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LF206: Molecular Cell Biology > Lecture 2. DNA Repair, Transcription Regulation > Flashcards

Lecture 2. DNA Repair, Transcription Regulation Flashcards

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1
Q

What is DNA constantly exposed to?

A

Damage

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2
Q

What is DNA damage?

A

Damage is any change from the normal nucleotide sequence and supercoiled double helical state

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3
Q

What can cause DNA damage?

A

Physical and chemical agents in the environment e.g. UV light, free
radicals produced during metabolism etc
Errors in DNA replication

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4
Q

What two general classes can DNA damage fall into?

A

Single base changes and structural distortions

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5
Q

What is damage caused by single base changes?

A

Produces mutations but have no effect on physical process of transcription or replication
Replication errors due to keto-enol type tautomerisation (alterong of a GC bond to form 2H instead of 3H)
Deamination of cytosine to uracil
Incorporation of U rather than T during replication
Chemical modification of bases (adding of methyl groups or longer chains of carbon (alkylation))

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6
Q

What is damage caused by structural distortions?

A

May impede transcription and/or replication
Single strand breaks
Covalent modification of bases e.g. alkylatio
Removal of a base
Interstrand and intrastrand covalent bonds

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7
Q

What is the best studied structural distortion?

A

Thymine dimer formation caused by UV light
Two adjacent thymines on the same strand become covalently linkedin a cyclobutane structure or a (6-4) photoproduct
Can become covalently linked when exposed to high energy
Forming of thymine dimer, making it harder for the structure distorted

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8
Q

How do cells sort out mismatches and structural distorions in the DNA?

A

Cells have systems which recognise mismatches and structural distortions in DNA and resolve these with a range of repair processes
Direct Repair - reversal or simple removal of the damage
Mismatch repair - detection and repair of mismatched bases
Excision repair - recognition of the damage followed by excision of a patch of DNA and its replacement by undamaged DNA
Tolerance systems - allow DNA replication to proceed through
damaged regions of DNA
Retrieval systems - recombinational processes to repair damaged DNA (less sffectove down the chain

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9
Q

What is the direct repair process of photolyase?

A

Photoreactivation is an example of direct repair (activated when cell in the light)
This process repairs any UV-induced intrastrand pyrimidine dimer (almost always thymine dimers)
The enzyme deoxyribopyrimidine photolyase binds specifically to pyrimidine (thymine) dimers in the dark
Photolyase contains two chromophores that absorb light energy in the range 300-600nm
Absorbed energy is used to split cyclobutane structures

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10
Q

What is the role of uracil DNA glycosylase in mismatch repair?

A

Uracil is occasionally incorporated into DNA instead of thymine (U more likely to bind to C than A, resulting in mismatch)
Uracil DNA glycosylase removes uracil base from the nucleotide making an AP site (apurinic or apyrimidinic) - doesn’t break the DNA, just removes a single base

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11
Q

What is the role of AP endonuclease in mismatch repair?

A

Makes a break in the phosphodiester backbone adjacent (5’) to the AP site (makes break where single base removed by uracil DNA glycosylase)

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12
Q

What is the role of DNA polymerase I in mismatch repair?

A

DNA polymerase I binds to the break (created by AP endonuclease) and lays down new DNA and the gap is sealed by DNA ligase

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13
Q

What is the mut system?

A

System in E. coli that recognises mismatches in the DNA or short insertions or deletions in the DNA (only does this on heavily methylated DNA)
Made up of MutS, MutL, MutH and MutU

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14
Q

What happens within the mut system?

A

MutS recognizes mismatches and short insertion/deletions (indels) on hemi-methylated DNA and binds to them
MutL binds and stabilises the complex
The MutS-MutL complex activates MutH
MutH locates a nearby methyl group and nicks the newly synthesised strand opposite the methyl group (assumes damage is on the newly synthesises strand)
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 ligaseseals the single strand break

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15
Q

In E. coli, what are the three excision repair modes?

A

Very short patch (deals with mismatches between bases)
Short patch ~20 nucleotides
Long patch 1,500 – 10,000 bps

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16
Q

What do both the short and long patch excision repair models utilise?

A

The repair endonuclease encoded by uvrA, uvrB and uvrC

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17
Q

What happens int he process of excision repair?

A

The enzyme (uvrABC) binds to damaged regions
Makes an incision on both sides of the damage (highlights where damage is)
UvrD (alias MutU, DNA helicase II) separates strands
As in mismatch repair DNA polI replaces the DNA and DNA ligase fills the gap

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18
Q

What do short patch repair account for?

A

99% of bulky lesions repair events

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19
Q

What is long patch repair?

A

An inducable activity

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20
Q

What are tolerance systems?

A

When a cell cannot fix it’s DNA as damage too severe - DNA Pol I and III cannot fix the DNA
Cell induces low-fidelity DNA polymerases (translesion synthesis polymerases (TSPs)) which can synthesise DNA past damaged bases

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21
Q

What is the downside in using translesion synthesis polymerase?

