Genetics Flashcards
Which phase can individual chromosomes be distinguished?
Metaphase
What is a karotype?
Organised representation of all the chromosomes in a eukaryotic cell at metaphase as they condense
Describe the different types of DNA coiling
(heterochromatin)- 30nm
(euchromatin)- 10nm
What histones are contained in a typical nucleosome?
2 H2A
2 H2B
2 H3
2 H4
What do the N-terminals of histone subunits do?
Interact with other proteins
Facilitate regulation and function of chromatin
What do linker histones (e.g H1) do?
Attach DNA onto histone octamers, limiting DNA movement
Stabilises formation of 30nm fibre which is transcriptionally silent
Rich in Lys and Arg, making it basic and can bind DNA
The chromatin scaffold can be remodelled to accommodate protein complexes for gene transcription and DNA replication
What does interphase chromatin contain which condense and decondense?
Set of fractal globules (globules within globules) which condense and decondese without knotting
What is contained in the nuclear periphery of interphase cells?
Transcriptionally inactive DNA
RNA transcripts are excluded
What do specialised DNA sequences ensure for complete interphase?
Facilitates complete DNA replication
Segregation of duplicated chromosomes in cell division
What does telomerase synthesise?
TTAGGG repeats on the 3’ overhang of chromosomes
How are centromeres defined?
171bp alpha satellite repeats that form condensed chromatin with histone octamers
What do kinetochore inner plate proteins bind to?
alpha satellite DNA
What do kinetochore outer plate proteins bind to?
protein components of the mitotic spindle
Describe a yeast centromere bound to a microtubule
Single nucleosome of centromeric chromatin is linked to a single microtubule with a basket structure
What increases with biological complexity?
Protein coding DNA
Non-protein coding DNA
What does non-protein coding DNA encode?
regulating transcription and organising access to protein-coding genes
Cis-regulatory information which determines when and where adjacent protein coding genes are transcribed
Describe transposons
Mobile genetic elements that jump around the genome
Can be non-replicative and replicative
Give three examples of transposons
P-element in flies, Activator/dissociator in maize where coloured kernals jump around the genome, Tn3/Tn10 in E.coli
Describe retroviral-like transposons
Replicate via RNA intermediates
Produce new DNA copies which integrate at new genomic locations
Self-encoded reverse transcriptase
Do not encode fully active infective viruses
Describe non-retroviral transposons
Abundant in vertebrate genomes
Replicates via an RNA intermediate with its own reverse transcriptase
Replicative
Give some examples of non-retroviral transposons
LINEs
SINEs
Human L1 elements
Human Alu elements
Mouse B1 elements
Describe an L1 insertion in humans
- L1 RNA synthase generates an ssRNA strand
- An endonuclease cleaves the first strand of target DNA
- DNA-primed reverse transcription takes place and a complimentary strand is synthesised
- The strand reintegrates with the inserted gene
What is an example of a disease caused by an L1 insertion?
Haemophilia
What initiates DNA synthesis at molecular level?
DNA helicase forming a replication fork
What machinery is involved in DNA replication?
DNA helicase- spliting DNA strands
DNA primase- synthesise short RNA primers
Sliding clamp
Clamp loader
DNA polymerase- adds nucleotides
What does DNA polymerase require to add nucleotides?
A pre-existing primer chain
Polymerase extends the RNA primer once it is in place
What removes the RNA primer from DNA?
Ribonuclease H
When the RNA pirmer is removed from DNA, what seals the nick?
DNA ligase
What can mutations in DNA helicase lead to?
Werner syndrome (premature aging)
Autosomal recessive in RecQ helicase gene WRN
What can a loss of function mutation in RecQ-family DNA helicase lead to?
Bloom syndrome- extreme sensitivity to sun exposure
Autosomal recessive
What increases the processivity of DNA polymerases?
Sliding clamp helps move DNA pol forward
When DNA pol interacts with the primer, template junction is maintained and nucleotides are rapidly added
What keeps the sliding clamp close to the primer:template junction?
Clamp loader
How is ssDNA kept available as a template during semi-conservative replication?
ssDNA binding proteins (SSBs) which keep the replication fork open and allow DNA pol to function
Describe DNA topoisomerases
Prevent DNA becoming tangled during replication, increasing processivity of DNA pol
This is because helicase unwinding parental DNA at the replication fork introduces tension in DNA
Topoisomerases nick and reseal DNA
What is the difference between type I and type II DNA topoisomerases?
Type I- nick and reseal 1 DNA strand (no ATP)
Type II- nick and reseal both DNA strands (requires ATP)
What is the difference between human and yeast origins of replication?
