Lecture 2: DNA Repair and Transcription Regulation

Question 1

What is DNA damage?

Answer 1

Any change from the normal nucleotide sequence n supercoiled double helical state

Question 2

Causes of DNA damage

Answer 2

• Physical n chemical agents in the environment
  
  • UV light, free radicals produced during metabolism
• Errors in DNA replication

Question 3

What 2 general classes does DNA damage fall into?

Answer 3

• Single base changes – produces mutations but have no effect on physical process of transcription or replication
  
  • Replication errors due to keto-enol type tautomerization
  • Deamination of cytosine to uracil
  • Incorporation of U rather than T during replication
  • Chemical modification of bases
• Structural distortions may impede transcription and/or replication
  
  • Single strand breaks
  • Covalent modification of bases e.g. alkylation
  • Removal of a base
  • Interstrand and intrastrand covalent bonds

Question 4

Cause of single base changes

Answer 4

• Replication errors due to keto-enol type tautomerization
• Deamination of cytosine to uracil → changes how they bind
• Incorporation of U rather than T during replication
• Chemical modification of bases

Question 5

Give an example of structural distorNon in the context of DNA damage

Answer 5

• Thymine dimer formation caused by UV light
  
  • 2 T on the same strand become covalently linked
  • Forms either
    
    • Cyclobutene structure
    • (6-4) photoproduct

Question 6

Cause of structural distortions

Answer 6

• Single strand breaks
• Covalent modifications of bases (e.g. alkylation)
• Removal of a base
• Interstrand n intrastrand covalent bands

Question 7

How are mismatches and structural distortions in DNA dealt with?

Answer 7

• Direct repair: doesn’t require nucleotide template, cleavage or synthesis
  
  • Reversal or simple removal of the damage
• Mismatch repair: bidirectional excision-resynthesis, detects + removes mismatch
  
  • Detection n repair of mismatched bases
• Excision repair: large parts of the DNA are removed n replaced (removal +synthesis)
  
  • Recognition of the damage followed by excision of a patch of DNA n its replacement by undamaged DNA
• Tolerance systems: allows DNA replication to proceed thru damaged regions of DNA
• Retrieval systems: recombinational processes to repair damaged DNA

Question 8

Give an example of direct repair

Answer 8

Photoreactivation

• Repairs UV induced T-T dimers
• Photolyase binds to T-T dimers in the dark
• Contains 2 chromophores that absorb light energy
• Uses this energy to split cyclobutene structures

Question 9

Give an example of mismatch repair

Answer 9

Uracil DNA glycosylase

• Context: U is sometimes incorporated into DNA instead of T
• Uracil DNA glycosylase removes U
  
  • RESULT: AP site (gap in DNA where there’s no base)
• AP endonuclease nicks the AP site
  
  • Makes a break in the phosphodiester backhone
• DNA Pol I binds to the break
  
  • Adds new nucleotide
• DNA ligase seals the gap

Question 10

What is the mut system?

Answer 10

• MutS: recognizes mismatches and short insertion/deletions (indels) on hemi-methylated DNA and binds to them
• MutL binds and stabilizes the complex
• MutS-MutL complex activates MutH
• MutH locates a nearby methyl group and nicks the newly synthesized strand opposite the methyl group
• MutU (Helicase II) unwinds the DNA from the nick in the direction of the mismatch
• DNA PolI degrades and replaces the unwound DNA and DNA ligase seals the single strand break

Question 11

What are the 3 excision repair modes found in E.coli?

Answer 11

• Very short patch (deals with mismatches between bases)
• Short patch: ~20 nucleotides
• Long patch: 1500 - 10,000 bps

Question 12

What does short and long patch repair utilize and what are they encoded by?

Answer 12

• Both short and long patch repair utilize the repair endonuclease
• Encoded by the uvrA, uvrB and uvrC genes

Question 13

Give an example of excision repair

Answer 13

• The enzyme (uvrABC) binds to damaged regions
  
  • Makes an incision on both sides of the damage
• UvrD (i.e. MutU, DNA helicase II) separates strands n removes damaged DNA
• DNA polI replaces the DNA and DNA ligase fills the gap
• Short patch repair accounts for 99% of bulky lesions repair events

Question 14

Give an example of tolerance systems

Answer 14

Inducible error prone repair

• Low-fidelity DNA polymerases (translesion synthesis polymerases (TSPs)) can synthesise DNA past damaged bases
• Not efficient at replicating undamaged DNA accurately
• Most lack proof-reading ability
• Two in E. coli, polymerases IV and V, and five in human cells
• Almost all are members of a new DNA pol family, the Y-family
• In some circumstances make many errors, so can generate mutations

Question 15

How does human polymerase η (eta) contribute to preventing UV-induced mutations and cancer?

Answer 15

• Human polymerase η (eta) efficiently bypasses the major UV photoproduct, typically inserting the correct nucleotides.
• It is less effective with other types of damage.
• In individuals with xeroderma pigmentosum, a highly skin-cancer-prone genetic disorder, polymerase η is defective.
• Its absence leads to an increased risk of UV-induced mutations and cancer.
• Although one of its counterparts may substitute for it in its absence, it is less efficient, resulting in increased mutations and cancer susceptibility.

Question 16

Give an example of a retrieval system

Answer 16

Daughter strand gap repair

• Doesn’t fix DNA damage
• Relies on other repair processes such as excision repair to repair the damage afterwards

Question 17

What is an SOS response?