A

Not efficient at replicating undamaged DNA accurately
Most lack proof-reading ability so more likely to make errors

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22
Q

How many translesion synthesis polymerases are there in E. coli and how many are in humans?

A

Two in E. coli, polymerases IV and V, and five in human cells
Can’t replicate undamaged DNA accurately but can replicate damaged DNA

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23
Q

What is the role of the TSP human polymerase η (eta)?

A

Can bypass the major UV photoproduct very efficiently, usually inserting the correct nucleotides. It is less efficient with most other types of damage (can cause more damage)

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24
Q

What is polymerase η?

A

TSP (translesion synthesis polymerase) defective in the variant form of a highly skin-cancer-prone genetic disorder, xeroderma pigmentosum. So η helps to prevent UV-induced mutations and cancer
In its absence, one of its cousins is thought to substitute for it, but does not do a very good job

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25
Q

What are retrieval systems?

A

Does not actually repair damage
Permits replication to occur successfully
Relies on other repair processes such as excision repair to repair the damage afterwards
If another copy of genome present in the cell, it can use the copy to try and fix the cell

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26
Q

What is the SOS response?

A

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

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27
Q

What genes are associated with the SOS response?

A

umuCD, dinB - TSPs
sulA - inhibitor of cell division (stops process of replication to repair the genome)
ssb - ssDNA binding protein
uvrA and B - involved in repairing UV dimers in DNA
polB - DNA polymerase II
dinD, dinF - ?
lexA - LexA repressor

28
Q

What are all genes and operons under SOS control subject to repression by?

A

LexA protein

29
Q

What is the LexA protein?

A

LexA has two domains – a dimerisation and a DNA-binding domain
There is a conserved binding site known as a ‘LexA box’ located within the promoter of genes regulated by LexA

30
Q

What is the role of the LexA box?

A

Binding of LexA to the LexA boxes represses expression of SOS operons

31
Q

What is the role of RecA proteins in the SOS response?

A

Inducing the SOS response (produced all the time in E. coli)
RecA responds to DNA damage (e.g. presence of ssDNA), changes conformation which activates it (RecA becomes RecA*)
RecA searches for damage and changes conformation if severe damage is found

32
Q

What does activated RecA (RecA*) do?

A

Inhibition of the 3’-5’ editing in DNA Pol III, allowing error-prone DNA replication
Interacts with LexA, which autocleaves becomes inactive leading to the SOS response

33
Q

What happens to RecA* and LexA after sulA is expressed, inhibiting cell division?

A

Once DNA damage is repaired RecA* converts back to RecA
LexA stops autocleaving and concentration increases
LexA represses SOS operons and cell division occurs

34
Q

What does the SOS response allow the cell to do?

A

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
Every organism does a process similar to this

35
Q

Besides the main function of the SOS response, what else does the SOS response acts as?

A

An antimicrobial resistance mechanism as well, particularly against quinolones

36
Q

What are genes that are always expressed known as?

A

Constitutive expression

37
Q

What are the two general transcriptional mechanisms for the control of gene expression?

A

Induction - the switching on of genes when they are required
Repression - the switching off of genes when they are not required

38
Q

What is an operon?

A

In bacteria and archaea some genes are transcribed together from a single promoter
Regulated like it was a single gene
Have a linked function and produce a polycistronic mRNA
More than one gene per mRNA

39
Q

When do induction and repression occur?

A

When a regulatory protein and the DNA interact:
Repressors are regulatory proteins which prevent transcription when bound to the DNA Activators (apoinducers) are regulatory proteins which activate transcription when bound to the DNA

40
Q

What are the roles of induces and co-repressors?

A

Regulatory proteins are often converted between inactive and active states by binding of small molecules (effectors)
Inducers activate activators or inactivate repressors, switch genes on
Co-repressors activate repressors or inactivate activators, switch genes off

41
Q

What are regulons?

A

Genes associated with a particular physiological function that are not in just one operon and together are controlled by a single regulatory protein

42
Q

What does the phosphate or PHO regulon consist of?

A

> 80 genes in many operons, all are controlled by one regulatory protein

43
Q

What are global control or global regulation systems?

A

Control systems that operate on such a wide basis because they regulate many genes associated with different metabolic functions in response to a single environmental factor

44
Q

What are examples of global regulatory systems in E. coli?

A

SOS response >30 genes
Heat shock >40 genes
Anaerobic respiration >20 genes
Catabolite repression >300 genes

45
Q

What is diauxic growth in E. coli?

A

E. coli will use glucose as its main carbon source, even if alternative carbon sources are present glucose is used first (e.g lactose)

46
Q

What is catabolite repression in E. coli?

