DNA Repair Flashcards
(33 cards)
Why is DNA repair important?
1 unprepared DSB is sufficient to kill a cell
Defects in repair pathways contribute to tumourigenesis- promote chromosomal translocations
What is the difference between mono- and multigenic diseases?
Multigenic- mutations in multiple genes, the combination causes the clinical phenotype
Monogenic- mutations in a single gene that are more severe, and tend to be recessive so they escape selection pressure
What is replication stress?
Any process that obstructs DNA replication or interfere with the replication forks
Unresolved replication stress may lead to genetic instability
Give specific examples of monogenic diseases in the DNA damage response
Fanconi anaemia- variations on the FANC gene- bone marrow failure, skeletal defects, genome instability, myeloid leukaemia
Seckel syndrome- ATR, ATRIP- growth retardation, dwarfism, microcephaly, mental retardation, genome instability
Ataxia Telangiectasia- ATM- genome instability, T/B cell leukaemia/lymphoma
Ataxia Telangiectasia-like disease- Mre11- neurodegeneration, ataxia, genome instability
Bloom syndrome- BLM- premature ageing, growth retardation, genome instability, leukaemia/lymphoma
Describe Seckel1
Seckel syndrome is a group of monogenic inherited disorders
-severe microcephaly, growth retardation, mental retardation, dysmorphic features, skeletal abnormalities
Rare autosomal recessive disorder
ATR kinase or ATRIP
Are functionally related proteins that form part of the DDR
Found at replication forks, are crucial in maintaining genome stability
Defects in phosphorylation if Chk1 and H2AX
Increased numbers of centrosomes
Rapidly proliferating CNS progenitor cells cannot deal with stress without ATR/ATRIP
Describe Bloom syndrome
Growth retardation, photosensitivity, Telangiectasia, hypo- and hyper pigmented skin, predisposition to malignancies, chromosomal instability
BLM is a helicase involved in resolving intermediates of DNA repair and removing proteins from DNA
➡️ reflects on replication, genome instability, increased genetic exchNge between chromosomes
How would you identify where a disease gene is?
Use genetic markers to analyse inherited regions- linkage
Linkage is the non-random association of traits passed from parents to offspring including restriction enzyme markers and SNPs
Requires at least two generations with affected and unaffected individuals
Genotype using marked and analyse regions conserved between affected individuals and carriers
Evaluate all genes in that region to identify the causative mutation
What is the principle behind using linkage when identifying disease genes?
If two sequences are close together they are likely to be inherited together- less likely to split up during recombination
Use restriction sites (trace inheritance of a particular pattern of restriction fragments) SNPs (SNP arrays- anneal to probes- measure fluorescent signal) and microsatellites
What are the cyclin and Cyclin dependent kinases involved at the different stages of cell cycle?
G1- Cyclin D1-3 Cdk2,4,5,6 G1-S- Cyclin E Cdk2 S- Cyclin A Cdk2 G2 and M- Cyclin A and B Cdk2
What are cell cycle checkpoints?
A point in eukaryotic cell cycle where progress through the cycle can be halted until conditions are suitable for the cell to proceed to the next stage
They prevent the propagation of deleterious genetic errors
Describe the G1-S checkpoint
Prevents entry into S phase and the replication of damaged DNA
Targets G1 phase Cdk activity
Controlled by ATM
DSB➡️MRN➡️ ATM-P➡️
➡️p53-P➡️p21- cdk2 inhibitor
➡️Chk2-P➡️cdc25A-P➡️ degraded so it does not remove the phosphates from Cdk2➡️ cdk2-P inactive
Describe the regulation of p53
Normally MDM2, bound to USP7 and inactive p53, targets and ubiquitinates p53 for destruction
If there is a DSB ATM-P inactivates MDM2 and blocks the interaction with USP7, it is ubiquitinated and degraded
ATM also activates Chk2 which activates p53 (WIP1 also removes phosphorylations on p53)
p53 promotes the transcription of p21 but build up of p53 in the cell activates MDM2 in a negative feedback loop
Name two important serine residues on p53 for phosphorylation
Serine 15 and 20
Regulated by ATR, ATM, DNA-PK, chk1 and chk2
Decrease interaction with MDM2 increase transcriptional activity
Describe the intra-S phase checkpoint
Prevents replication of damaged DNA
Both elongation and late origin firing are inhibited
Target replication associated cdk activity (cdk2/7)
Controlled by ATM/ATR dependent DDR pathway
DSB➡️MRN ➡️ MDC1➡️ ATM-P➡️ chk2-P➡️
Single strand DNA of the replication fork➡️ 9-1-1/TOPBP1➡ ATR/ATRIP-P➡️ chk1-P➡️
➡️cdc25A-P is degraded➡️ cdk2-P prevents exit out of S phase
Name some proteins, sites of phosphorylation and associated diseases
ATM- S367, T1885, S1981, S2996- AT RAD50- S635- NBS-like disorder BRCA1- S1387- Fanconi anaemia complementarity group S ATR/ATRIP- T1989 on ATR- Seckel syndrome Chk1- S317, S345
Describe the G2/M phase checkpoint
Prevents entry into mitosis with DNA damage
Targets G2/M phase cdk activity eg. Cyclin B and Cdk2
Controlled by ATM/ATR dependent DDR
Resected DSB (single DNA strand)➡️
9-1-1/TOPBP1 ➡ATR/ATRIP-P ➡️
MRN➡️ MDC1➡️ ATM-P➡️
➡️chk1 and 2-P➡️ cdc25B/C-P➡️ cdk1-P (inactive)
(➡14-3-3 exported from the nucleus)
What can replication cause?
Replication forks that are not moving properly can be converted into double strand breaks
Most likely to happen during S phase and also mitosis
How are replication forks converted into DSBs?
Stalled forks means that the polymerase on the lagging strand can fall off
Then endonuclease processes it into a one ended DSB by cutting the single strand DNA at the ‘mouth’ of the fork
When are regressed replication forks found in normal cell division?
They are holiday junctions that make up chiasmata during recombination in meiosis
Endonuclease normally cuts Holliday junctions and a regressed fork is seen as the same structure and so it is cut
Describe the direct/alternative replication fork collapse
Single strand break on the template that is not repaired
Enzyme encounters break and falls off template
SSB transformed into a DSB
How are collapsed replication forks dealt with?
DSB➡️ ATM pathway
ssDNA➡️ ATR pathway
Degradation of cdc25A and phosphorylated cdk1, 2 lead to cell cycle arrest
Either homologous recombination or apoptosis
Describe homologous recombination of a one ended DSB
A 3’ over hang is required EXO1, Mre11 and BRCA1
Rad51 and BRCA2 finds the homologous sequence in the sister chromatid strand
Forms a D-loop
HJ resolution by HJ resolvases (GEN1, MUS81/EME1, SLX1/SLX4)
Re-establishes the replication fork
How would you target homologous recombination for cancer therapy?
Familial cancers with mutation in enzymes such as BRCA1,2 and FANCN,J have a HR defect
Tumours are sensitive to PARP1 inhibitors (SSB repair enzyme) normal somatic cells still have one wild type copy
Cancer cells die of unrelated spontaneous SSBs
What is the significance of oncogenes in replication stress?
Oncogenes slow down replication forks- spontaneous replication stress demonstrated by cancer cells- eventually the cell will go into mitosis with replication unfinished- anaphase bridge between chromatids
Leading to genomic instability, fragmentation and other damage
