9.2 Genetic disorders

Question 1

Types of mutations

Answer 1

Types of simple mutation

1. Transitions (pyrimidine-to-pyrimidine and purine-to-purine)
2. Transversions (pyrimidine-purine and purine-to-pyrimidine)
3. Insertions and deletions (a nucleotide or a small number of nucleotides)

Other mutations:

• Missense mutation
  
  • Incorrect aa (as different single nucleotide) whichg may produce a malfunctioning protein
• Nonsense mutation
  
  • Replacement of single nucleotide –> shortening of protein (non-functional)
  • Deletion mutation
    
    • Incorrect amino acid sequence as deletion of one nucleotide
  • Frameshift mutation
    
    • Inserted DNA base resulting in shift and abnormal amino acid sequence

Question 2

What is polymorphism?

Answer 2

Genetic polymorphism can be when there are different alleles for a gene within a population

Question 3

What is achondroplasia?

Answer 3

• Autosomal dominant change in FGFR3 that affects bone growth
• Inheriting two copies of affected gene is embryonic lethal

Question 4

DNA repair mechanisms

Answer 4

• Direct repair
• Base excision repair
• Nucleotide excision repair
• Mismatch repair
• Double stranded break repair
  
  • Non-homologous end-joining
  • Homology-directed repair

Question 5

Explain direct repair

Answer 5

Direct repair

• O6-methylguanine-DNA methyltransferase I and II (MGMT)
• Also called DNA-alkyltransferases:
  
  • remove modified bases O6-alkylguanine and O4-alkylthymine
  • often involved in cancer
  • results in abnormal base pairing as well as the excision of these bases
  • in turn leads to strand breakage

Question 6

Explain excision DNA damage repair

Answer 6

1. Base excision repair (BER)
2. Nucleotide excision repair (NER)
3. Mismatch repair (MMR)

Question 7

What are some spontaneous reactions?

Answer 7

1. Depurination (C–>T)
2. Deamination (amine group replaced)

Question 8

Explain base excision repair

Answer 8

1. BER initiated by DNA glycosylases
2. At the AP site where AP endonucleases (enzyme cuts duplex DNA)
3. AP exonucleases (AP lyase) (cut ribose-phosphate backbone) –> removed
4. DNA polymerase & DNA ligase catalyses incorporation new base

Question 9

Explain NER (nucleotide excision repair)

Answer 9

1. Thymine- thymine dimers are initially recognized by a complex of the XP-C (xeroderma pigmentosum C protein) and 23B proteins.
2. This complex then recruits transcription factor II human (TFIIH) complex (part of RNA polymerase II preinitiation complex), whose helicase subunits, powered by ATP hydrolysis, partially unwind the double helix. XP-G and RPA proteins then bind to the complex and further unwind and stabilize the helix until a bubble of about 25 bases is formed.
3. XP-G (now acting as an endonuclease) and XP-F, a second endonuclease, cut the damaged strand at points 24–32 bases apart on each side of the lesion.
4. This releases the DNA fragment with the damaged bases, which is degraded to mononucleotides. Finally the gap is filled by DNA polymerase exactly as in DNA replication, and the remaining nick is sealed by DNA ligase.

Question 10

Explain proofreading by DNA polymerase

Answer 10

• Nucleotide-excision repair proceeds most rapidly in cells whose genes are being actively transcribed on the DNA strand that is serving as the template for transcription.
• If RNA polymerase II, tracking along the template (antisense) strand), encounters a damaged base, it can recruit other proteins, to make a quick fix before it moves on to complete transcription of the gene.
• All DNA polymerases have a similar three-dimensional structure, which resembles a half-opened right hand.
• The “fingers” bind the single-stranded segment of the template strand, and the polymerase catalytic activity (Pol) lies in the junction between the fingers and palm.
• As long as the correct nucleotides are added to the 3′ end of the growing strand, it remains in the polymerase site.
• Incorporation of an incorrect base at the 3′ end causes melting of the newly formed end of the duplex. As a result, the polymerase pauses, and the 3′ end of the growing strand is transferred to the 3′ → 5′ exonuclease site (Exo) about 3 nm away, where the mispaired base is removed.
• Subsequently, the 3′ end flips back into the polymerase site and elongation resumes.

Question 11

What is mismatch repair (MMR) and explain

Answer 11

Corrects errors introduced during replication

1. A complex of the MSH2 and MSH6 proteins (bacterial MutS homologs 1 and 6) binds to a mispaired segment of DNA in such a way as to distinguish between the template and the newly synthesized daughter strand.
2. Binding recruits MLH1 and PMS2 (both homologs of bacterial MutL). The resulting DNA-protein complex then binds an endonuclease that cuts the newly synthesized daughter strand. Next a DNA helicase unwinds the helix, and an exonuclease removes several nucleotides from the cut end of the daughter strand, including the mismatched base.
3. As with base excision repair, the gap is filled in by a DNA polymerase (Pol δ, in this case) and sealed by DNA ligase.

Question 12

Explain how to repair double stranded breaks?

Answer 12

Repairing Double-Strand Breaks

1. Homology-directed repair (HR)
2. Nonhomologous End-Joining (NHEJ)

Question 13

Explain homology-directed repair

Answer 13

• In HR (also called homologous recombination) the broken ends are repaired using the information on the intact:
• •sister chromatid (during mitosis or meiosis), or on the
• •homologous chromosome
• •same chromosome if there are duplicate copies of the gene on the chromosome oriented in opposite directions (head-to-head or back-to-back).
• •Two of the proteins used in HR are encoded by the genes BRCA1 and BRCA2.
• •Inherited mutations in these genes predispose women to breast and ovarian cancers.
• Repair of dsDNA breaks during the late S phase and G2 phase of the cell cycle often depends on the ability of the repair apparatus to consult the sequences in the undamaged sister chromatid.
• •Begins with the resection (removal) by an exonuclease of one of the two DNA strands at each of the ends formed by a dsDNA break.
• •Each of the resulting ssDNA strands (blue, red) then invades the undamaged sister chromatid, whose double helix (green, grey) has been unwound by the repair apparatus in order to accommodate the pairing of the invading ssDNA strands with complementary sequences in the undamaged sister chromatid.
• •ssDNA strands from the damaged chromatid are then elongated in a 5’ to 3’ direction by a DNA polymerase, using the strands of the sister chromatid’s DNA as templates.
• •Extended ssDNA strands are released from the sister chromatid and pair with one another. Further elongation by a DNA polymerase and a ligase
• •Reconstruction of a double helix possessing wild-type DNA sequences.

Question 14

Explain Non-homologous end-joining

Answer 14

• •Used to restore a DNA double helix following a double-strand break when the nucleotide sequences from a sister chromatid are not available.
• •In NHEJ, the resection of single strands from both broken ends results in ssDNA overhangs that can then be joined to one another
• •Subsequent filling in of single-strand gaps and the ligation of any remaining ssDNA breaks results in the reconstruction of a double helix that lacks some of the base pairs that were present in the original undamaged DNA helix.