A

Glucose represses the synthesis of enzymes that metabolise less preferred carbon sources
If lactose is not available or glucose is available an E. coli cell will have very few (<5) molecules of the enzymes that metabolise lactose

47
Q

What happens when glucose is not available for the E. coli?

A

When the cells runs out of glucose there is a very rapid induction of the enzymes of lactose metabolism
Protein synthesis can be detected within 2-3 minutes, ultimately leading to about 5000 β-galactosidase molecules per cell

48
Q

What two systems control the operating of diauxic growth?

A
  1. Catabolite repression by glucose
  2. Induction by lactose
49
Q

What is in the lac operon?

A

Encodes for 3 different enzymes, lacZ (β-galactosidase) , lacY (gene that allows lactose into the cell) and lacA (removes toxic compounds that are similar to lactose) and all are expressed at the same time

50
Q

What does lacI encode for?

A

Produces lacI repressor, which binds to the operator site when the cell doesn’t want to use lactose, interfering with RNA binding to the promotor, no gene expression

51
Q

What happens in the lac operon when lactose is present in the cell?

A

Lactose is converted into allolactose
Allolactose binds to the lacI repressor, causing it to fall off allowing RNA polymerase to bind and produce RNA form the operon
This produces the three proteins that allows more lactose to enter the cell and produce more β-galactosidase to break this lactose down

52
Q

How did Jacob and Monod show that specific proteins are expressed to regulate gene expression using the lac operon found in gene expression?

A

Used a merodipliod (partially diploid) E. coli by inserting lac operon genes into an F’ plasmid to complement mutated lac operon genes on the chromosome (not naturally occurring/genetically engineered)
The effect of the mutation helped the determine that some genes encoded for diffusible products that regulated gene expression on both DNA molecules (trans-acting elements) whilst other genes did not produce a product but regulated genes on the DNA molecule they were encoded upon (cis-acting elements)

53
Q

Why is “trans/cis-acting” not relevant to the lacZ, Y and A proteins?

A

All transcribed into mRNA, then translated to protein
Not regulatory proteins
They can be complemented in merodiploid cells

54
Q

What happens if lacZ and lacY are not present?

A

Allolactose induces expression on both molecules
Chromosome produces a functional
LacZ F’ plasmid produces a functional LacY
These complement the two mutated genes, so phenotype is “normal”

55
Q

Is lacI trans or cis-acting?

A

Trans-acting element
Not transcribed and both are binding sites for proteins

56
Q

What does trans-acting element mean?

A

Gene product diffuses to affect both DNA molecules
Phenotype: normal Lac⁺ inducible

57
Q

What does cis-acting element mean?

A

Mutated lacO has no gene product so only affects DNA molecule it is on
No repression occurs on chromosome operon
Phenotype: mutant Lac⁺ constitutive

58
Q

What is an easy way to tell the difference between trans-acting and cis-acting elements?

A

Elements of DNA that are mutated and don’t produce products tend to be cis-acting elements
Elements that do produce gene products (like protein) tend to be trans-acting

59
Q

Why is the paradigm that DNA makes RNA makes DNA not always true?

A

DNA makes RNA that regulates DNA

60
Q

What are non-coding RNA (ncRNA) molecules?

A

Small, non-coding RNA molecules to regulate gene expression

61
Q

What is an example of ncRNA regulation involving RNA degradation?

A

One type of addiction cassette produces a very stable mRNA of a lethal membrane protein, but also makes a ncRNA, a highly unstable anti-sense RNA molecule that binds to mRNA to prevent translation of lethal mRNA
So long as the plasmid is passed to a daughter cell in replication the lethal
mRNA is repressed (when plasmid is present, RNA degradation non lethal)

62
Q

What happens when a plasmid-free cell is produced during RNA degradation?

A

The unstable antisense RNA cannot be produced (no plasmid)
It degrades rapidly and the lethal RNA is translated leading to cell death (lethal)

63
Q

What is the archetypal member of RNA-based addiction cassettes?

A

The hok (host killing) sok system of plasmid R1 is the archetypal member of such RNA-based addiction cassettes
Regulation of expression by ncRNA (uses stable mRNA and unstable ncRNA control)

64
Q

How does E. coli control it’s iron usage?

A

Using the ncRNA, RhyB

65
Q

What happens in E. coli when iron is freely available?

A

Global regulator FUR binds Fe²⁺ and represses ryhB
All iron-requiring proteins are made
Any free iron is bound to protein

66
Q

What happens in E. coli when iron is limited?

A

Without iron, FUR stops repressing rhyB (FUR requires iron to repress)
RyhB ncRNA binds to mRNA of nonessential iron proteins which degrades mRNA
Iron need of cell falls
Self regulates as if iron concentration rises ryhB repression reactivated

67
Q

What does RhyB regulate?

A

Many mRNAs, classic “ncRNA degrades mRNA” regulation

LF206: Molecular Cell Biology (18 decks)

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