MRE11 uses its nuclease activity to remove Spo11 and process the ends.
3. Strand exchange begins with a RecA-like protein
4. DNA synthesis generates new strands
5. Crossover junctions resolve (with or without crossover)


90% of homologous recombination is resolved as ___. Explain.

Non-crossovers; the two original DNA duplexes separate in an unaltered form, except for the region of heteroduplex that formed near the site of the original strand-break.


Describe resolution as crossovers.

A double Holliday junction is formed and cleaved by a specialized enzyme. The two original portions of each chromosome upstream and downstream from the 2 Holliday junctions are swapped, creating 2 chromosomes that have crossed over.


What is gene conversion? When can it occur?

Apparent nonreciprocal recombination during meiosis; when maternal and paternal alleles in a heteroduplex joint contain slight differences in DNA sequence. MMR can result in restoration of the parental sequence or in gene conversion.


What happens in loss of heterozygosity (LOH)?

Produces daughter cells that contain one or another homozygous allele in a diploid state following cell division; it follows somatic recombination


LOH can also result from repair of the homologous chromosome following ___.

DSB during S phase