IMED2004 FULL SET

Define dna damage

a physical abnormality in dna- can block transcription and hence translocation.

What is a consequence of dna damage in frequently dividing cells

Can give rise to mutations which can allow cells to proliferate without control/evade apoptosis- cause of cancer

What is a consequence of dna damage in infrequently dividing cells

Accumulates over time and contributes to aging

Name the 6 common dna repair mechanisms

1. Mismatch. 2. direct. 3. base excision. 4. nucleotide excision. 5. homologous recombination. 6. non-homologous end joining

Outline the 3 steps in mismatch repair technique

1. recognise mismatched bases by detecting dna distortions.
2. Determine which base is the correct one.
3. Excise incorrect base and repair

Outline step 1 in mismatch repair in E. coli

1. during dna replication, MutS recognises and binds to mismatches

Outline step 2 in mismatch repair in E. coli

2. MutH recognises the newly synthesised daughter strand by identifying hemi-methylated CATG sites and nicks the DNA

Outline step 3 in mismatch repair in E. coli

3. MutL recruits a helicase, the entire MuSHL comples moves along the dna, separating the dna strand to be removed. The segment of this strand including the incorrect nt is degraded by exonuclease. Gap is filled by polymerase and sealed by ligase.

Name the three mismatch repair proteins

MutS, MutH and MutL

Name four ways that bases can be damaged

Oxidation, deamination, alkylation and hydrolysis

What is the name of the non-human direct reversal protein

Photolyases

What is the name of the human direct reversal protein + give example

Alkyl transferase. E.g. MGMT

How does MGMT work

irreversible transfers the errant methyl group onto itself and is subsequently degraded

What type of damage is base-excision repair used for

lesions that do not distort the double helix

What proteins initiate base-excision repair

DNA glycoslyases

Outline step 1 of base-excision repair

1. Each dna glycoslyase recognises and removes a specific type of damaged base

Outline step 2 of base-excision repair

AP endonuclease cleaves the phosphodiester bond on the 5' side of the AP site

Outline step 3 of base-excision repair

AP endonuclease removes the deoxyribose sugar

What are the two forms of excision repair

Short and long patch

What polymerase performs short-patch repair

DNA polymerase beta

What polymerase performs long-patch repair

DNA polymerase delta or epsilon

What type of dna damage is nucleotide-excision repair used for

Bulky lesions that distort the double helix, including pyrimidine dimers

What are the two sub-pathways of NER

I. Global genomic NER and II. Transcription couples NER

Outline the first step of GG-NER

damage sensing proteins constantly scan the genome and recognise distortion in the dna helix

Outline the first step in TC-NER

Inititated when dna damage causes rna pol to stall at the bulky lesion

Outline step 2 of NER

Assembly of multi-protein complex at site, including helicase and CCB protein to stabilise the single strands

Outline step 3 of NER

On affected strand, dna around the lesion is excised by cleaving the sugar-phosphate backbone

Outline step 4 of NER

dna Pol fills the gap using undamaged strand as template and ligase seals the backbone.

Name the human disease caused by mutation in GG-NER

Xeroderma pigmentosum

Name the human disease caused by mutation in TC-NER

Cockayne syndrome

What type of dna damage is homologous recombination used for

DS breaks in actively dividing cells

What type of dna damage is non-homologous end joining used for

DS break in inactive cells

Which of the two forms of DS break repair systems is usually error prone

Non-homologous end joining

When can homologous recombination occur

Only during S phase when dna is replicating to sister chromatid can be used

Briefly outline the steps in homologous recombination

Resection, formation of nucleoprotein filament, strand invasion, dna synthesis from free 3' end of invading strand, separation from template and re-annea with original strand.

Outline the steps in non-homologous end-joining

1. Recognising dna damage. 2. binding of broken ends by KU20 + 80. 3. trimming the ends to get blunt 5'P and 3'OH. 4. Ligation by DNA ligase IV

What us a transition substitution

Purine replaced by a purine

what is a transversion substitution

Purine replaced by a pyrimidine

What two bases are purines

A + G

What two bases arep yrimidines

T + C

What is a forward mutation

Mutation that alters wild type

What is a reverse mutation

Changes mutant back to WT

What is a suppressor mutation

A genetic change that hides or suppresses the effect of another mutation

What is depurination

Loss of purine base from nucleotide. Results when covalent bond connecting purine to 1' carbon atom of deoxyribose sugar breaks, producing apurinic site (A-P)

How can an apurinic site lead to transversion mutation

Apurinic site cannot provide a template, nt with incorrect base will be incorporated into the new strand, at the next round the incorrect base will be used as a template leading to a permanent mutation.

What mutagens can induce an A-P site

radiation, hydroxyl radicals, enzymatic activity, dna adducts

Define mycotoxin

Toxic compounds that are naturally produced by certain types of moulds that grow on cereals, dried fruits and nuts.

