lecture 4

Question 1

What are mutations

Answer 1

• permanent change in DNA
• in germ cells -> hereditary diseases and natural diversity
• somatic cells -> carcinogenesis
• most mutations are not harmful

Question 2

types of mutations

Answer 2

1. genomic: changes in DNA quantity (numerical chromosomal abnormality)
2. chromosomal: changes in DNA quality (structural chromosomal abnormality)
3. gene: changes in nucleotide sequence of a gene

Question 3

Origin of gene mutations

Answer 3

• errors occurring during DNA replication
• spontaneous or induced environmental changes

Question 4

Errors during DNA replication

Answer 4

• addition of the wrong base from DNA polymerase, mainly in loop-shaped areas
• DNA replication slippage:
  DNA polymerase can skip bases in template DNA (template strand loops out) by slipping forward -> unbound bases, nucleotides missing
  DNA polymerase can add extra bases in new strand when the new strand loops out

Question 5

Errors due to environmental factors

Answer 5

• radiation, chemical mutagens, temperature, etc
• due to accidental endogenous events -> chemical cell processes -> are called spontaneous mutations
• majority of these are not repaired -> permanent mutations in DNA

1. modifications of individual bases
2. cross-linkage between two DNA chains
3. chromosome breakage and nucleotide modifications
4. incorrect base connections - thymine dimers (T-T)
5. recombination of segments -> destruction or transcriptional silencing or over-activation of genes

Question 6

Molecular Basis of mutations

Answer 6

• nucleotide substitution or point mutation in DNA that alters the genetic code on a triple base -> causes substitution of an amino acid

point mutations
- silent : no change
- missense : change in coding sequence of the gene (conservative or non-conservative)
- nonsense : termination of transcription

INDEL mutations
splice mutations
repeats (or duplications)

Question 7

point mutations

Answer 7

• silent: produces synonym codons -> no change in protein structure or function, does not affect phenotype
• missense (conservative): new codon -> no or minimal changes in structure and function of protein, some are noticed and affect the phenotype
• missense (nonconservative): change in structure and function of protein, noticed and affect the phenotype
• nonsense: generates a stop codon (-> premature termination of translation) or destroys a stop codon (-> continuation of translation)
  -> creation of stop codons in the wrong place
  -> cause significant changes in protein structure and function, affects the phenotype

Question 8

INDEL mutations

Answer 8

• insertions
• deletions
• can be small size (one or more bases) or large-scale (part of gene or whole gene)
• frame shift when INDEL includes a few base pairs, which is not a multiple of three -> mutation changes the open reading frame
  -> usually leads to premature termination of translation -> non-functional protein, affects the phenotype
  -> more harmful than point mutations
• mutation including a multiple of three -> no shift in reading frame
• microdeletion syndrome: when more than one gene is involved -> large deficiencies and interferences at chromosome level, probably due to unequal crossing over between multiple copies of similar or identical DNA sequences

Question 9

splice mutations

Answer 9

2 types
- affect the bases in splice region -> problems in splicing process
- substitution of bases within introns -> alternative splicing sites

• significant changes in protein structure or function -> protein is unstable and destroyed -> affects the phenotype

Question 10

Repeats and Duplications

Answer 10

• more rare than deletions
• large duplications may lead to duplication of a gene or part of DNA -> affects the amount of the gene product -> overexpression
• repeat mutation increases the number of a small DNA sequence in the genome
  -> most common: nucleotide repeats (small DNA sequences repeated in sequence)
  -> change in structure and function of protein -> affects phenotype

Question 11

Huntington’s disease

Answer 11

• autosomal dominant
• neurodegenerative disorder
• caused by a heterozygous expanded trinucleotide repeat (CAG)n, encoding glutamine, on Chromosome 4p16

Question 12

Mutation sensitive points

Answer 12

• Transition: replacing one purine with another (A and G) or pyrimidine with another ( C and T)
• Transversion: replacing a purine with a pyrimidine or vice versa
• transitions are more frequent
• C->T or C -> A transients affect methylation of cytosine residues
  -> CG islets are mutation sensitive sites

Question 13

molecular diagnostic methods used to detect mutations

Answer 13

• southern blotting
• allele specific oligonucleotide technique (ASO)
• next-generation sequencing
• polymerase chain reaction

Question 14

frequency of errors in DNA replication

Answer 14

10^-10

• corrected by repair enzymes
• <99% of errors are repaired by various DNA repair mechanisms

Question 15

Types of DNA repair mechanisms

Answer 15

1. direct repair mechanisms: immediate removal and restoration
   - repair during replication
   - 0-6-methylguanin repair in bacteria
   - thymine dimer repair in plants
2. indirect repair mechanisms: damage is recognized and repair enzymes are recruited, incorrect part is removed and new segment is synthesized by repair polymerases
   - base excision repair
   - nucleotide excision repair
   - homologous recombination repair
   - non homologous end joining
   - mismatch repair

Question 16

Repair during Replication

Answer 16

• DNA pol (roh) and other proteins of the replication complex
• recognize and remove wrong nucleotide with exonuclease activity
• select the right nucleotide and introduce it into the new chain

Question 17

Indirect repair mechanism: general process of removing the damage

Answer 17

1. identification and labelling of the error
2. recruitment of repair enzymes to the area
3. removal of the damage
4. synthesis of a new segment and restoration

Question 18

Base excision repair

Answer 18

• in modified or absent bases and breaks of one strand
• main mechanism of protection agains damage of internal origin

1. modified base is recognized and removed by DNA glycosylases
2. apurinic/apyrimidinic AP site is recognised by endonucleases and they create an opening in DNA strand (removal of backbone)
3. DNA pol beta adds nucleotides, dna ligase synthesises the new segment

Question 19

Nucleotide excision repair

Answer 19

• to remove bulky complexes
• deals with exogenous factors

two variants:
- genomic NER: removes bulky complexes from the entire genome
- transcription coupled NER; removes bulky complexes from the RNA polymerase II pathway

1. XPC recognises helix distortions
2. special endonucleases (XPG and XPF) are recruited and create an opening in the strand
3. gap in chain is synthesised by DNA polymerases and sealed by DNA ligase

Question 20

Homologous Recombination repair

Answer 20

• to repair breakage or damage at the same site on both DNA strands (double strand)
• ATM kinase, ATR kinase, DNA-PK
• cell cycle is stopped:
  cell in S phase: homologous repair enzyme RAD are activated -> uses intact strand as a template for synthesis of the damaged strand
  cell in G1 phase: HR cant repair. ATM connects the cut edges and brings them together so ligase joins them -> cut edge joining

Question 21

Non-homologous End joining

Answer 21

• primary pathway for repair of DNA double-strand breaks throughout cell cycle, including S- and G2-phase
• relies on Ku protein to thread onto each broken DNA end
• Ku recruits enzymes and complexes to trim (nucleases) or to fill in (polymerases) to ends to make them optimally ligatable by the DNA ligase IV complex

Question 22

mismatch repair

Answer 22

• to repair small loops in DNA
• can recognise the parent strand from the newly synthesised strand
• usually activated immediately after replication -> corrects remaining errors
• interacts and cooperated with DNA replication system and NER and HR repair systems
• malfunction -> carcinogenesis

Question 23

DNA Repair and Carcinogenesis

Answer 23

• repair mechanisms incomplete or inefficient
• incident or error / mutation accumulation -> increased likelihood that genes for carcinogenesis are altered
• damage to functionality of repair enzymes -> dramatic increase in chromosomal and genetic mutations