Answer 17

If E. coli suffers severe DNA damage it activates the expression of a large number of diverse unlinked genes involved in DNA repair, error-prone DNA replication, etc.

Question 18

Explain the SOS response mechanism

Answer 18

• All genes and operons under SOS control are subject to repression by the LexA protein
• LexA has two domains: a dimerization and a DNA-binding domain
• LexA box (conserved binding site) is located within the promoter genes regulate by LexA
• Binding of LexA to the LexA boxes represses expression of SOS operons
• RecA responds to DNA damage (e.g. presence of ssDNA) → RecA changes conformation which activates it (RecA*)
• RecA*
  
  • Inhibits 3’→5’ editing in DNA Pol III, allowing error-prone DNA replication
  • Interacts with LexA, which autocleaves becomes inactive leading to the SOS response
• sulA expression inhibits cell division
• Once DNA damage is repaired RecA* converts back to RecA
• LexA stops autocleaving and concentration increases
• LexA represses SOS operons and cell division occurs
• The SOS response allows the cell to survive severe DNA damage by allowing DNA replication but at the expense of fidelity. It is a last ditch effort by the cell to replicate with severe DNA damage

Question 19

What are the 2 general transcriptional mechanisms for gene expression control?

Answer 19

• Induction: switching on genes when required
• Repression: switching off of genes when not required

Question 20

What is an operon?

Answer 20

A cluster of genes transcribed from a single promoter to give a single mRNA but encodes several proteins

Question 21

What are the 2 types of regulatory proteins?

Answer 21

• Repressors
• Activators (apoinducers)

Question 22

What are repressors?

Answer 22

Regulatory proteins which prevent transcription when bound to the DNA

Question 23

What are activators?

Answer 23

Regulatory proteins which activate transcription when bound to the DNA

Question 24

What are 2 types of effectors bind to regulatory proteins n activate/inactivate them?

Answer 24

• Inducers: activate activators in activate repressors (switch genes on)
• Co-repressors: activate repressors or inactivate activators (switches genes off)

Question 25

What are regulons?

Answer 25

Genes associated w particular physiological function may not be in just one operon

Question 26

What is global regulation systems / global control?

Answer 26

Control systems that operate on a wide basis

Question 27

Why is there diauxic E.coli growth when glucose n lactose are both present?

Answer 27

- *E. coli* prefers to use glucose as its carbon source - Catabolite repression - Glucose represses the synthesis of enzymes that metabolize lactose - If both glucose and an alternative carbon source is available the glucose is used first → **Diauxic growth** - If lactose is not present/glucose is present, E.coli will only hv few enzymes that metabolize lactose - E.coli only has enzymes that synthesize glycose in medium containing both glucose + lactose - When cells run out of glucose, there is rapid induction of enzymes for lactose metabolism

Question 28

How did Jacob n Monad show that specific proteins are expressed to regulate gene expression in the lac operon?

Answer 28

- Experiment - Inserted Iac operon genes into the F plasmid to complement mutated Iac operon genes to chromosome - Why? - Merodiploid shows how complementation works - Recessive mutations as a working copy in the plasmid can correct mutation in the chromosome as they can diffuse across the cell - RESULT - Some genes encoded diffusible products that regulate gene expression of both DNA molecules - Transacting elements - Other genes didn’t produce a product but regulated genes on the DNA molecule they were encoded upon - Cis-acting elements

Question 29

What is gene complementation?

Answer 29

- Mutations in two separate genes result in non-functional proteins, but when these mutated genes are present together in a cell, they compensate for each other's deficiencies - RESULT: restoration of a normal phenotype.

Question 30

Describe gene complementation in the context of lac mutations

Answer 30

- Both operons (sets of genes) are repressed initially. - Allolactose, an inducer, activates expression of both molecules. - The chromosome produces a functional LacZ gene, while the F' plasmid carries a functional LacY gene. - These functional genes complement the two mutated genes, resulting in the restoration of normal phenotype due to the presence of functional proteins.

Question 31

What is a super repressor?

Answer 31

- Repressor which won’t come off the operator - Leads to gene repression - Overrides the ability to complement - Dominant mutations

Question 32

What’s the difference b/w trans- and cis- acting elements?

Answer 32

- Trans-acting - Product affects both DNA molecules - Cis-acting - Products only affects the DNA it’s on

Question 33

Give 2 examples of regulation by non-coding RNA (ncRNA)?

Answer 33

- Addiction cassettes in plasmids - Plasmid makes stable mRNA which is lethal - Plasmid also makes ncRNA (unstable antisense RNA) that binds to lethal mRNA n prevents translation - If daughter cell has the plasmid - Lethal mRNA is repressed - Daughter cell doesn’t hv plasmid - ncRNA isn’t produced - Lethal mRNA is translated - RESULT: cell death - EXAMPLE: hok sok system in plasmid R1 - Regulation of iron-containing genes in E.coli - RhyB (ncRNA) controls Fe2+ use - Iron freely available - FUR (global regulator) binds Fe2_ n represses rhyB - Iron requiring proteins made - Any free iron is attached to protein - Limited iron - FUR doesn’t bind Fe2+ → stops repressing rhyB - RhyB binds to mRNA of nonessential iron proteins - mRNA degrades - Need of Fe2+ in cell fals - Self regulates: as Fe2+ ↑, rhyB repression reactivates