Yeast- autonomously replicating sequences (ARS)
Human- sequences near LMNB2, MYC
Why is initiation of eukaryotic DNA replication biphasic?
Two stages:
1. Replicator selection in G1 phase where pre-replicative complex forms
2. Origin activation occurs at the start of S-phase where DNA unwinds and DNA polymerase is recruited
What does temporal selection when initiating DNA replication ensure?
Each origin is used and each chromosome replicates once per cell cycle
What does an origin recognition complex bind to?
Replicator sequence in the origin, e.g ARS in yeast
Outline formation of the pre-recognition complex in G1 phase?
- The ORC binds to the replicator sequence
- Cdc6 and Cdt1 bind to the ORC
- Helicase Mem2-7 binds and the pre-recognition complex forms
What activates the pre-recognition complex?
Cyclin-dependent kinase (Cdk) in S-phase. This inhibits new pre-recognition complexes forming
What ensures chromosomes are replicated exactly once per cell cycle?
There is close relationship between pre-RC function, Cdk levels and cell cycle
How is DNA replication finished with the unsealed gap?
At chromosome ends, pol or ligase haven’t sealed a gap. This is next to an RNA primer which is removed by ribonuclease H, shortening newly synthesised DNA strands at the 5’ ends
This can risk losing valuable coding information- premature aging and mutations
Outline how telomerase functions
- Adds TTAGGG repeats on 3’ end to compensate for loss of telomere sequences from RNA primer removal
- DNA primase binds and initiates RNA primer synthesis
- DNA polymerase extends this as an extra okazaki fragment
What is the telomerase shuffle?
Telomerase RNA component acts as a template where the telomere repeat sequences are synthesised in a step-wise process
When there are errors in DNA, what can happen to it?
DNA repair
cell death
Stay mutated and replicate
What are some endogenous causes of DNA damage?
Reactions with other molecules in the cell
Hydrolysis
Reactive oxygen species
What are some exogenous causes of DNA damage?
UV
X-rays
Carcinogens
Chemotherapies
What are some consequences of endogenous DNA damage?
Depurination
Deamination
Methylation
Replication errors
What are some consequences of exogenous DNA damage?
Pyrimidine dimers
Double strand links
Interstrand crosslinks
Are transversions or transition mutations more likely>
Transitions (pyrimadines to pyrimidine, vice versa)
Explain the outcome of a depurination of adenine
A purine is cleaved off by the N-glycosidic bond, causing a wobble base
A frameshift can occur, generating missense proteins
What mutations can UV light cause?
Pyrimidine dimers due to DNA to distorting
Interstrand DNA crosslinks- DNA cannot unwind for replication
DNA-protein crosslinks
What can induce single strand breaks in DNA?
X-rays
Ionising radiation
Topoisomerase II inhibitors
What can induce double strand breaks in DNA?
ROS
Hydroxyurea
Camptocthecin
Outline base excision repair (BER)
- DNA glycosylase removed damaged base
- AP endonuclease and phosphodiesterase remove the sugar phosphate
- Pol and ligase add a new nucleotide and seal the nick
Repairs abasic sites and deamination
Outline nucleotide excision repair (NER)
Endonuclease cuts phosphate bonds
DNA helicase removes short stretches of ssDNA.
Pol and ligase seal the nick
Outline how translesion DNA polymerase replicates damaged DNA
- When polymerase encounters DNA damage, the sliding clamp is covalently modified
- DNA pol is released and translesion polymerase is loaded onto DNA
- DNA is synthesised and covalent modifications are removed.
- Translesion DNA pol is replaced by replicative DNA pol again
What is the double strand break repair that happens in G1?
Non-homologous end joining:
Error prone
Results in loss of nucleotides around break site
Ku70/80 and DNA PKes facilitate end joining
Ligase IV seals gaps
What is the double strand break repair that happens in S phase?
Homologous recombination:
Error free repair that uses sister chromatids as a template
How is DNA damage initially detected?
- ATM/ATR kinase associate with the site of DNA damage
- Chk1/Chk2 kinase activates and phosphorylates p53
- p53 dissociates from mdm2 and binds to a regulatory region of the p21 gene
- This inactivates G1/S-CDK and S-CDK complexes, preventing the cell cycle progressing
What is xeroderma pigmentosum?
Autosomal recessive condition that increases skin cancer risk 2000 fold
Associated with a defective NER pathway (UV damage)
How are cells with DNA damage studied?
Comet assay- electrophoresis to look at overall damage and find marks of DNA damage