How does aflatoxin B1 promote liver cancer

Promotes A-P site formation that forms DNA adducts

What is deamination

Loss of amino group (NH2) from a base

What is the dominant mutation of aflatoxin b1

G > T

What is the dominant mutation caused by UV radiation

C > T

What is the aetiology of cancer type signature 1

Endogenous mutational process initiated by spontaneous deamination of 5-methylcytosine

What is the aetiology of cancer type signature 7

Due to UV light exposure, large numbers of CC>TT mutations

What occurs to the epigenome during embryogenesis

A global wave of dnamethylation that increases H3K27 and H3K4

What gene is a mutation found in that causes obesity and yellow coat colour in mice

agouti

How does a mutated agouti gene cause yellow coat colour

Agouti encodes paracrine signalling molecule that promotes follicular melanotytes to produce yellow phaeomelanin rather than black

At what number of cells in an embryo do cells choose one X to remain on

1000 cell stage

What inactivates X

XIST and X-chromosome-encoded IncRNA. XIST coats X chr leading to heterochromatin spreading and dna hypermethylation

What is genomic imprinting

When two copies of a gene are inherited (maternal and paternal) only one is expressed, the non-expressed one is imprinted. It is epigenetic because it is a heritable change of gene expression not in the dna sequence.

How many imprinted genes have been identified in the human genome

70

What body functions can imprinted genes have an effect on

Growth regulation, brain function, behaviour

angleman syndrome symptoms

Severe retardation, microcephaly, lack of spech

What causes angelman syndrome

Deletion of maternal 15q11-a13 causing no maternal UBE3A

Symptoms of Prader syndrome

Mild mental retardation, obseity, short stature.

What causes Prader-Willi syndrome

Deletion of paternal 15q11-q13 causing decreased snoRNA

Symptoms of Beckwith-Wiedemann syndrome

Embryonic and placental overgrowth, predisposition to childhood tumours

What causes Beckwith-wiedemann syndrome

genetic and epigenetic changes in a regions of 1 megabase on chr11p

what is the first molecular type of beckwith-wiedemann syndrome

Gain of methylation at ICR1- controlling H19 non-coding rna expression

what is the second molecular type of beckwith-wiedemann syndrome

loss of methylation at ICR2 controlling KCNQ11OT1 non-coding rna expression

what is the third molecular type of beckwith-wiedemann syndrome

Paternal uniparental disomy

what is the fourth molecular type of beckwith-wiedemann syndrome

CDKN1C mutations

what is the fifth molecular type of beckwith-wiedemann syndrome

Chromosomal rearrangements

IGF2 and CDKN1C- which is paternal and maternal

IGF2 is normally on paternal chromsomes and CDKN1C is maternally expressed.

What gene variant is obesity in humans associated with

FTO gene

What epigenetic biomarker can be used to determine gestational age

DNA methylation

What happens to dna methylation as we age

Progressive methylation loss

What epigenetic marker does cancer normally show

global hypomethylation and focal hypermethylation of CpG islands

What is a CpG island

In humans, genes that have GC rich areas of DNA in promoter regions

What does methylation of C residues in CpG islands cause

Gene silencing/repression

What is DNMT1 responsible for

The maintenance of established patterns of dna methylation- follows the replication fork adding methylation marks to newly synthesised dna

What is DNMT3a and b responsible for

Mediate establishment of new or de novo dna methylation patterns

What enzyme mediates active demethylation

Ten-eleven translocation (TET 1,2 and 3)

How does passive DNA demethylation occur (TET-independent demethylation)

Inhibiting maintenance DNMT1 during division

What is an epigenetic mutation

Inherited defects that have an effect through epigenetic mechanisms

What is an imprinting disorder

can result from abnormal methylation of key genes in growth and development

What mode of inheritance is fragile X

x-linked dominant

What symptoms does fragile x cause

Intellectual disability, behavioural and learning challenges, physical characteristics

What is the most common cause of fragile x syndrome

Expansion of CGG triplet repeat region in 5'UTR of FMR1 gene which leads to silencing of the gene

What is Rett syndrome

Neurological disorder of the brain grey matter leading to deceleraton of the head growth and small hands, mostly affects females

What is the main cause of rett syndrome

mutations in MECP2 gene on X chromosome

What is epigenetics

Factors that cause stable and heritable, yet reversible, changes in the way genes are expressed without changing their original dna sequence.

What are two mechanisms of epigenetic regulation

DNA methylation and histone modification

What is the histone code hypothesis

Modifications of the histone tails act as epigenetic marks that control the expression of chromosomal regions. The marks are heritable , they act sequentially to form the histone code.

What is the nucleosome comprised of

Histone octamer- H2A, H2B, H3 and H4 (2 units of each)

What is chromatin composed of

DNA and histones

What is the name of condensed chromatin

Heterochromatin

What is the name of extended chromatin

Euchromatin

What charge are histone proteins and why

Positive due to arginine and lysine aa's.

Is acetylated chromatin transcriptionally active or repressed

Active- in extended form

How does acetylation promote transcription

Acetylation of histone tails neutralises their positive charge and releases the negatively charged dna